Field of the Invention

The present invention relates to disaccharides, and to their use in treating and preventing inflammatory disorders.

Background of the Invention

Carbohydrates were previously thought of as only providing energy. However, it is now known that carbohydrates are involved in many other biochemical processes. Some examples of these processes are immune recognition of microbes and cellular signaling during the inflammatory . response.

A disaccharide is a carbohydrate composed of two monosaccharides. 'Disaccharide' is one of the four chemical groupings of carbohydrates (monosaccharide, disaccharide, oligosaccharide, and polysaccharide). There are two basic types of disaccharides: reducing disaccharides, in which the monosaccharide components are bonded by hydroxyl groups; and non-reducing disaccharides, in which the components bond through their anometric centers. Disaccharides are formed when two sugars are joined together and a molecule of water is removed. The two monosaccharides are bonded via a dehydration reaction (also called a condensation reaction or dehydration synthesis) that leads to the loss of a molecule of water and formation of a glycosidic bond. The glycosidic bond can be formed between any hydroxyl group on the component monosaccharide. So, even if both component sugars are the same (e.g., glucose), different bond combinations (regiochemistry) and stereochemistry (alpha- or beta-) result in disaccharides that are diastereoisomers with different chemical and physical properties. For example; milk sugar (lactose) is made from glucose and galactose whereas cane sugar (sucrose) is made from glucose and fructose.

It has been known for some time that carbohydrates, including disaccharides can limit immune reactivity (for example see Lider et al., 1995; Mazmanian et al., 2005). Indeed, more recently, the disaccharide trehalose has been shown to limit pro-inflammatory activity both in vitro and in animal models (Minutoli et al., 2008). The present invention describes a synthesised disaccharide compound that inhibits activation of the inflammatory response.

Statements of the Invention

According to one aspect of the invention there are provided disaccharides comprising the structure shown in Fig. 1 (DSl) and the structure shown in Fig. 7 (DS2).

According to another aspect of the invention there is provided use of the disaccharide of the invention as a medicament.

According to another aspect of the invention there is provided use of the disaccharide of the invention in the preparation of a medicament for treating or preventing undesirable inflammatory activity.

According to another aspect of the invention there is provided use of the disaccharide of the invention in the preparation of a medicament for treating or preventing undesirable gastrointestinal inflammatory activity.

In one embodiment the gastrointestinal inflammatory activity is Crohn's disease, ulcerative colitis, irritable bowel syndrome, pouchitis, post infection colitis, Clostridium difficile associated diarrhoea, Rotavirus associated diarrhoea or post infective diarrhoea.

According to another aspect of the invention there is provided use of the disaccharide of the invention in the preparation of a medicament for treating or preventing rheumatoid arthritis.

According to another aspect of the invention there is provided use of the disaccharide of the invention in the preparation of a medicament for treating or preventing autoimmune disorders.

The invention also provides the use of the disaccharide in the preparation of a medicament for prophylaxis or treatment of sepsis. The invention further provides the use of the disaccharide in the preparation of a medicament for prophylaxis or treatment of LPS or TLR-4 mediated inflammatory responses.

In a further aspect the invention provides the use of the disaccharide in the preparation of a medicament for reducing LPS stimulated TNF-α production.

According to another aspect of the invention there is provided a pharmaceutical composition comprising the disaccharide of the invention and a pharmaceutically acceptable carrier.

In a further embodiment the invention also provides a foodstuff comprising the disaccharide. For example the foodstuff may be one or more selected from the group comprising: yogurts, cereals, beverages and the like.

Brief Description of the Figures

Further preferred features and embodiments of the present invention will now be described by way of non-limiting example and with reference to the accompanying figures.

Fig. 1 shows the structure of a disaccharide (DSl);

Fig. 2 illustrates that the disaccharide (DSl) induces IL-10 secretion from human peripheral blood mononuclear cells;

Fig. 3 illustrates that the disaccharide (DSl) reduces release of TNF-α from human peripheral blood mononuclear cells following LPS stimulation;

Fig. 4 illustrates that TNF-α secretion is reduced following LPS and LPS + DSl injection in mice;

Fig. 5 illustrates that LPS induced serum TNF-α levels are reduced following LPS +DSl injection in mice;

Fig. 6 is an outline of the process used to prepare the disaccharide DSl; - A - Fig. 7 shows the structure of another disaccharide (DS2);

Fig. 8 illustrates that the disaccharide DS2 induces IL-IO secretion from human peripheral blood mononuclear cells;

Fig. 9 illustrates that the disaccharide DS2 reduces release of TNF-α from human peripheral blood mononuclear cells following LPS stimulation;

Fig. 10 illustrates that LPS induced serum TNF-α levels are reduced following LPS +DS2 injection in mice;

Fig. 11 is an outline of the process used to prepare the disaccharide DS2;

Fig. 12 shows the structure of N-acetyl-D-Lactosamine; and

Fig. 13 illustrates that LPS induced serum TNF-α levels are not reduced following N-acetyl-D- Lactosamine injection in mice.

Detailed description

Various preferred features and embodiments of the present invention will now be described by way of non-limiting example.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature.

We have identified disaccharides which have immuomodulatory properties.

Treatment It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment. The treatment of mammals is particularly preferred. Both human and veterinary treatments are within the scope of the present invention.

Inflammation

Inflammation is a local response to cellular injury that is marked by capillary dilatation, leukocytic infiltration, redness, heat, pain, swelling, and often loss of function. Control of the inflammatory response is exerted on a number of levels (for review see Henderson B., and Wilson M. 1998). The controlling factors include cytokines, hormones (e. g. hydrocortisone), prostaglandins, reactive intermediates and leukotrienes.

Cytokines are low molecular weight biologically active proteins that are involved in the generation and control of immunological and inflammatory responses, while also regulating development, tissue repair and haematopoiesis. They provide a means of communication between leukocytes themselves and also with other cell types. Most cytokines are pleiotrophic and express multiple biologically overlapping activities. Cytokine cascades and networks control the inflammatory response rather than the action of a particular cytokine on a particular cell type (Arai KI, et al., 1990). Waning of the inflammatory response results in lower concentrations of the appropriate activating signals and other inflammatory mediators leading to the cessation of the inflammatory response. Tumor narcrosis factor alpha (TNFα) is a pivotal proinflammatory cytokine as it initiates a cascade of cytokines and biological effects resulting in the inflammatory state. Therefore, agents which inhibit TNFα are currently being used for the treatment of inflammatory diseases, e. g. infliximab.

Pro-inflammatory cytokines are thought to play a major role in the pathogenesis of many inflammatory diseases, including inflammatory bowel disease (IBD). Current therapies for treating IBD are aimed at reducing the levels of these proinflammatory cytokines. The exopolysaccharide of the present invention may have potential application in the treatment of inflammatory disorders. This may be achieved, for example, by increasing the concentration of non-inflammatory cytokines such as, but not limited to IL-10, and/or decreasing the concentration of inflammatory cytokines.

Inflammatory Bowel Disease Inflammatory bowel disease (IBD) is characterised by a chronic relapsing intestinal inflammation. IBD is subdivided into Crohn's disease and ulcerative colitis phenotypes. Crohn's disease may involve any part of the gastrointestinal tract, but most frequently the terminal ileum and colon. In approximately 10% of cases confined to the rectum and colon, definitive classification of Crohn's disease or ulcerative colitis cannot be made and are designated 'indeterminate colitis.' Both diseases include extra-intestinal inflammation of the skin, eyes, or joints.

Crohn's disease and ulcerative colitis are commonly classified as autoimmune diseases as both illnesses are marked by an abnormal response by the body's immune system resulting in chronic inflammation in the lining of the intestines. The prevalence of inflammatory bowel disease is increased in individuals with other autoimmune diseases, particularly ankylosing spondylitis, psoriasis, sclerosing cholangitis, and multiple sclerosis.

Crohn's disease

Crohn's disease is a chronic disorder that causes inflammation of the digestive or gastrointestinal wherein the immune system attacks the intestine.

Although Crohn's disease most commonly affects the end of the ileum and the beginning of the colon, it may involve any part of the gastrointestinal tract. Bowel inflammation is transmural and discontinuous; it may contain granulomas or be associated with intestinal or perianal fistulas. The CARD 15 gene and an allele of the ABCBl gene are thought to be associated with susceptibility to Crohn's disease.

Ulcerative Colitis

Ulcerative colitis is a disease that causes inflammation and sores in the lining of the large intestine. It is a nonspecific chronic inflammatory disease affecting the bowel. Ulcers form and bleed in places where the inflammation has killed the cell lining. In contrast to Crohn's disease, the inflammation is continuous and limited to rectal and colonic mucosal layers; fistulas and granulomas are not observed. Both genetic and environmental factors seem to be important in its etiology. Fuss et al. examined lamina propria T cells from patients with ulcerative colitis and found that they produced significantly greater amounts of IL 13 and IL5 than control or Crohn's disease cells and little IFN- gamma. They concluded that ulcerative colitis is associated with an atypical Th2 response mediated by nonclassic NKT cells that produce IL 13 and have cytotoxic potential for epithelial cells.

Pouchitis

Chronic and/or acute inflammation of the ileal reservoir, so-called "pouchitis", is a frequently observed long-term complication of the ileo-anal pouch anastomosis. In ulcerative colitis patients, the prevalence of pouchitis varies from less than 10% to higher than 40%. The definition of "pouchitis" includes clinical symptoms, macroscopic inflammatory lesions at endoscopy and histological evidence of intense acute inflammation of the reservoir mucosa.

Clostridium difficile associated diarrhoea

Clostridium difficile is an anaerobic, gram-positive spore forming bacillus first isolated in 1935 from faecal flora of healthy neonates. It was not until 1978 that its association with antibiotic induced pseudomembranous colitis (PMC) was established. Almost all antibiotics have been linked with C. difficile diarrhoea and colitis, including vancomycin and metronidazole (which are used for its treatment) and cancer chemotherapy. The frequency of association is related to frequency of use, the route of administration and the impact of that antibiotic on the colonic microflora.

Irritable bowel syndrome

Irritable bowel syndrome (IBS) is a chronic disorder that interferes with the normal functions of the large intestine (colon). It is characterised by a group of symptoms - crampy abdominal pain, bloating, constipation, and diarrhoea.

IBS causes a great deal of discomfort and distress, but it does not permanently harm the intestines and does not lead to intestinal bleeding or to any serious disease such as cancer. Signs and symptoms of IBS vary widely from one person to another and often occur with many other diseases.

Other active ingredients

It will be appreciated that the disaccharide of the present invention may be administered prophylactically or as a method of treatment either on its own or with probiotic and/or prebiotic materials, hi addition, the bacteria may be used as part of a prophylactic or treatment regime using other active materials such as those used for treating inflammation or other disorders, especially those of the gastrointestinal tract. Such combinations may be administered in a single formulation or as separate formulations administered at the same or different times and using the same or different routes of administration.

Pharmaceutical compositions

A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent. It preferably includes a pharmaceutically acceptable carrier, diluent or excipients (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

Where appropriate, the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in a mixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.

Example 1 — Synthesis of disaccharide DSl

One method for synthesis of the GaINAc βl-3 GIc disaccharide is as follows:

(a) 1 -benzyl glucose was reacted with benzaldehyde/zinc chloride to form the 4,6 benzylidene derivative.

(b) This was selectively 2-benzoylated with benzoyl chloride at -20C in DCM/Pyridine.

(c) This was glycosylated with 1-thioethyl 3,4 6 tri-0-acetyl galactosamine N-trichloroethyl carbamate in DCM with N-iodosucinimide/triflic acid activation.

(d) The benzylidene protecting group was removed with 95% TFA in DCM.

(e) The amine protecting group was removed with zinc/acetic acid.

(f) The intermediate was N- and O- acetylated with acetic anhydride/pyridine. (g) After purification the O-acetyl groups and the O-benzoyl group were removed with NaOMe/MeOH

(h) The benzyl groups were removed by hydrogenation with Pd-C catalyst.

(i) Confirmation of the disaccharide structure was determined using ion chromatography and nuclear magnetic resonance.

(j) Lyophilized material was stored at -8O ⁰ C.

Various steps in a method for preparing the disaccharide DSl are illustrated in Fig. 6.

Example 2 -Disaccharide DSl has immunomodulatory activity when co-incubated with human immune system cells in vitro.

The disaccharide (DSl) was assayed using the PBMC (peripheral blood mononuclear cell) cytokine induction assay, hi this assay, PBMCs are isolated from blood by density gradient separation and incubated for 72 hours at 37 ⁰ C (in the presence of penicillin and streptomycin) with control media, or with increasing concentrations of synthesised DSl. Supernatants were assayed for IL-10 and TNF-α levels using mesoscale discovery (MSD) kits and analysed using an MSD plate reader.

Fig. 2 illustrates the results of this assay. DSl stimulated the secretion of the anti-inflammatory cytokine IL-10 in a dose dependent manner when PBMCs were stimulated with greater than 25 μg/ml DSl.

In addition to testing resting PBMCs, the TLR-4 ligand lipopolysaccharide (LPS) was used to activate PBMCs with or without DSl stimulation. With increasing concentrations of DSl ₅ LPS- stimulated secretion of TNF-α is significantly reduced. These results are illustrated in Fig. 3 as the mean % secretion of TNF-α stimulated with LPS alone. Example 3 - Disaccharide DSl has anti-inflammatory activity when injected into a murine model of sepsis.

DSl was injected i.p. into healthy mice and these mice were observed for 24 hours. No obvious signs of distress were noted suggesting that this disaccharide was well tolerated by the animals and DSl did not induce sepsis or a pro-inflammatory response. Following the 24 hour observation period, animals were injected i.p. with lipopolysaccharide (LPS) in order to induce a sepsis-like response. All animals were culled 2 hours later and splenocyte cytokine secretion measured in vitro. Splenocytes isolated from DS1+LPS treated mice released significantly less TNF-α when compared to mice that received LPS alone (Fig. 4).

Example 4 - Disaccharide DSl has anti-inflammatory activity when injected into a murine model of sepsis. Balb/C female mice at 10-12 weeks of age were sourced from Harlan laboratories Ltd. and housed in 12h light/dark cycle and provided standard laboratory chow and water ad libitum.

Mice of similar weight were randomised into 4 groups and administered as follows: Group 1 PBS (carrier control ), Group 2 DSl 20mg/kg, Group 3 LPS 2mg/kg +PBS , Group 4 LPS 2mg/kg + DSl lmg/kg, each group (n=8) LPS administration where references occured 24 hours after treatment (Sigma, L4391) via intra peritoneal injection.

4 hours after LPS administration blood was sampled by puncturing the face vein of each animal, serum was then extracted and analysed using the Meso Scale Discovery mouse TNF-α Ultrasensitive kit. Results are presented in Fig. 4. These data show that DSl significantly reduces the levels of circulating TNF-α in mice.By approximately 560 pg/ml.

The data in example 3 and 4 clearly demonstrate that DSl, has immuno-modulatory activity and protects against LPS induced (TLR-4) mediated inflammatory responses. (Fig. 5)

Example 5 - Synthesis of disaccharide DS2 GalpNac βl-4 GIc disaccharide DS2 (see Fig. 7) is prepared by an analogous process to that described above in Example 1.

Various steps in a method for preparing the disaccharide DS2 are illustrated in Fig. 11.

Example 6 - Disaccharide DS2 has immunomodulatory activity when co-incubated with human immune system cells in vitro.

The disaccharide DS2 was assayed using the PBMC (peripheral blood mononuclear cell) cytokine induction assay. In this assay, PBMCs are isolated from blood by density gradient separation and incubated for 72 hours at 37 ⁰ C (in the presence of penicillin and streptomycin) with control media, or with increasing concentrations of synthesised DS2. Supernatants were assayed for IL-IO and TNF-α levels using mesoscale discovery (MSD) kits and analysed using an MSD plate reader.

Fig. 8 illustrates the results of this assay. DS2 stimulated the secretion of the anti-inflammatory cytokine IL-IO in a dose dependent manner when PBMCs were stimulated with 2.5 ng/ml DS2.

In addition to testing resting PBMCs, the TLR-4 ligand lipopolysaccharide (LPS) was used to activate PBMCs with or without DS2 stimulation. With the same concentration of DS2, LPS- stimulated secretion of TNF-α is significantly reduced. These results are illustrated in Fig. 9 as the mean % secretion of TNF-α stimulated with LPS alone.

Example 7 - Disaccharide DS2 has anti-inflammatory activity when injected into a murine model of sepsis.

Balb/C female mice at 10-12 weeks of age were sourced from Harlan laboratories Ltd. and housed in 12h light/dark cycle and provided standard laboratory chow and water ad libitum.

Mice of similar weight were randomised into 2 groups and administered PBS (carrier control n= 6), lmg/kg DS2 (n=8) via single intra peritoneal injection. 24 hours later all mice were challenged with 2mg/kg lipopolysaccharide (Sigma, L4391) also via intraperitoneal injection. 2 hours after LPS administration blood was sampled by puncturing the face vein of each animal, serum was then extracted and analysed using the Meso Scale Discovery mouse TNF-α Ultrasensitive kit. Results are presented in Fig. 10. These data show that DS2 significantly reduces the levels of circulating TNF-α in mice by approximately 811 pg/ml.

Taken together, the data in Examples 6 and 7 clearly demonstrate that DS2 has immunomodulatory activity and protects against LPS (TLR-4) mediated inflammatory responses.

Tumor necrosis factor (TNF) promotes the inflammatory response, which in turn causes many of the clinical problems associated with autoimmune disorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and refractory asthma. A compound which can significantly inhibit TNFα levels may have potential as therapeutic.

Taken together, these data demonstrate that the disaccharides DSl and DS2 have immunomodulatory activity and protect against LPS or TLR-4 mediated inflammatory responses.

Example 8 - N-acetyl-D-lactosamine does not show anti-inflammatory activity when injected into a murine model of sepsis. [Comparative Example]

N-acetyl-D-Lactosamine (see structure Fig. 12) is a compound of similar structural charater as DSl and DS2.

Balb/C female mice at 10-12 weeks of age were sourced from Harlan laboratories Ltd. and housed in 12h light/dark cycle and provided standard laboratory chow and water ad libitum.

Mice of similar weight were randomised into 2 groups and administered PBS (carrier control n=10), or lmg/kg N-acetyl-D-Lactosamine (n=10) via single intra peritoneal injection. 24 hours later all mice were challenged with 2mg/kg lipopolysaccharide (Sigma, L4391) also via intra peritoneal injection.

2 hours after LPS administration blood was sampled by puncturing the face vein of each animal, serum was then extracted and analysed using the Meso Scale Discovery mouse TNFα Ultrasensitive kit. Results are presented in Fig. 13. These data show that N-acetyl-D-Lactosamine does not reduce the levels of circulating TNF-α in mice.

The invention is not limited to the embodiments hereinbefore described which may be varied in detail.

References

Arai KI, et al., Amu Rev Biochem 59: 783-836, 1990

Fuss et al., "Nonclassical CD Id-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis." Inflamm Bowel Dis. 2005 Jan; 1 l(l):74-5

Henderson B., and Wilson M. In"Bacteria-Cytokine interactions in health and disease", Lider et al., 1 : Proc Natl Acad Sci U S A. 23;92(11):5037-41, 1995 Mazmanian et al., Cell 122:107-118, 2005 Merkle and Poppe Methods Enzymol. 230:1-15, 1994 Minutoli et al., Eur J Pharmacol. 28;589(l-3):272-80, 2008.

Remington's Pharmaceutical Sciences, Mack Publishing Co. 1985 (Editor: A.R. Gennaro) York et al., Methods Enzymol. 118:3-40, 1986