WO2015075486A2 - Medicament - Google Patents

Abstract

This invention relates to glycosaminoglycan (GAGs) polysaccharides, and oligosaccharides derived from GAGs, suitably sulphated heparosan saccharide wherein the SO₃- / COO- ratio of the sulphated heparosan is in the range 1 to 3.8, which have been determined to inhibit keratinocyte inflammatory activation, for use in the treatment of inflammatory disorders of the skin, in particular psoriasis, eczema and atopic dermatitis.

Description

MEDICAMENT

FIELD OF THE INVENTION

This invention relates to glycosaminoglycan (GAGs) polysaccharides, and oligosaccharides derived from GAGs, which have been determined to inhibit keratinocyte inflammatory activation, for use in the treatment of inflammatory disorders of the skin, in particular psoriasis, eczema and atopic dermatitis.

BACKGROUND OF THE INVENTION

Glycosaminoglycans (GAGs) are a class of polysaccharides that have been shown to be present in a very wide phylogenetic range of organisms. GAGs are classified as heparin, heparan, chondroitin, dermatan, keratan, and hyaluronic acid, according to their structure. They are characterised as polymers of a disaccharide repeating unit of a hexuronic acid linked to an amino sugar, wherein both sugar residues can be substituted with sulphate groups. In vivo, GAGs are commonly found linked to core proteins to form proteoglycans. These proteoglycans are present on cell surfaces and in connective tissues.

GAGs, and oligosaccharides derived from GAGs, have been described as having many biological effects, mediated through their abilities to interact with a range of active molecules such as plasma proteins (e.g. anti-thrombin, heparin co-factor II), cytokines and chemokines (e.g. IL8, CCL5), growth factors (e.g. FGF2, VEGF), adhesion molecules (e.g. MAC-1, L-selectin), tissue degrading enzymes (e.g. elastase, Cathepsin G) and cytotoxic peptides (e.g. eosinophil cationic proteins).

Anticoagulant and antithrombotic activity (US4303651, KR20030073145,

US5817645A), antiviral activity (EP035505A1, EP295956, AA61K31737FI), anti- cancer activity (US7790700, US5541166A, US20080125393), and activity against cardiovascular disease (W09911273A1) have been described for particular GAG molecules. Certain bacteria derived semi-synthetic GAGs are described as having anticoagulant (US2011281820A1), anti-viral (US2005009780), and anti-cancer (WO02083155) activity, as well as other potential medical uses (US2004077848A1, review by Oreste & Zopetti 2012). Anti-inflammatory activity of specific GAGs, and oligosaccharides derived from GAGs, has been discussed (Lever et al. 2001; Lever & Page 2002), particularly heparin (US5037810, WO9530424, EP1300153A1, WO2007014049,

US2005282775, US4916219), which was first discovered in 1916 and entered clinical trials as an anti-coagulant in the 1930's. Most of the effects associated with specific disease models are related to heparin and heparin derivatives. These include treatment effects for conditions including asthma, chronic obstructive pulmonary disease, inflammatory bowel disease and wound healing / tissue repair (reviewed by Lever & Page 2012). The use of orally administered chondroitin and chondroitin derivatives to treat joint inflammation is also described (US7816329B2, WO03015799). Further, the use of particular sulphated oligosaccharide GAG derivatives (chemically modified, or fragmented) as anti-inflammatories has also been discussed (W09633726A1, US7332480).

The use of GAGs or oligosaccharides derived from GAGs, to treat inflammatory disorders of the skin are limited to the use of heparin for wound healing, including psoriatic plaques (included in US5037810) and allergic dermatitis (included in WO2007014049), the use of hyaluronic acid in psoriasis (included in US5631242A) and the description of chondroitin sulphate and amino sugar glucosamine for the treatment of psoriasis (WO2005014012A1; US2007020218A1; Andres et al. 2013; Moller 2010; Verges et al 2005). There remains a need to determine further treatments, which provide for an anti-inflammatory activity able to allow treatment of psoriasis.

SUMMARY OF THE INVENTION

Despite the body of knowledge that exists regarding the anti-inflammatory activity of heparin and its derivatives, it is not believed that other GAGs or oligosaccharides derived from GAGs have been evaluated for their specific effects on the

immunomodulatory mediators associated with psoriasis. Given the large number of structural variants of GAGs that can be generated, the advantageous properties of the particular saccharide structures of the invention, namely activity in the treatment of inflammatory skin disorders, in particular psoriasis, eczema and atopic dermatitis, have not been previously identified. Further, a lack of understanding of GAG structure and function, where structure, composition, chain length and charge may all play a role in conferring activity has meant the ability of specific GAG molecules to modulate inflammatory response has not been predictable. For example, whilst the anti-inflammatory activity of GAGs, and oligosaccharides derived from GAG, has been discussed, it is also the case that some GAGs and GAG derivatives can exhibit pro-inflammatory activity. This has been illustrated with highly sulphated chondroitin sulphate (McKee et al. 2010) and with some types of hyaluronic acid (a non-sulphated GAG) (Stern et al. 2006). Further some GAGs may show both pro- and anti-inflammatory effects depending on the model system used (Severin et al. 2012, Schlorke et al. 2012). Thus GAGs and GAG derivatives can elicit a complex profile of possible biological responses.

Psoriasis, an autoimmune disease that affects the skin, has multiple underlying inflammatory drivers. Evidence now indicates that a major part of the proinflammatory process is sustained by the activation of THl7cells, which in turn produce IL-17A and IL-22, stimulating keratinocytes to adopt a pro-inflammatory and hyper-proliferative response is sustained by the production of numerous chemokines and cytokines (Di Cesare et al. 2008; Nickoloff 2007).

The inventors have determined GAGs, and oligosaccharides derived from GAGs capable of targeting the key inflammatory pathways in psoriasis, by interacting with specific mediators such as cytokines, chemokines and proteolytic enzymes, and preventing their activity. These GAGs, and oligosaccharides derived from GAGs, which have anti-inflammatory activity, specifically relevant for use in the treatment of inflammatory skin disorders, in particular, psoriasis, eczema and atopic dermatitis, are discussed herein. Whilst some responses of GAGs have been related to certain structural features, for example, a specific pentasaccharide sequence is necessary for binding of heparin to anti-thrombin, conferring anti-coagulant activity (Olson et al. 2010), and at least 19 monosaccharides are required for heparin to bind Factor Ila (Xu et al. 2012) there is yet to emerge a clear structure-activity relationship, which allows one to predict the anti-inflammatory activity of a particular GAG structure in a particular disease (Raman et al. 2005; Rudd et al. 2010). As different GAGs, and oligosaccharides derived from GAGs, have different binding affinities to pro-inflammatory mediators, such as chemokines and cytokines, and these cannot yet be predicted by structure (de Paz et al. 2007) evaluation of these interactions can only be tested on a case-by- case basis, against those inflammatory mediators, which are key drivers in the target disease. It has been determined that the particular saccharides of the invention have a profile of activity suitable for the reduction of the key pro- inflammatory mediators, which sustain skin inflammation and in particular psoriasis.

According to a first aspect of the present invention, there is provided a saccharide, in particular a glycosaminoglycan with a repeating subunit structure comprising an N-acetylglucosamine linked β1→4 to hexuronic acid, suitably glucuronic acid, wherein the number of repeating disaccharide subunits (n) is in the range 2 to 30, (equivalent to 1.2-18kDa) linked al→4, as depicted in formula I

, and sulphated at between 1 to 5 of the positions a, b, c, d, and e for use in the treatment of an inflammatory skin disorder. As will be understood, such compounds may contain a hexuronic acid, suitably a glucuronic acid, or glucosamine group as the terminal monosaccharide at the reducing or non-reducing end of the saccharide. In embodiments the hexuronic acid can be glucuronic acid at each position in the saccharide chain, typically such molecules would be considered as heparosan saccharides isolated from K5 E. coli (K5 heparosan) or from Pasteurella multicida wheren the glucuronic acid can be possibly sulphated at any of position a or b as indicated in formula I and the glucosamine molecule can be sulphated at any of position c or d or e as indicated in formula I.

As will be understood, from the present disclosure the saccharides of the present invention can relate to N, 0 sulphated heparosans, wherein preferably the degree of sulphation of the N, 0 sulphated heparosans expressed as a sulphate to carboxyl ratio is around 1 to 3.8, suitably 1.4 to 3.8. As used herein, the term heparosan will be understood to refer to the natural biosynthetic precursor of heparin and heparan sulphate which is unepimerized and unsulphated or for example the capsular polysaccharide of Paseurella multocida type D and Escherichia coli. In embodiments the degree of sulphation of the N, 0 sulphated heparosans expressed as a sulphate to carboxyl ratio can be about 3.8. In embodiments, the N,0 sulphated heparosans of the present invention can consist of identical polysaccharide chains of well-defined molecular mass, being in the range of 1.2-18kDa, suitably 3-15kDa, suitably 6 to 9kDa. In alternative embodiments they can also consist of a mixture of chains of variable molecular masses, these molecular masses being in the range of 1.2-18kDa, suitably 3-15kDa, suitably 6 to 9kDa. In embodiments where a mixture of chains is provided the chains may only differ from each other by a molecular weight of about 300Da. In alternative embodiments, the chains may differ from each other across the range of molecular weights provided in the range, for example 1.2-18kDa, suitably 3-15kDa, suitably 6 to 9kDa.

In embodiments, the skin disorder can be selected from psoriasis, atopic dermatitis and / or eczema. As will be understood, the invention provides a method of reducing inflammation of the skin, in particular for use in treating an inflammatory skin disorder, for example psoriasis, atopic dermatitis and / or eczema comprising the step of administering a therapeutically effective amount of a saccharide with a repeating subunit structure comprising an N-acetylglucosamine linked β1→4 to a hexuronic acid, suitably glucuronic acid, wherein the number of repeating disaccharide subunits (n) is in the range 2 to 30, (equivalent to 1.2-18kDa) linked al→4, as depicted in formula I, and sulphated at between 1 to 5 of the positions a, b, c, d, and e to a subject in need thereof.

Naturally occurring GAG polysaccharide heparosan compounds are known, such as K5 polysaccharide from E. coli and heparosan from Pasteurella multicida. Such heparosan saccharides with the correct subunit structure, but lacking the correct sulphate substitutions may be chemically depolymerised and sulphonated to provide the correct chain length and structure as required to provide saccharides of the present invention. These structures, known as heparosans, can relate to heparan sulphate structures. Heparan sulphate can be distinguished from heparin. Heparan sulphate's disaccharide units are organised into distinct sulphated and non-sulphated domains. In particular, heparan sulphate glycosaminoglycans (GAG) are typically a polydisperse mixture of linear polysaccharides consisting primarily of N-acetylated [→4>p-D-GlcAp-(l→4)-oc-D-GlcNpAc-(l→] and N-sulphated disaccharides [→4)-β- D-GlcAp-(l→4)-a-D-GlcNpS-(l→or→4)-a-L-IdoAp-(l→] that are arranged mainly in segregated domains (where GlcNp is 2-amino-2-deoxyglycopyranose, IdoAp is idopyranosyluronic acid, GlcAp is glycopyranosyluronic acid, and S is sulphate).

As will be understood by those of skill in the art epimerization resulting in conversion of D -glucuronic acid to L-iduronic acid can occur such that epimerized portions of the heparan sulphate are provided heterogeneously throughout the heparan sulphate. Heparan sulphate may be distinguished from heparin, a related GAG, by its ratio of GlcNpAC to GlcNpS, wherein in heparin the ratio of GlcNpAC to GlcNpS can be greater than 3.0. Typically, heparan sulphate can have a sulphate content of less than 20%. Typically the ratio of D -glucuronic acid to L-iduronic acid can be greater than 2 in heparin. Typically, heparan sulphate can have a carbazole to orcinol ratio of less than 2. Typically, heparan sulphate can differ in deposition of O-sulphates and uronic acid epimers when compared to heparin. In embodiments, heparan sulphate can have O-sulphates found in proximity to N-sulphates. This acts to enhance the clustering of the sulphate residues and the heterogeneous chemical composition and charge density of heparan sulphate.

Suitably, the GAGs discussed herein may be homogenous molecules with sulphation occurring at the same positions at each disaccharide repeating subunit. Suitably they may be produced synthetically, such they do not have the heterogeneity of naturally occurring heparan sulphate or heparin.

In embodiments, GAGs as discussed herein are not disulphated on the uronic acid. In embodiments, highly sulphated molecules as discussed herein can have 75-80 % disulphated uronics. As will be understood this means one of more OH groups in the glucuronic acid residues of the polysaccharide is / are replaced by an OSO3H group, also the glucosamine N-acetyl group is replaced by an N-sulphate group.

In embodiments, a saccharide of or for use in the invention can down-regulate IL8 and IL6 produced by primary human keratinocytes in vitro, which have been stimulated with TNFa, IL17A and histamine. In embodiments the saccharides of or for use in the invention can down-regulate the histology score in an imiquimod- induced mouse model of psoriasis. In embodiments the saccharides of or for use in the invention can inhibit the activity of neutrophil elastase. In embodiments the saccharides of or for use in the invention can inhibit monocyte chemoattractant protein-1 induced monocyte chemotaxis. In embodiments the saccharides of or for use in the invention show no or reduced anti-coagulant activity compared to heparin or low molecular weight heparin in an APTT assay. In embodiments oligosaccharides can be semi-synthetic molecules prepared by the fractionation and modification of naturally occurring GAGs found in the tissues of vertebrates, invertebrates, or produced by selected prokaryotic organisms. Suitably, downregulation may be about 70 to 90%.

GAG's, and oligosaccharides derived from GAGs, have a multiplicity of activities, including anti-coagulant, pro- and anti-proliferative, anti-metastatic, anti-viral, and anti-oxidant activity. Any consideration of development of GAG's for an anti- inflammatory therapeutic application must take account of the potential for such activity, which may manifest themselves as side effects. The saccharides for use in this invention have been chosen to have low anticoagulant activity as shown the example in Figure 8. The development of GAGs, and oligosaccharides derived from GAGs, for therapeutic applications must also consider the issue of delivery, since GAGs are large highly charged molecules, which have poor oral availability; in the case of heparin for anticoagulant applications, delivery is intra venous. There is preliminary evidence that inhaled heparin is able to deliver anti-inflammatory activity in vivo (Seeds et al. 1995), but this has not been demonstrated for other GAGs. Topical application of GAGs such as hyaluronic acid on the skin for cosmetic purposes is well developed, but there is limited evidence of topical application of sulphated GAGs eliciting an anti-inflammatory response in the skin. Due to the lack of prior art it would not be obvious to someone skilled in the art that the specific saccharides of the invention, GAGs or oligosaccharide derived from the GAGs would have efficacy in topical application against psoriasis, or that any GAG, or oligosaccharide derived from a GAG, will penetrate the skin to reach pro-inflammatory targets.

In embodiments, the repeating subunit structure of a saccharide can comprise N- acetylglucosamine linked β1→4 to a glucuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 5 to 25, (equivalent to 3-15kDa) linked al→4, as depicted in formula I, and is sulphated at between 1 to 5 of the positions a, b, c, d, and e.

In embodiments of the invention the saccharide can be an oligosaccharide with a repeating structure N-acetylglucosamine linked β1→4 to a glucuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 10 to 15

(equivalent to 6-9kDa), linked al→4, as depicted in formula I and sulphated at between 1 to 5 of the positions a, b, c, d, and e. In embodiments the saccharide can be an oligosaccharide with a repeating structure N-acetylglucosamine linked β1→4 to a glucuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 5 to 25 (equivalent to 3-15kDa), linked al→4, as depicted in formula I, and sulphated at positions a, b, c, and e, and optionally sulphated at position d.

In embodiments the saccharide can be can an oligosaccharide with a repeating structure N-acetylglucosamine linked β1→4 to a glucuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 10 to 15 (equivalent to 6-9kDa), linked al→4, as depicted in formula I, and sulphated at positions a, b, c, and e, and optionally sulphated at position d.

In embodiments, molecules sulphated at positions a, b, c and e with variable sulphation at position d, are preferred. In embodiments a saccharide of the invention can be selected from any of the first 6 molecules of Table 1. The final molecule shown in Table 1, which has the glucuronic acid reside epimerised to iduronic acid, retains anti-inflammatory activity, but is not preferred for use in the invention because of the difficulty and cost of manufacture of this molecule. As would be understood by those of skill in the art, the heparosan polysaccharide compound can be isolated, for example as described in Eur. J. Biochem. 116 (1984), 359-364, and as described in Carbohydrate Research 337 (2002), 1547-1552. Sulphation of the polysaccharide, for example a K5 heparosan polysaccharide or the capsular polysaccharide of Pasteurella multocida can be achieved as known in the art, for example by means of an (alkyl)3N-S03 complex, for example (CH3)3N-S03, (C2Hs)3NS03 or an SO3 complex of an aromatic heterocyclic compound which has an N atom in the ring, such as, for example, pyridine-S03. The reaction can be carried out at a temperature of 15 to 80 degrees C. the reaction time is typically in the range of 1 hour to 40 days dependent of the degree of the sulphation desired. Suitably the reaction may be carried out in an organic solvent such as dimethyl formamide. The amount of complex used may vary within wide limits, but typically will be around 0.01 to 5 mol per mole eq of OH groups in the polysaccharide, the OH groups in the COOH groups not being included. Synthesis of sulphated heparosans, in particular the K5 E. coli heparosan may be undertaken as described for example in EP 0333243.

Table 1

Saccharide Description

K5/NS polysaccharide (repeat unit N-acetylglucosamine and glucuronic acid [→4 p-D-GlcAp-(l→4)-a-D-GlcNpAc- (l→]j for example, but not limited to as produced by E.coli - N -sulphated

K5/OS (L) polysaccharide (repeat unit N-acetylglucosamine and glucuronic acid [→4 p-D-GlcAp-(l→4)-a-D-GlcNpAc- (l→]j for example, but not limited to as produced by E.coli - O-sulphated at C6 of GlcNAc, low level of sulphation at C2 and C3 of GlcA.

K5/OS (H) polysaccharide (repeat unit N-acetylglucosamine and glucuronic acid [→4 p-D-GlcAp-(l→4)-a-D-GlcNpAc- (l→]j for example, but not limited to as produced by E.coli - O-sulphated at C6 and C3 of GlcNAc, C2 and C3 of GlcA.

K5/NS,OS (L) K5 polysaccharide (repeat unit N-acetylglucosamine and glucuronic acid [→4)- -D-GlcAp-(l→4)-a-D- GlcNpAc-(l→]) for example, but not limited to as produced by E.coli - N-sulphated, O-sulphated at C6 of GICNSO3, low level of sulphation at C2 and C3 of GlcA.

K5/NS,OS (H) polysaccharide (repeat unit N-acetylglucosamine and glucuronic acid [→4 p-D-GlcAp-(l→4)-oc-D-GlcNpAc- (l→]j for example, but not limited to as produced by E.coli - N-sulphated, O-sulphated at C6 of GICNSO3, C2 and C3 of GlcA, variable O-sulphation at C3 of GICNSO3. K5/NS,0S (H) 8.3kDa polysaccharide (repeat unit N-acetylglucosamine and glucuronic acid [→4 p-D-GlcAp-(l→4)-a-D-GlcNpAc- (l→]j for example, but not limited to as produced by E.coli - ragment of K5/NS,0S (H) 8.3kDa molecular weight.

E-K5/NS,0S(H) polysaccharide (repeat unit N-acetylglucosamine and glucuronic acid [→4 p-D-GlcAp-(l→4)-a-D-GlcNpAc- (l→]j for example, but not limited to as produced by E.coli - Epimerised K5. O-sulphated at C6 of GICNSO3, and C2 and C3 of GlcA and IdoA, variable sulphation at C3 of GlcNSOs.

where GlcAp = glucuronic acid, GlcNpAc = n-acetylated glucosamine, IdoA = iduronic acid, where GICNSO3 = N-sulphated glucosamine

Suitably, in embodiments, sulphated heparosan of the invention, suitably a

heparosan with a SO3^" / COO^" ratio of 1 to 3.8, in particular highly sulphated

heparosan, for example with a SO3^" / COO^" ratio of about 3.8, specifically molecules of Table 1 can be of a molecular weight (unless indicated otherwise) of 20kDa or less, or 18kDa or less or in the range 3 to 15kDa, or 6 to 9kDa.

The percentage sulphation of the polysaccharides as discussed above are indicated further below in Table 2 as typical approximate ratios.

Table 2

In embodiments, the saccharides of the present invention can be prepared by chemical modification of naturally occurring polysaccharides isolated from the tissues of organisms including mammals and marine invertebrates. The saccharides can also be prepared by chemical modification of naturally occurring

polysaccharides isolated from microorganisms. In embodiments saccharides of the invention can be prepared from GAG molecules by enzymatic or chemical depolymerisation to provide the desired chain length, followed by fractionation to generate purified saccharides of the structure described. Where such molecules are insufficiently sulphated they can be chemically sulphonated to provide the correct structure. In embodiments, the saccharides can be homogenous structures.

In embodiments, naturally occurring GAG polysaccharides, such as K5

polysaccharide from E. coli and heparosan from Pasteurella multicida, with the correct subunit structure, but lacking the correct sulphate substitutions may be chemically depolymerised and sulphonated to provide the correct chain length and structure.

N- and O-sulphonation of K5 polysaccharide and subsequent depolymerisation to prepare oligosaccharide of the desired structure is described in patents

US2004077848 (Al) and US2008146793 (Al).

Saccharides of the present invention have utility for the treatment of psoriasis through their anti-inflammatory activity. In embodiments of the invention the saccharides are capable of down-regulating inflammatory markers relevant to psoriasis, including IL8, and of reducing the histological scoring in an imiquimod- induced in vivo mouse model of psoriasis.

In embodiments, highly sulphated saccharides are those that have 4 or 5 sulphates per disaccharide subunit (annotated by H in table 1) and low sulphated

polysaccharide (annotated by L in table 1) are those which contain fewer than 4 or 5 sulphates on the disaccharide subunit. The position or number and position of the sulphates on the disaccharide backbone relative to each other are also important in describing the overall sulphate structure of a GAG.

In embodiments the saccharides of the invention can be applied topically to the skin of a subject suffering from an inflammatory skin disorder, for example psoriasis, atopic dermatitis, and eczema to allow for the treatment of such inflammatory skin disorders including psoriasis, atopic dermatitis, and eczema.

A saccharide of the present invention may be provided as a pharmaceutically acceptable salt or pharmaceutically acceptable solvate. In particular, because the carboxyl and sulphate groups in the saccharides of the invention may occur in an ionised state in solution depending on the pH, the saccharides according to the invention may be provided as a salt. In embodiments the polysaccharide or a derivative thereof can be administered alone, or in admixture with a pharmaceutical carrier, excipient or diluent selected with regard to the intended route of administration and standard pharmaceutical practice. A pharmaceutical carrier can be a physiologically acceptable carrier, either organic or inorganic, natural or synthetic with which the saccharide or derivative thereof of the present invention can be combined to facilitate the application.

In embodiments the saccharide of the invention can be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), carrier(s), or buffer stabiliser(s). The saccharide can be formulated in a hydrogel composed of polyacrylate Na salt, glycerol, paraben and imidazolidinyl urea. In embodiments, the saccharide can be provided as part of a liposomal formulation. The liposomal formulation utilised can be any liposomal formulation as known in the art. Accordingly, a second aspect of the present invention provides a topical

composition for use in the treatment of an inflammatory skin disorder, the composition comprising a saccharide as described herein.

Suitably, in embodiments, the saccharide of the invention comprises at least 50% of the active component of the composition, more suitably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, suitably 100% of the active component of the composition. Compositions for topical administration may be provided, for example, as a gel, cream or ointment. Such compositions can be applied directly to the skin or carried on a suitable support, such as a bandage, gauze, mesh or the like that can be applied to an area to be treated. Suitably a topical composition may conveniently be presented in a unit dosage form and may be prepared by any of the methods well- known in the pharmaceutical industry.

A topical composition of the invention may also contain one or further active compounds selected as necessary for the condition being treated. For example a composition may comprise a further active compound, which targets distinct pathways or mechanisms from that targeted by the product of the invention. This may provide improved efficacy, for example a synergistic effect. In embodiments the oligosaccharide of the present invention can be provided in combination with corticosteroid such as Clobetasol propionate, or other widely used topical agents.

Treatment

A saccharide of the invention or a composition containing the saccharide of the invention thereof may be used to treat a number of skin conditions, including psoriasis, atopic dermatitis, and eczema. Treatment includes any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviation or prophylactic effects. According to a further aspect of the present invention there is provided a saccharide of the present invention or a composition comprising such a saccharide for use as a medicament. In particular, there is provided a saccharide of the present invention or a composition comprising such a saccharide for use in the treatment of an inflammatory skin condition, in particular a skin condition selected from psoriasis, atopic dermatitis, and eczema. Administration

A medicament comprising a saccharide of the present invention may be for human usage or veterinary usage. Suitably in veterinary usage the animal patient may be a terrestrial animal, more suitably a companion or performance animal. Suitably a patient may be a human.

Preferred features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness. Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.

Throughout the specification, unless the context demands otherwise, the terms 'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes' or 'including' will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

Embodiments of the present invention will now be discussed by way of example only with reference to the figures in which:

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 illustrates example disaccharide repeating subunit structures of the saccharides of the invention wherein Figure 1(1) illustrates a GlcA containing unit

[→4 p-D-GlcAp-(l→4)-a-D-GlcNpAc-(l→ where GlcNAc = N-acetylglucosamine and GlcA = glucuronic acid], Positions of possible sulphate substitutions are indicated by arrows and marked as a (GlcA 30S), b (GlcA 20S), c (GlcN60S), d (GlcN30S) or e (GlcNS).

Figure 2 illustrates example saccharides and a description of their structure and a comparative example E-K5/NS,OS(H).

Figure 3 illustrates the effects of saccharides on BHK cell viability wherein there is provided example data for the effects of saccharides on BHK cell viability (all samples tested at O.lmg/ml) after 18 hours incubation. The data illustrates the mean cell viability, % relative to control. Treatment with saccharides results in no effects on observed cell viability in this model. Doxorubicin is shown as an assay control. Low molecular weight (LMW) heparin (5-8kDa), LMW hyaluronic acid (10- 18kDa) and heparan sulphate are shown for comparison. K5 lOkDa is K5 polymer with reduced chain length (K5 = approximately 70kDa, depending on culture conditions).

Figure 4A illustrates the effects of saccharides on human neutrophil elastase activity (wherein activity may be affected by inhibition of release of the elastase, inhibition of the elastase enzyme directly) wherein there is shown example data for the effects of saccharides on human neutrophil elastase activity (all agents tested at O.lmg/ml). Data illustrates elastase activity, % relative to control. Treatment with sulphated saccharides results in a reduction of elastase activity at this dose, but there is little or no reduction with non-sulphated saccharides (K5, K5 lOkDa, LMW hyaluronic acid). LMW heparin, LMW hyaluronic acid and heparin sulphate are shown for comparison. K5 lOkDa is K5 polymer with reduced chain length.

Figure 4B illustrates the dose-dependent effects of saccharides on human neutrophil elastase activity wherein there is shown example data for the effects of saccharides on human neutrophil elastase activity. Data illustrates the elastase activity, % relative to control. Treatment with mono-sulphated or unsulphated saccharides does not significantly reduce elastase activity at the doses tested, but treatment with highly sulphated saccharides results in a dose-dependent reduction of elastase activity. LMW heparin is shown for comparison.

Figure 5A illustrates the dose-dependent effects of saccharides on IL8 release by primary human keratinocytes wherein there is provided example data for the effects of saccharides on IL8 release from keratinocytes stimulated with lOng/ml TNFa, 50ng/ml IL17A & lOmicroM histamine. The data indicates that sulphated saccharides inhibit the release of IL8 in a dose-dependent fashion and have greater potency at similar doses than the NF-κΒ inhibitor (SC-514), and the MAPK p38 inhibitor (SB203580). The data indicates that non or low sulphated saccharides (K5, K5/NS, K5/NS,OS(L)) do not reduce IL8 release as significantly as more highly sulphated saccharides at these doses, but treatment with more highly sulphated saccharides at such doses inhibits IL8 release. LMW heparin is shown for comparison.

Figure 5B illustrates the dose-dependent effects of saccharides on IL6 release by primary human keratinocytes wherein there is provided example data for the effects of saccharides on IL6 release from keratinocytes stimulated with lOng/ml TNFa, 50ng/ml IL17A & lOmicroM histamine. The data indicates that non or low sulphated saccharides (K5, K5/NS, K5/NS,OS(L), K5 DP10) result in little reduction in IL6 release at these doses, but treatment with more highly sulphated saccharides inhibits IL6 release. LMW heparin is shown for comparison.

Figure 6 illustrates the dose-dependent effects of saccharides on PMA stimulated human neutrophil reactive oxygen species (ROS) production wherein there is shown example data for the effects of saccharide on oxygen ROS production. Data illustrates ROS produced, % relative to control. The saccharides show no inhibition of ROS release. LMW heparin is shown for comparison. Figure 7A and 7B illustrates the dose-dependent effects of saccharides on THP-1 (monocyte) chemotaxis wherein there is provided example data for the effects of saccharides on MCP-1 (monocyte chemoattractant protein) stimulated THP-1 (pro monocytic cell line) chemotaxis. Figure 7 A illustrates a dose-dependent inhibitory effect of sulphated saccharides on MCP-1 stimulated monocyte chemotaxis; Figure 7B illustrates little or no effect of non-sulphated saccharides on MCP-1 stimulated monocyte chemotaxis, although moderate inhibition by saccharide K5 at higher doses may be due to a non-specific effect due to the large size of the molecule. LMW heparin and LMW hyaluronic acid are shown for comparison.

Figure 8 illustrates the effects of saccharides on activated partial thromboplastin time (APTT), wherein there is provide example data for the effects of saccharides on APTT. Saccharides all exhibit reduced or no anti-coagulant effect when compared to heparin or LMW heparin. Hyaluronic acid is shown for comparison.

Figure 9 illustrates the dose-dependent effect of saccharides on mouse skin inflammation wherein there is provided example data for the effects of saccharides on imiquimod (IMQ) induced mouse skin inflammation after 6 days of IMQ treatment and saccharide dosing. Inflammation is measured by scoring of histological parameters including epidermal and keratinocyte proliferation, and leukocyte infiltration. The saccharides show inhibition of skin inflammation at the doses tested. Clobetasol propionate (a potent topical corticosteroid used to treat inflammatory conditions such as Eczema and dermatitis) and cyclophosphamide (a treatment for autoimmune disease) are shown as controls.

* denotes statistical significance by AN OVA with Dunnett's post hoc analysis.

DETAILED DESCRIPTION OF THE INVENTION

The following methods as set out in each of the following examples were used to generate the results as indicated in the attached figures.

The inventors have demonstrated that sulphated K5 heparosan saccharides and fragments thereof, in particular highly sulphated K5 heparosan saccharides and fragments thereof, reduce / inhibit elastase activity and can inhibit IL8 and IL6 release. Where sulphation is discussed, it will be understood that sulphation of K5 heparosan or K5 heparosan fragments or other heparosan polysaccharides such as those produced by Pasteurella multocida or those prepared by chemical synthesis of smaller saccharide units relates to sulphation of one or more OH groups in the heparosan saccharide or fragments thereof, not including the OH of the COOH group and that sulphation typically replaces an OH group with an OSO3H group and / or replacement of the acetyl group of N-acetylglucosamine to N-sulphate. Fragments may be prepared by fragmentation of the K5 heparosan saccharide or sulphated K5 heparosan saccharide or other heparosan polysaccharides such as those produced by Pasteurella multocida and also by synthesis of smaller saccharide units as would be understood in the art.

As described for example in Figure 2, the inventors have determined that sulphated K5 heparosan polysaccharide and fragments thereof, (K5 polysaccharide being the capsular polysaccharide present in some Escherichia coli bacteria), which has a repeating structure GlcA containing unit [→4)-p-D-GlcAp-(l→4)-a-D-GlcNpAc-(l→ where GlcNAc = N-acetylglucosamine and GlcA = glucuronic acid), inhibits neutrophil elastase and to inhibit IL8 and IL6 release.

Typically, the molecular weight of a K5 polysaccharide is about 10^s to 2. 10⁶ Da. If fully sulphated, a K5 polysaccharide may have a molecular weight of up to about 4. 10⁶ Da. The inventors have determined that fragments of sulphated K5 polysaccharide are also able to inhibit elastase and further to inhibit IL8 and IL6 release, In particular, as an example, a 8.3kDa fragment of a sulphated K5 polysaccharide compound (K5/NS, OS) has been demonstrated to inhibit elastase activity.

As indicated in Figure 4A elastase activity has been determined to be inhibited to a greater extent by K5 sulphated compounds which are highly sulphated (as indicated by H). Further, as indicated by Figure 4B, highly sulphated K5 saccharide treatment provides for dose dependent reduction of elastase activity. Interleukin-8 is often associated with inflammation. As an example, it has been cited as a proinflammatory mediator of psoriasis. As illustrated in Figure 5A, sulphated K5 saccharides, in particular highly sulphated K5 saccharides inhibit the release of IL8 in a dose dependent fashion. Interleukin 6 (IL6) is an interleukin that acts as a pro-inflammatory cytokine and as illustrated by Figure 5 B, highly sulphated K5 saccharides inhibit IL6 release.

As would be understood by those of skill in the art, the K5 saccharide can be isolated, for example as described in Eur. J. Biochem. 116 (1984), 359-364. Sulphation of the K5 saccharide can be achieved as known in the art, for example by means of an (alkyl)3N-S03 complex, for example (CH3)3N-S03, (C₂H5)3NS03 or an SO3 complex of an aromatic heterocyclic compound which has an N atom in the ring, such as, for example, pyridine-S03. The reaction can be carried out at a temperature of 15 to 80 degrees C. the reaction time is typically in the range of 1 hour to 40 days dependent of the degree of the sulphation desired. Suitably the reaction may be carried out in an organic solvent such as dimethyl formamide. The amount of complex used may vary within wide limits, but typically will be around 0.01 to 5 mol per mole q. of OH groups in the K5 saccharide, the OH groups in the COOH groups not being included. Suitably N-sulphation would be undertaken using processes as would be known in the art. Synthesis of sulphated K5 heparosan compounds and fragments as described herein may be undertaken as described for example in EP 0333 243. Example 1 - Determination of cytotoxicity.

Various different cell-based screening assays can be used to determine the cytotoxicity of the saccharides of the invention. Specifically cytotoxicity is examined by measuring the effects of saccharides of the invention on the metabolic activity of a BHK cell line (hamster kidney fibroblast ECACC 85011433). 90% confluent BHK cells were harvested and plated in a 96-well white microplate at lxlO⁴ cell/well in 100 microL freshly prepared culture media (Glasgow Minimum Essential Medium (GMEM), 10 % Foetal Calf Serum, 5 % Tryptose Phosphate Broth, 2 mM L- Glutamine). They were left for 1 hour at 37 °C 5% C0₂ to allow >80% adhesion to the well. 11 microL of lmg/ml saccharide sample in Hanks Balanced Salt Solution (HBSS), HBSS only control, fucoidan (lmg/ml in HBSS) control, and doxorubicin (lOmicrog/ml, lmicrog/ml in HBSS) controls were added to triplicate wells and the plate incubated for 16-18 hours at 37 °C 5% C0₂. The plate was allowed to come to room temperature for 30 minutes before additions of 100 microL Cell titre glow reagent (Promega). The plate was mixed for 2 minutes on a plate shaker and then incubated for 10 minutes at room temperature. The resulting luminescence for each well was measured on plate reader (BioTek, Synergy 3) using Gen5 software. Mean luminescence for each sample or control was calculated. The HBSS control well was designated as 100% metabolic activity and sample luminescent values were calculated against this % activity = (test well / control well) * 100. The fucoidan and doxorubicin controls should be within established values.

Results for this example are provided by figure 3 which indicates treatment with saccharides as discussed herein results in no effects on observed cell viability in this model. Example 2 - Effects on neutrophil elastase activity

Different protocols are possible for the measurement of the effect of the saccharides of the invention on neutrophil elastase enzyme activity. Specifically elastase activity was measured by incubation of saccharide with stimulated freshly isolated human neutrophils followed by reaction of released enzyme with a labeled substrate and colourimetric measurement on a plate reader. Freshly isolated human neutrophils were resuspended in HBSS (without Ca and Mg) and cells counted on a

haemocytometer. The cells were centrifuged and resuspended in HBSS to give a concentration of 2.5 x 10⁶ cells/ml. 22 microL of sample, controls or HBSS were added to a microtube followed by: 25 microL of cytochalasin B (at 40 mg/ml in HBSS to give a final concentration 5 mg/ml); 25 microL of TNF a (at 80 ng/ml in HBSS to give a final concentration of 10 ng/ml, with 25 microL HBSS used in place of TNF a for a non-stimulated control); 150 microL of neutrophil suspension (or for a media only control group add 150 microL of HBSS in place of cells). Contents were gently mixed and the tubes incubated at 37 °C for 30 minutes. After incubation 25 microL of fMLP (at lmicrog/ml in HBSS to give a final concentration of lOOng/ml) was added, or HBSS to the non-stimulated control group. Tubes were incubated for a further 45 minutes at 37 °C. Tubes were centrifuged at 5000rpm for 5 minutes on a Heraeus Biofuge to pellet the cells and 25 microL of the supernatant is transferred into triplicate wells of a 96 well black microplate. 150 microL of Tris HC1 pH 7.5 and 20 microL of neutrophil elastase substrate 1 (0.5mg/ml in Tris-HCl pH 7.5) were added to each well, except for a blank well which contains no substrate. The plate was transferred to a pre warmed (37 °C) plate reader (BioTek Powerwave HT) and readings are taken at 405nm every 5 minutes for 1 hour using Gen5 software. Vmax was calculated over 4 data points between 10 minutes and 1 hour. Mean Vmax was calculated for each sample, control or blank. The control well (stimulated cells with substrate, but no test samples) Vmax was designated as 100% and samples and controls are calculated against this to generate % elastase activity: % activity = (test well Vmax / control well Vmax) * 100.

Results for this Example are provided by figures 4A and 4B.

Example 3 - Effects on keratinocyte cytokine release.

The effects of saccharides of the invention on keratinocyte cells may be assessed using a range of different cell lines or primary cells, in various different growth media with or without pro-inflammatory stimulus. The resulting cytokine release can be assessed by different methods such as multiplex arrays or ELISA's.

Specifically primary keratinocytes (Promocell C12003) were grown in full keratinocyte growth media with calcium and supplements (Promocell C20011) at 37 °C, 5% C0₂ until 70-90% confluent. They were harvested by trypsinisation, washed and seeded in the wells of a 24 well plate tissue culture plate at 30,000 cells per well. Cells were grown until ~80-90% confluent (~56 hours) and the media was then changed to basal media (Promocell C20211) with 0.5mM calcium, for a further 16-18 hours. Samples (lmg/ml in HBSS), controls (fucoidan lmg/ml in HBSS) or blanks (HBSS vehicle only) were then added to the wells (X10 dilution) for 6-8 hours before addition of pro-inflammatory stimulus, or 1-2 hours in the case of SC514 and SB203580 (NfkB and MAPK p38 inhibitor respectively). Pro- inflammatory stimuli were lOng/ml TNFalpha and 50mg/ml IL17A in combination, or lOng/ml TNFalpha, 50mg/ml IL17A and lOmicroM histamine in combination. Cells were incubated for a further 16-18hours at which point the supernatant was collected and stored at -80°C. The collected supernatant was analysed for human IL8 or IL6 content by ELISA (Peprotech). The assay was read on a microplate reader (BioTek PowerWave HT using Gen5 software) at A450-630nm, and quantification was made by reading off the standard curve. The stimulated control well was designated as 100% secretion of IL8 or IL6 and samples were calculated against this % secretion = (corrected test well pg/ml / corrected control well pg/ml) * 100.

Results for this example are provided in figures 5A and 5B. Example 4 - Effects on oxidative burst from neutrophils.

There are numerous protocols to measure the production of reactive oxygen species from immune cells, using different cells, stimuli and substrates. Specifically inhibition of the oxidative burst by saccharides of the invention was measured using human neutrophils, which were stained with the reagent DCFH-DA. Freshly isolated human neutrophils were resuspended in HBSS (without Ca and Mg) and cells counted on a haemocytometer. Cells were resuspended at lxlO⁶ in HBSS, mixed with an equal volume of DCFH-DA at 40microM in HBSS and incubated for 30mins at 37 °C, 5% C0₂. 100 microL of stained cells were added to each well of a black 96 well microplate, apart from triplicate wells of a blank (HBSS only) and unstained cells control. 20 microL of lmg/ml of samples, HBSS or controls (diphenyleneiodium chloride (DPI) ImicroM concentration in HBSS) were added to triplicate wells containing stained cells. Cells were stimulated to produce ROS by the addition of 50 microL of PMA (4nM in HBSS), except for no stimulation control wells. Fluorescence generated by the oxidation of DCFH-DA by ROS was measured on a fluorescent plate reader (Biotek Synergy 3) at 37°C, 485/528nm kinetic read every 10 minutes for 2.5 hours. Mean fluorescence is calculated, and blanked. Mean fluorescent data for the PMA stimulated cells was designated as 100% response and samples and controls were evaluated against this: % oxidative burst = (sample fluorescence / PMA stimulated cells fluorescence) *100.

Results from this example are provided by figure 6.

Example 5 - Effects on blood cell chemotaxis

A chemotaxis assay can be carried out using different types of immune cell, with different chemotactic agents. Specifically, the effects of saccharides of the invention on the chemotaxis of monocytes, using THP-1 human pro-monocytic cell line (HPA 88081201) were also assessed. Saccharides (triplicate wells) at selected

concentrations were mixed with THP-1 cells at 2xl0⁶ /ml in RPMI 1640 medium (PAA) with 25mM HEPES, 2mM Glutamine and 0.1% BSA (Sigma) in a 96 v-well polypropylene (PP) plate (Greiner). A cells only control was also set up in triplicate. The cells and saccharides were incubated at 37°C 5% C02 for 30 minutes.

235microL of lOng/ml of monocyte chemoattractant protein-1 (MCP-1) (Peprotech) in the same medium was added to the lower chamber of a 96 well 5micron mesh chemotaxis plate (Corning), using assay medium as a negative control. The upper plate was refitted and the plate incubated at 37°C 5% C02 for 30 minutes to pre- equilibrate media. The assay was carried out by transferring 75microL of THP-1 cells (~150,000cells) and test samples from the PP plate into the upper chamber of the MCP-1 containing chemotaxis plate. Care was taken to ensure cells were fully suspended and mixed well before transfer. The plate was incubated at 37°C 5% C02 for 120 minutes. Chemotaxis was measured by removing media from the upper chamber, transferring the membranes to a plate containing 180microL of Accutase enzyme (Sigma), shaking for 5 minutes and discarding membranes. 100 microL CellTiter-Glo reagent (Promega - as for neutrophil chemotaxis) was added to the lower well of the assay plate and to the Accutase containing plate. They were shaken for 2 minutes, incubated for 10 minutes and then 200microL of the well contents was transferred to a white 96-well plate and the luminescence measured on a Synergy 2 plate reader (Biotek) using Gen5 software. Data was blanked using the media and cells only control, values were pooled from the lower chamber and accutase samples, and % chemotaxis was calculated by comparison to the cells only MCP-1 control wells (100% chemotaxis).

Results from this example are provided by figure 7A and 7B.

Example 6 - Effects on activated partial thromboplastin time (aPTT) (coagulation).

Various protocols can be used to assess the effects of saccharides on blood coagulation pathways. Specifically, the effects of saccharides of the invention on blood coagulation were measured using an activated Partial Thromboplastin Time assay using an ACL9000 automatic coagulometer (Instrumentation Laboratories). This assay was used to measure the effects of saccharides on both the intrinsic (involving Factors XII, XI, IX, VIII) and common (Factors X, V, II, fibrinogen) blood coagulation pathways. The ACL9000 was calibrated daily using a normal control, and abnormal control plasma (Instrumentation Laboratories). A standard curve was prepared using 5^th International heparin standard (NIBSC) at 0.5-5units/ml in water, in 0.5 unit increments. 30 microL of test saccharides at lmg/ml in water, a water blank, a heparin control (H3393 Sigma) at O.Olmg/ml in water, and heparin standards, were pipetted into reaction vessels. 270 microL of human plasma (TCS reagents), which had been previous thawed and filtered at room temperature, was added to each reaction vessel. The reaction vessels were placed in the carousel of the ACL9000. The aPTT programme was selected, with APTT-SP reagent (colloidal silica dispersion with phospholipids to stimulate contact activation, Factor XII production, Factor X and prothrombin activation) and calcium, being added automatically to the samples. The time to clotting (from the addition of calcium to thrombus formation, measured by optical density) in seconds was displayed. Any samples out of the standard curve range were further diluted in water and rerun. A standard curve was generated by plotting time to clot against heparin standard concentration (IU), and the aPTT value (IU/mg) for the test saccharides was calculated.

Results from this example are provided by figure 8.

Example 7 - Effects on skin inflammation in imiquimod (IMQ) treated BALB/c mice.

There are numerous protocols to assess the effects of saccharides of the invention on skin inflammation in mouse models, using different types of mice, genetic inducers and external stimuli. Specifically the effects of saccharides of the invention on skin inflammation were assessed using an IMQ-induced BALB/c mouse model. Test groups included a naive plus vehicle group, an IMQ only group, an IMQ plus vehicle group, a saccharide plus vehicle group at 3 different concentrations (1, 0.1, 0.01%), a cyclophosphamide control (lOmg/kg in 0.5% CMC), and a clobetasol control (30mg per application), with 8 mice per group. Saccharides were dissolved in an aqueous gel containing a polyacrylate sodium salt, glycerol, paraben and imidazolidinyl urea (=vehicle). 500microL of test gels of 30mg of clobetasol were applied daily to the shaved backs of mice, 4 hours prior to the application of 50mg of IMQ cream (5%). Vaseline was used in the case of naive mice test group and the cyclophosphamide was dosed orally once a day. Observations of skin appearance

(scaling, folding, erythema) were made each day to provide a disease activity index. Dosing was repeated for 6 days when all mice were sampled. Skin samples from all mice were fixed in formalin, embedded, sectioned and stained with haematoxylin and eosin. Slides were scored for epithelia hyperplasia, keratinocyte proliferation (hyperkeratosis), leukocyte infiltration and increased vascularisation. Scores were plotted against test agents to determine the effects of saccharide treatment compared to IMQ plus vehicle controls.

Results from this example are provided in Figure 9.

Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.

REFERENCES

Andres RM et al. 2013 Potential antipsoriatic effect of chondroitin sulphate through inhibition ofNF-kB and STAT3 in human keratinocytes. Pharmacological Research 20: 20-26. De Paz JL et al. 2007 Profiling heparin-chemokine interactions using synthetic tools. ACS Chemical Biology 2 (11): 735-744. Di Cesare A et al. 2009 The IL-23/Thl 7 axis in the immunopathogenesis of psoriasis. J. Invest. Dermatol. 129(6): 1339-1350. Casu B et al. 1994 Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E.coli K5. Carbohydrate Res. 263 (2): 271-284.

Ceccarelli M et al. 2009 Anti-inflammatory effects of low molecular weight heparin derivative in a rat model of carrageenan-induced pleurisy. J Cell Mol Med 13 (8B): 2704-2712. DeAngelis et al. 2002 Identification of the capsular polysaccharides of Type D and F Pasteurella multocida as unmodified heparin and chondroitin,

Respectively Carbohydrate Research 337, 1547-1552. Ferreras C et al. 2012

Endothelial heparan sulphate 6-O-sulphation levels regulate angiogenic response of endothelial cells to fibroblast growth factor 2 and vascular endothelial growth factor. JBC 287 (43): 36132-36146. Iovu M et al. 2008 Anti-inflammatory activity of chondroitin sulfate. Osteoarthritis and Cartilage 16(Supplement 3): p. S14. Lever R et al. 2001 Role of glycosaminoglycans in inflammation. Inflammopharmacology 9(1- 2): p. 165-169. Lever R & Page CR 2002 Novel drug development opportunities for heparin. Nat. Rev. Drug Dis 1 (2):140-148. Lever R & Page CR 2012 Non- anticoagulant effects of heparin: an overview. In: Heparin - A century of progress. Handbook fo Experimental Pharmacology 207 (eds Lever, Mulloy, Page) Springer, Berlin, pp 218-305. McKee J et al. 2010 Structure elucidation and biological activity of the oversulphated chonodritin sulphate contaminant in Baxter heparin. J. Clin. Pharmcol. 50 (10): 1159-1170. Medeiros GF et al. 2000 Distribution ofsulphated glycosaminoglycans in the animal kingdom: widespread occurrence of heparin-like compounds in invertebrates. Biochimica et Biophysica Acta - Genera 1475 (3): 287. Moller I et al. 2010 Effectiveness of chondroitin sulphate in patients with concomitant knee osteoarthritis and psoriasis: a randomized, double-blind, placebo-controlled study. Osteoarthritis Cartilage. 18 Suppl 1: p. S32-40. Nickoloff BJ 2007. Cracking the cytokine code in psoriasis. Nat. Med. 13(3): 242-244. Olson ST et al. 2010

Molecular mechanisms of antithrombin-heparin regulation of blood coagulation proteinases. Biochimie 92 (11): 1587-1596. Oreste P & Zopetti G 2012 Semisynthetic heparinoids In: Heparin - A century of progress. Handbook fo Experimental Pharmacology 207 (eds Lever, Mulloy, Page) Springer, Berlin, pp 403-422. Raman R et al. 2005 Structural insights into biological roles of protein-glycosaminoglycan interactions. Chem. Biol. 12: 267-277. Rudd TR et al. 2010 The conformation and structure ofGAG's: recent progress and perspectives. Curr. Opin. Structural Biol. 20: 567-574. Schlorke D et al. 2012 The influence of glycosaminoglycans on IL8 mediated functions of neutrophils. Carbohydrate Res. 356: 196-203. Seeds EA et al. 1995 The effect of inhaled heparin and related glycosaminoglycans on allergen- induced eosinophil infiltration in guinea-pigs. Pulmonary Pharmacology 8: p. 97-105. Severin IC et al. 2012 Glycosaminoglycan analogs as novel anti-inflammatory strategy. Frontiers in Immunology 3 (293): 1-12. Stern R et al. 2006 Hyaluronan fragments: an information rich system. Eur. J. Cell Biol. 85: 699-715. Vann, W. et al. 1981 Eur. J. Biochem. The Structure of the Capsular Polysaccharide (K5 Antigen) of Urinary-Tract-Infective Escherichia coli 010 : K5 : H4 A Polymer Similar to Desulfo- Heparin 116, 359-364. Verges et al. 2005 Clinical and histopathological

improvement of psoriasis with oral chondroitin sulphate: a serendipitous finding. Dermatology Online Journal 11:31. Xu Y et al. 2012 Chemo enzymatic synthesis of heparin oligosaccharides with both anti-factor Xa and anti-factor Ha activities. JBC 287 (34): 29054-29061.

Claims

Claims

A sulphated heparosan saccharide for use in the treatment of an inflammatory skin disorder wherein the SO₃- / COO- ratio of the sulphated heparosan is in the range 1 to 3.8.

A heparosan saccharide for use in the treatment of an inflammatory skin disorder as claimed in claim 1 wherein the saccharide is selected from at least one of an N-sulphated polysaccharide with a repeat unit N-acetylglucosamine and glucuronic acid [→4 p-D-GlcAp-(l→4)-a-D-GlcNpAc-(l→]; an O-sulphated at C6 of GlcNAc polysaccharide with a repeat unit N- acetylglucosamine and glucuronic acid [→4)- -D-GlcAp-(l→4)-a-D-GlcNpAc [1→] and sulphation at C2 and C3 of GlcA; an O-sulphated at C6 and C3 of GlcNAc, C2 and C3 of GlcA polysaccharide with a repeat unit N-acetylglucosamine and glucuronic acid [→4)- -D-GlcAp (l→4)-ct-D-GlcNpAc-(l→]; an N-sulphated, O-sulphated C6 of GICNSO3, wherein there is provided sulphation at C2 and C3 of GlcA polysaccharide with a repeat unit N- acetylglucosamine and glucuronic acid [→4)- -D-GlcAp-(l→4)-a-D-GlcNpAc

(i→] ; an N-sulphated, O-sulphated at C6 of GICNSO3, C2 and C3 of GlcA, 0- sulphation at C3 of GICNSO3 polysaccharide with a repeat unit N- acetylglucosamine and glucuronic acid [→4)- -D-GlcAp-(l→4)-a-D-GlcNpAc (1→]; and a 8.3 kDa fragment of K5/NS,0S (H) an N-sulphated, O-sulphated at C6 of GICNSO3, C2 and C3 of GlcA, O-sulphation at C3 of GICNSO3 polysaccharide with a repeat unit N-acetylglucosamine and glucuronic acid [→4)- -D-GlcAp (l→4)-a-D-GlcNpAc-(l→].

3. A saccharide with a repeating subunit structure comprising an N- acetylglucosamine linked β1→4 to a hexuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 2 to 30, linked al→4, as depicted in formula I

, and sulphated at between 1 to 5 of the positions a, b, c, d, and e for use in the treatment of an inflamatory skin disorder.

4. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of the preceding claims wherein the saccharide is capable of down-regulating IL8 and IL6 produced by primary human keratinocytes in vitro, which have been stimulated with TNFa, IL17A and histamine.

5. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of the preceding claims wherein the saccharide is capable of down-regulating the histology score in an imiquimod-induced mouse model of psoriasis.

6. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of the preceding claims wherein the saccharide is capable of inhibiting the activity of neutrophil elastase.

7. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of the preceding claims wherein the saccharide is capable of inhibiting monocyte chemoattractant protein-1 induced monocyte chemotaxis.

8. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of the preceding claims wherein the saccharide provides no or reduced anti-coagulant activity compared to heparin or low molecular weight heparin in an APTT assay.

9. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of claims 3 to 8 wherein the saccharide is about 3 to 15 KDa and has a repeating subunit structure comprising N-acetylglucosamine linked β1→4 to a glucuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 5 to 25, linked al→4, as depicted in formula I,

and is sulphated at between 1 to 5 of the positions a, b, c, d, and e.

10. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of claims 3 to 9 wherein the saccharide is an

oligosaccharide of about 6 to 9kDa, with a repeating structure N- acetylglucosamine linked β1→4 to a glucuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 10 to 15, linked al→4, as depicted in formula I and sulphated at between 1 to 5 of the positions a, b, c, d, and e.

11. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of claims 3 to 9 wherein the saccharide is about 3 to 15 kDa, has a repeating structure N-acetylglucosamine linked β1→4 to a glucuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 5 to 25, linked al→4, as depicted in formula I , and sulphated at positions a, b, c, and e, and optionally sulphated at position d.

12. The saccharide for use in the treatment of an inflammatory skin disorder as claimed in any one of claims 3 to 9 wherein the saccharide is about 6 to 9 kDa, has a repeating structure N-acetylglucosamine linked β1→4 to a glucuronic acid wherein the number of repeating disaccharide subunits (n) is in the range 10 to 15 , linked al→4, as depicted in formula I, and sulphated at positions a, b, c, and e, and optionally sulphated at position d.

13. The saccharide for use in the treatment of a skin disorder as claimed in any one of claims 1 to 12, wherein the saccharide has a homogeneous structure such that each disaccharide repeating subunit of the saccharide has identical sulphated positions.

14. A saccharide for use in the treatment of a skin disorder as claimed in any one of claims 1 to 12 wherein the saccharide has a heterogeneous structure such that each disaccharide repeating subunit of the saccharide has variable sulphated positions.

15. A saccharide for use in the treatment of a skin disorder as claimed by claim 13 or claim 14 wherein the sulphation provides a highly sulphated saccharide.

16. A saccharide for use in the treatment of a skin disorder as claimed by claim

13 or claim 14 wherein the sulphation provides a low sulphated saccharide.

17. A saccharide for use in the treatment of a skin disorder as claimed by claim

14 wherein the sulphation of C2 and C3 of GlcA is low.

18. A saccharide for use in the treatment of a skin disorder as claimed by any one of claims 14 to 17 wherein the o-sulphation at C3 of GlcN03 is variable in the saccharide.

19. A pharmaceutical composition comprising a saccharide of any one of claims

1 to 18.