WO2011089446A1 - Use of pharmaceutically active compounds - Google Patents

Abstract

The present invention comprises use of polysulphated chondroitin sulphates (p-CS) for the treatment of diseases or conditions related to collagen fibril formation. The invention particularly relates to a polysulphated chondroitin sulphate (p-CS) with a sulphation degree of 2.8 to 3.8, or a pharmaceutically acceptable salt thereof, for the treatment of conditions or diseases related to collagen fibril formation.

Description

USE OF PHARMACEUTICALLY ACTIVE COMPOUNDS

This invention relates to the use of polysulphated glycosaminoglycans. More particularly it relates to the use of polysulphated chondroitin sulphate for the treatment of diseases or conditions related to collagen fibril formation.

Many proteins of the extracellular matrix (ECM) are modified post- translationally by addition of oligosaccharide chains and are thus known as

glycoproteins. The oligosaccharides are linked either O-glycosidically to serine or threonine residues, or N-glycosidically to an asparagine residue. Proteoglycans are glycoproteins that are substituted with a particular class of carbohydrate polymers, known as the glycosaminoglycans (GAGs). Proteoglycans are found in the ECM, at the cell surface and intracellularly in storage granules. In the ECM, they contribute to its structure and organisation, and at the cell surface often function as receptors and/or co-receptors. All glycosaminoglycans (with the exception of hyaluronan) are synthesised on a core protein acceptor and are thus an integral component of proteoglycans.

Glycosaminoglycans (GAGs) are named to indicate that one of the

monosaccharides in the repeating sequence of disaccharides is an amino sugar. The other monosaccharide is an uronic acid (glucuronic acid or iduronic acid), with the exception of keratan sulphate where it is a galactose. While other oligosaccharide substituents may be branched, GAG chains are linear (again, with the exception of keratan sulphate). Proteoglycans may be substituted with one (e.g. decorin) and up to some hundred (e.g. aggrecan) GAG chains.

There are 4 types of glycosaminoglycans: hyaluronic acid, chondroitin sulphate/ dermatan sulphate (CS), heparan sulphate/heparin (HS) and keratan sulphate. The disaccharides in all glycosaminoglycan chains except hyaluronan are sulphated, increasing their negative charge and leading to an extended conformation of the chain. These molecules will occupy large solvent domains; this results in high viscosity solutions. This property is essential in cartilage and is the basis on which the tissue's resistance lies.

The repeating disaccharide sequence in CS is glucuronic acid-N-acetyl- galactosamine (GlcA-GalNAc; see Figure 1). Chondroitin sulphate is found naturally in several forms, named chondroitin-4 sulphate (also denoted CS-A), chondroitin-6 sulphate (also denoted CS-C) and -D and -E forms, respectively. These forms differ in the extent of sulphation of the saccharides. CS-E is the highest sulphated variant with around 1.6 sulphate groups per disaccharide unit. (See for example Sugahara, K and Yamada, S., Trends in Glycoscience and Glycotechnology, 12, 67, 321-349 (2000) for the definitions of the different forms of CS.)

Mature collagen fibres may contain several different types of bound accessory proteins. They play a part in the organisation of these fibres and regulate links to other molecules thereby contributing to the architecture of the fibrillar collagen network. A recent concept is that of modulator molecules, which regulate the early steps in the assembly of collagen monomers to fibres. Cartilage oligomeric matrix protein (COMP) is such a modulator and accelerates the formation of fibres from monomers (Halasz, K. et al. (2007) J. Biol. Chem. 282, 31166-31173). Other molecules have the opposite effect and slow down fibre formation in vitro, e.g. decorin (Vogel, K. G. et al. (1984) Biochem.J. 223, 587-597) and fibromodulin (Hedbom, E. and Heinegard, D. (1989) J. Biol. Chem. 264, 6898-6905). Gene targeting of these molecules lead to abnormal collagen fibrils and disturbed mechanical properties of the tissues (Danielson, K. G. et al. (1997) J. Cell Biol. 136, 729-743; Svensson, L., et al. (1999) J. Biol. Chem. 274, 9636-9647). Together these proteins in the vicinity of the cell regulate the early stages of collagen fibre formation.

Perlecan exists as HS and CS substituted forms and it has been shown that these forms can be used to facilitate collagen fibril formation (Kvist, A.J. et al. (2006) J. Biol. Chem. 281, 33127-33139). To our surprise synthetically modified CS was effective in promoting collagen fibril formation, whereas non-modified CS variants show significantly less effect. The synthetically modified CS was also effective in different wound healing studies in vivo.

A number of publications describing the effect of chondroitin sulphate on wound healing and for treating arthrosis exist (e.g. US5929050, JP10120577 and RU2216332 US5,955,578; Gilbert et al, Laryngoscope, 114, 1406 (2004); Suyama et al, Jpn. J. Exp. Med. (1971); Kirker et al, Biomaterials, 23, 3661 (2002); Zou et al, J. Dent. Res. 83(11), 880 (2004); Ruszczak, Adv. Drug Del. Rev. 55, 1595 (2003); Kosir et al, J. Surg. Res. 92, 45 (2000); WO 2005/102362; Kasavina, B.S. et al, Bull. Exp. Bio. Med. 718-720 (1961). The invention relates to the use of polysulphated chondroitin sulphates (referred to herein also as p-CS) with improved effects in the promotion of collagen fibril formation for the treatment of diseases and conditions related to collagen fibril formation. In some embodiments, the p-CS facilitates the formation of collagen fibril formation in a wound or ulcer.

Although synthetically-prepared polysulphated chondroitin sulphates (p-CS) with sulphation degrees varying from 1.6 to 4 have previously been reported in the literature, the effects of p-CS on collagen fibril formation are new and unexpected.

According to one aspect, the invention provides the use of polysulphated chondroitin sulphates (p-CS), with a sulphation degree of 2.8 to 3.8 or a

pharmaceutically acceptable salt thereof, for the treatment of conditions and diseases related to collagen fibril formation, including but not limited to wound healing.

As mentioned above, p-CS is a polymer made up of repeating GlcA-GalNAc disaccharide units. The p-CS polymers referred to herein will generally contain a heterogeneous mixture of chains which may be characterised by the range of molecular weights and the average degree of sulphation of the chains.

In some embodiments of the invention, the range of sizes of the p-CS is 5000- 60000 Da. In other embodiments, the preferred range of sizes of the p-CS is 20000- 59000 Da. The sizes of the p-CS may be determined by gel chromatography in 0.2M NaCl on calibrated columns of Sephadex G-200 (e.g. Wasteson, A. J Chromatogr. 59, 87-97 (1971b)]. This method separates the polymer into fractions containing different molecular weights, giving the range of molecular weights which are present in the sample. The molecular weight determination can also be made by densitometry or by polyacrylamide gel electrophoresis in gel slabs (e.g. Dietrich & Nader, Biochim.

Biophys. Acta, 343, 34 (1974)) .

Preferably, the molecular weight is determined using the method and conditions described in Michelacci and Dietrich, Int. J. Biol. Macromol. 8, 108-113 (1986) under the heading "Experimental" (the contents of which are specifically referred to and incorporated herein by reference).

Any of the hydroxy positions in the GlcA-GalNAc disaccharide may be sulphated in the p-CS used herein. As can be seen from Figure 1, there are four possible sulphation sites. As used herein the term "degree of sulphation" refers to the average number of sulphate groups per disaccharide unit. The degree of sulphation may be from 2.8 to 3.8 sulphate groups per disaccharide unit. In other embodiments, the degree of sulphation is 3.0 to 3.4 or 3.4 to 3.8 sulphate groups per disaccharide unit. In other embodiments, the degree of sulphation is above 2.8, 3.0, 3.2, 3.4 or 3.6 sulphate groups per disaccharide unit. In some embodiments, the degree of sulphation is 2.4- 4.0, 2.6-4.0, 2.6-3.8, 2.8-4.0, 2.8-3,8, 3.0-3.8, 3.0-4.0, preferably 3.5-3.8, more preferably 3.6-3.7, sulphate groups per disaccharide unit.

The sulphur content may be determined by any suitable means, for example from elemental analysis. An alternative method involves paper chromatography and toluidine blue staining of an acid hydrolysed sample (e.g. Michelacci and Dietrich, Int. J. Biol. Macromol. 8, 108-113 (1986)).

A further way to characterise the p-CSs described herein is by S/C % ratio, i.e. sulphur: carbon ratio. This may be determined by standard methods, e.g. by elemental analysis. Preferably, the S/C % ratio is 34 to 75, more preferably 40 to 70.

Other ways to characterise the p-CS of the invention include FT-I , NMR('H) and ESI-TOF MS. The invention includes p-CSs as defined herein wherein the structure or composition of the p-CS and/or the positions of the sulphate groups and/or the degree of sulphation is characterised by a spectrum or profile as shown in any one of Figures 2-4.

Methods of preparing p-CS are known in the art. These methods may be based on treatment of an existing available chondroitin or chondroitin sulphate with a sulphating agent. Alternatively, polysulphated chondroitin sulphates may be prepared synthetically. For example, synthetically-prepared polysulphated chondroitin sulphates (p-CS) with sulphation degrees varying from 1.6 to 4 have previously been reported in the literature. (See, for example, Maruyama, T. et al. Carbohydr. Res., 306, (1-2), 35- 43 (1998)).

One convenient sulphating agent that will not otherwise affect the CS molecule is an amine-S03 complex. Suitable amines include both aliphatic and aromatic amines. A preferred complex is a pyridine-S03 complex, but other complexes that could be used are for example S0₃-trimethylamine and S0₃-tributylamine.

The degree of sulphation can be controlled by any suitable means, e.g. by: a) Changing the molar equivalence of the sulphating agent, e.g. pyridine-sulphur trioxide or other amine-S03 complex; b) Changing the reaction temperature (a higher temperature yielding a higher degree of sulphation);

c) Changing the reaction times (longer times yielding a higher degree of

sulphation);

either on their own or in combination, a) and b) are the preferred methods.

The invention is not limited, however, to any particular method of

manufacturing the p-CS.

Preferably, the p-CS used herein is produced by a method comprising polysulphating CS using a SCVpyridine complex, particularly preferably wherein the starting CS is in the form of tributylammonium CS-A or CS-C in DMF.

p-CSs are more highly sulphated than natural CSs and thus may be charged compounds. The invention also comprises the use of pharmaceutical acceptable salts of the pCSs, such as alkali metal salts (sodium, potassium, caesium) or alkaline earth salts (e.g. magnesium, zinc, calcium, strontium) and ammonium, as well as organic salts.

The invention also relates to the use of pharmaceutical compositions or formulations comprising p-CS for the treatment of conditions and diseases related to collagen fibril formation.

Pharmaceutical compositions or formulations comprising a p-CS or mixture of p-CSs disclosed herein or salts thereof as the active ingredient may additionally comprise one or more carriers, fillers and other additive agents which are generally used in the preparation of medicines.

The administration may be by oral administration by tablets, pills, capsules, granules, powders, solutions and the like, or parenteral administration by injections (e.g., intravenous, intramuscular and the like), suppositories, percutaneous preparations, transnasal preparations, inhalations and the like or by topical administration.

Preferably, the pharmaceutical composition is formulated for topical use.

The amount of p-CS which is used for the treatments described herein is an amount which is effective to treat the relevant condition or disease.

With regard to treatments of diseases which can be treated by promoting collagen fibril formation, the amount of p-CS which is used in such treatments will be an amount which is effective to promote collagen fibril formation.

The dose is generally decided in response to each case by taking symptom, age, sex and the like of the subject to be administered into consideration, but in the case of oral administration, it is generally approximately from 0.001 mg/kg to 100 mg/kg per day per adult, and this is administered once or by dividing into 2 to 4 times. When intravenously administered, it is generally administered once to two or more times a day within the range of from 0.0001 mg/kg to 10 mg/kg per day per adult. In the case of transnasal administration, it is generally administered once to two or more times a day within the range of from 0.0001 mg/kg to 10 mg/kg per day per adult. In addition, in the case of inhalation, it is generally administered once to two or more times a day within the range of from 0.0001 mg/kg to 1 mg/kg per day per adult.

In some embodiments, the pCS composition may be administered at a dose of 50 ng/mL - 200 μg/mL vehicle. In some embodiments, the pCS composition may be administered at a dose of 50 ng/mL - 1 μg/mL, 1 μg/mL - 25 μg/mL, 10 μg/mL - 100 μg/mL, 50 μg/mL - 100 μg/mL or 100 μg/mL to 200 μg/mL. Such dosages are particularly preferred for wound area reduction.

In other embodiments, the pCS composition may be administered at a dose of 25 ng/mL - 50 μg/mL vehicle. In some embodiments, the pCS composition may be administered at a dose of 25 ng/mL - 1 μg/mL, 1 μg/mL - 25 μg/mL, 10 μg/mL - 100 μg/mL, 50 μg/mL - 100 μg/mL or 100 μg/mL to 200 μg/mL. Such dosages are particularly preferred for improving wound tissue tensile strength.

In a solid composition for oral administration, one or more of the compounds referred to herein may be mixed with at least one inert filler such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, aluminium magnesium silicate or the like. The composition may also contain inert additives such as lubricants (e.g. magnesium stearate), disintegrators (e.g. carboxymethylstarch sodium), and solubilizing agents, and the like. The tablets or pills may be coated with a sugar-coating or a gastric- or enteric-coating.

With regard to injections for parenteral administration, sterile aqueous or nonaqueous solutions, suspensions and emulsions may be included. As the aqueous solvent, for example, distilled water for injection and physiological saline may be included. Examples of a non-aqueous solvent include propylene glycol, polyethylene glycol, plant oils (e.g. olive oil or the like), alcohols (e.g. ethanol or the like), polysorbate 80, and the like. Such a composition may further contain tonicity agents, antiseptics, moistening agents, emulsifying agents, dispersing agents, stabilizing agents and/or solubilizing agents. These are generally sterilized by, for example, filtration through a bacteria retaining filter, formulation of bactericides or irradiation. In addition, they can also be used by producing a sterile solid compositions and dissolving or suspending them in sterile water or a sterile solvent for injection prior to use.

Inhalations, transmucosal preparations transnasal preparations and the like may be used in a solid, liquid or semisolid form and can be produced in accordance with conventionally known methods. For example, excipients such as lactose, starch or the like, as well as a pH-adjusting agent, an antiseptic, a surfactant, a lubricant, a stabilizer, and/or a thickener and the like, may be optionally added. An appropriate device for inhalation or blowing may be used for the administration. For example, using a conventionally-known device such as a measured administration inhalation device or the like or a sprayer, a compound can be administered alone or as a powder in a prescribed mixture, or as a solution or suspension by a combination with a medicinally- acceptable carrier. The dry-powder inhaler or the like may be for single or multiple administration use, and a dry-powder or a powder-containing capsule may be used. Alternatively, it may be in a form such as a pressurized aerosol spray or the like, which uses suitable gas such as chlorofluoroalkane, hydrofluoroalkane, carbon dioxide or the like.

Pharmaceutical compositions or formulations for topical administration may contain stabilising agents, buffering agents and/or additional gelating agents such as but not limited to hyaluronan, PEG, HMPC, EHEC and carboxymethyl cellulose to obtain controlled release and/or elimination.

As used herein, the term "topical" refers to the application of a composition to the site where it is intended to have its effect. The site may be external (e.g. the skin) or an internal site which is accessed externally (e.g. nasal passage, anus, vagina, GI tract) or an internal site which is accessed during a surgical procedure (e.g. application to an internal organ). In one embodiment, the invention relates to topical

administration to a duodenal ulcer.

In some embodiments, the pCS composition is administered in the form of a gel for topical application.

The invention provides the use of p-CS as defined herein for treatment of various conditions and diseases related to excessive, insufficient or in other ways abnormal collagen fibrillogenesis by the facilitation or prevention of collagen fibril formation (CFF). In particular, the invention provides the use of p-CS as defined herein or a pharmaceutically acceptable salt thereof for the treatment of conditions and diseases related to collagen fibril formation.

Collagen fibers are important structural components of the extracellular matrix of tissues and organs, including but not limited to skin, bone and cartilage. As shown herein (e.g. Figure 5 and Figure 6), p-CS stimulates the formation of collagen fibrils from collagen monomers more effectively than naturally-occurring CS variants. Thus, p-CS or active fragments thereof can be used to stimulate the regeneration of damaged tissues in which collagen fibers, formed by fibril forming collagens, are structural components.

During wound healing, the collagen fibres play an essential role in the regulation of cellular and molecular events, e.g. by forming the substrate upon which cells proliferate and migrate, and by regulating the activities of secreted factors, e.g. growth factors, cytokines and proteases. In healthy wounds, the dermal extracellular matrix supports the processes of re-epithelialisation and angiogenesis by acting as a scaffold for migrating cells. In chronic ulcers, these processes are severely disturbed due to a deficient composition and reorganization of the extracellular matrix. Thus, the facilitation of collagen fibril formation provides a means to promote healing of wounds in general, and chronic ulcers in particular. For instance, p-CS or active fragments thereof could be applied topically to the wound or ulcer in an appropriate formulation to enable the interaction of the compound with endogenous collagen monomers, thereby accelerating the processes of collagen fibril formation and extracellular matrix re-organization.

Indications comprised in this application for which the compounds may be useful include conditions and diseases related to collagen fibriUogenesis, including but not limited to acute and chronic wound healing, fibrotic disorders (e.g. pulmonary fibrosis, fibrosis caused by toxins and fibrosis as a result of ischemic conditions), skeletal reconstruction, skeletal repair and cartilage repair.

Some of the p-CS compounds referred to herein can be used to treat different types of wounds by promoting the collagen fibril formation thereby shortening the wound closure time and/or for increasing the strength of the scar tissue.

p-CS may be useful for treatment of non-healing, chronic wounds (ulcers) including but not limited to diabetic ulcers, venous ulcers and pressure ulcers. Also, the compounds may be useful to promote healing of skin wounds resulting from various sorts of injury or trauma caused by accidents, disease and surgical procedures (e.g., burn injuries, incisions, lacerations, surgical wounds).

In some embodiments, the invention particularly relates to the treatment of wounds or ulcers which are bleeding. In other embodiments, the invention particularly relates to the treatment of wounds or ulcers which are not bleeding.

Furthermore, p-CS may be useful to promote healing of tissues following various sorts of injury or trauma to internal organs or tissues, exemplified but not limited to injuries caused by accidents, diseases or surgical procedures (e.g. bone fractures, cartilage destruction, hernias, herniorraphy, surgical anastomosis).

Some of the p-CS referred to herein can be used to inhibit the formation of collagen fibrils, thereby reducing scar formation. This property can be useful in the treatment of conditions and diseases in which excessive scar formation or fibrosis is a component.

Scarring is a natural response of the body to trauma and injury. In fibrotic conditions the normal wound healing response continues out of control, with excessive production and deposition of collagen. This leads to a loss of function when normal tissue is replaced with scar tissue. Fibrosis can affect virtually all organ systems in the body.

There are many different causes of fibrosis, e.g. trauma, surgery, infection, environmental pollutants and toxins (including alcohol). Some examples of fibrotic conditions are formation of scar tissue following heart attack, kidney fibrosis as a complication of diabetes, lung fibrosis and surgical scar tissue formation between internal organs.

Acute fibrosis is a response to various forms of trauma, such as injury, infections, surgery, burns, radiation damage and chemotherapeutic treatments. Many chronic conditions, e.g. diabetes, viral infection and hypertension, induce a progressive fibrosis causing continuous loss of tissue function. The liver, kidney and lung are commonly affected. Systemic fibrotic diseases include diabetic nephropathy, scleroderma, idiopathic pulmonary fibrosis and reactive fibrosis following myocardial infarct.

The invention further provides modified collagen fibres comprising p-CS as defined herein. Such fibres may be produced by cross-linking a collagen fibre to p-CS, preferably using glutaraldehyde or a carbodiimide. Such fibres may be used in transplantation and/or used in vivo.

The invention also provides a matrix scaffold or hydrogel comprising p-CS as defined herein, optionally additionally comprising cells, e.g. fibroblasts.

The matrix scaffold or hydrogel comprises a hydrogel-forming polymer, an interstitial liquid, and optionally cells retained within the hydrogel. The term

"hydrogel-forming polymer" refers to a polymer which is capable of forming a cross- linked or network structure or matrix under appropriate conditions, wherein an interstitial liquid and optionally cells may be retained within such a structure or matrix. Initiation of the formation of the cross-linked or network structure or matrix may be by any suitable means, depending in the nature of the polymer.

In one embodiment of the invention, the hydrogel-forming polymer is collagen. In this embodiment, the collagen hydrogel comprises a matrix of collagen fibrils which form a continuous scaffold around an interstitial liquid and optionally the cells. For example, dissolved collagen may be induced to polymerise/aggregate by the addition of dilute alkali to form a gelled network of cross-linked collagen fibrils. The gelled network of fibrils supports the original volume of the dissolved collagen fibres, retaining the interstitial liquid. General methods for the production of such collagen gels are well known in the art (e.g. WO2006/003442, WO2007/060459 and

WO2009/004351).

The collagen which is used in the modified fibres or gel may be any fibril- forming collagen. Examples of fibril-forming collagens are Types I, II, III, V, VI, IX and XI. The gel may comprise all one type of collagen or a mixture of different types of collagen. Preferably, the fibres or gel comprise or consist of Type I collagen. In some embodiments of the invention, the modified fibre or gel is formed exclusively or substantially from collagen fibrils, i.e. collagen fibrils are the only or substantially the only (non- pCS) polymers in the modified fibres or gel.

In other embodiments of the invention, the hydrogel-forming polymer is alginic acid or a alginate salt of a metal ion. Preferably, the metal is a Group 1 metal (e.g. lithium, sodium, or potassium alginate) or a Group 2 metal (e.g. magnesium, calcium, strontium or barium alginate). Preferably, the polymer is sodium alginate or calcium alginate. In yet other embodiments of the invention, the hydrogel-forming polymer is a cross-linked acrylic acid-based (e.g. polyacrylamide) polymer, e.g. Carbopol^®

(Lubrizol).

In yet further embodiments, the hydrogel-forming polymer is a polymerizable cellulose derivative, a hydroxyl ether polymer (e.g. a poloxomer) or a natural gum.

The interstitial liquid may be any liquid in which polymer may be dissolved and in which the polymer may gel. Generally, it will be an aqueous liquid, for example an aqueous buffer or cell culture medium.

The p-CS of the invention may merely be embedded within the hydrogel or matrix scaffold, or chemically bonded thereto (e.g. cross-linked to the hydrogel or matrix scaffold, preferably using glutaraldehyde or a carbodiimide). Crosslinking helps to prevent elution of the p-CS from the matrix or gel. Additionally, the hydrogel or matrix (e.g. collagen) may be cross-linked with other GAGs, e.g. chondroitin-6- sulphate.

Such matrices and gels can be used for treating conditions and diseases related to collagen fibril formation by transplantation of cell-containing or cell-recruiting scaffolds or gels into or onto the patient.

In particular, the modified collagen fibres or matrix scaffold or hydrogel can be inserted into or applied onto a patient in order to promote healing of skin or other tissues, for example after an injury caused by accidents or diseases, or after a surgical procedure (e.g. ulcers, skeletal fractures, cartilage erosions, hernias, herniorrhaphy, surgical anastomosis). For instance, p-CS could be used to produce modified collagen fibres or a matrix scaffold or a hydrogel that can be applied to severe burn injuries, e.g. a burn affecting a large area of the skin.

The matrix scaffold or hydrogel may be in any form, e.g. a membrane, a film, a tube or a sponge. It may be in dehydrated or lyophilised form, or not.

The cells which are optionally entrapped within the matrix scaffold or hydrogel are preferably mammalian cells, particularly preferably human cells. Examples of such cells include cells involved in wound repair, for example dermal and epidermal cells (e.g. fibroblasts and keratinocytes) and stem cells.

The invention also provides a process of producing a scaffold matrix or hydrogel comprising the steps: precipitating a hydrogel-forming polymer from a hydrogel-forming polymer solution in the presence of p-CS as defined herein, and optionally dehydrating or lyophilising the resultant precipitate. Preferably, the hydrogel forming polymer is collagen.

In some embodiments, the process additionally involves the step of cross- linking the hydrogel-forming polymer (e.g. collagen) to the p-CS, preferably using glutaraldehyde or a carbodiimide. The cross-linking may be carried out before or after precipitation.

The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety. Figure 1. Basic structure of a chondroitin sulfate. Repeating dimeric units of

GlcA β 1-3 GalNAc. All hydroxy positions may be sulphated or/and epimerised. The various positions open for sulphation are numbered.

Figure 2. FT-IR. IR spectra of compounds 1 (a) and 2 (b).

Figure 3. Proton NMR of Compounds 1 (a) and 2 (b) where the change in sulphation degree and homogeneity of the sample can be seen.

Figure 4. ESI-TOF MS shows the fragmentation of different batches for Compounds 1 (a) and 2 (b).

Figure 5: Effect of various natural (CS-E, CS-A, C6S, CS-C) and synthetic p- CS on collagen fiber formation in vitro. The figures after each p-CS indicate the number of sulphate units per saccharide. Each point represents the mean of three measurements. Degree of sulphation of remaining CS^'s are CS-C 1.05; CS-A 0.89; CS-E 1.57; C6S 1.20. Figure 6: Comparison of the stimulatory effects of natural CS-E and

Compound 1 on collagen fibre formation in vitro. Each point represents the mean of three measurements.

Figure 7. Effect of treatment with a p-CS on wound area reduction in healthy mice. Compound 1 or vehicle (0.5% carboxymethylcellulose/PBS, pH 7.4), were applied topically to the wound once daily for eleven consecutive days. The wound area reduction was significantly (P<0.05) increased upon treatment with the p-CS compared to vehicle treatment, on day 11 for 0.001 μg dose, day 7-11 for 0.01 μg dose, and on day 3, 7-11 for 0.1 μg dose. Data are presented as means + SEM. The statistical evaluation was performed with ANOVA followed by Dunnett's test (N=5/group).

Figure 8. Effect of treatment with p-CS compound 1 on wound breaking strength in diabetic mice, 14 days after wounding. Compound 1 or vehicle (0.5% carboxymethylcellulose/PBS, pH 7.4) were applied topically to the wound once daily for 14 consecutive days. *P<0.05 vs. vehicle. Data are presented as the mean of two measurements + SD. The statistical evaluation was performed with ANOVA for multiple comparison of results; the Duncan multiple range test was used to compare group means. N=6/group.

Example 1: Compound 1

Sulphation of CS was essentially carried out in a manner analogous to that as described in Bocker et al. Carbohydrate Research, 230 (1992), 245-256, which describes the sulphation of carbohydrates, in particular glucose.

Before sulphation was carried out, the starting CS (either CS-A or CS-C obtained from Sigma-Aldrich) was run through an ion-exchange column to exchange sodium with tributylammonium. An acidic ion-exchange resin (IRA- 120) was activated using 200 ml chilled 3 M HC1 and washed with water (approx. 600 ml) until the eluant was neutral. 0.374 g CS was dissolved in 6 ml water and cooled to +5°C. The CS solution was placed on the column and the ion exchange was made in a top-fed freezer. The column was washed with water (approx. 600 ml) until the pH of the eluant was ~6. The eluate was then neutralized using 10% tributylamine in ethanol while cooling in an ice-bath. The water solution was then extracted with 3x100 ml diethyl ether. The aqueous solution was dried to yield a white powder. Sulphation was carried out as follows: To a stirred solution of 200 mg tributylammonium CS in 12 ml dimethyl formamide (DMF) at room temperature was added 455 mg SOs/pyridine complex dissolved in 20 ml DMF at a rate at which the temperature was maintained. The reaction mixture was heated to 50°C and stirred an additional one hour after which the reaction mixture was cooled to room temperature and 33 ml water was added and pH was adjusted to ~9 by adding 0.1 M NaOH. 190 ml saturated NaOAc in ethanol was added and the mixture was stirred for two hours at room temperature after which the mixture was centrifuged at 1500 rpm for 18 min. The supernatant was removed. The remainder was dissolved in 12.5 ml water and desalinated by gel chromatography (NAP-25 disposable column, Amersham

Biosciences). 2.5 ml solution was eluted with 3.5 ml water and the column was regenerated with 25 ml water. The combined eluates (35 ml) were freeze-dried yielding approximately 60 mg of a polysulphated CS product having around 3.63 sulphate groups per disaccharide unit. This corresponds to around 69.1% as a calculated S/C% ratio.

Example 2: Compound 2

The pre-treatment before sulphation was carried out in the same way as in Example 1 above.

A lower degree of sulphation was obtained by using the same procedure as above, but the addition of the pyridine-SC>3 complex was added at 0°C and the reaction mixture was stirred at +2°C for 30 minutes before quenching the reaction as described above. This produced a sulphated CS product with around 1.85 sulphate groups per disaccharide unit. This corresponds to around 35.2% as a calculated S/C % ratio.

The products were characterized by elementary analysis to establish the degree of sulphation, shown in Table 1 below, as well as FT-IR, NMR (1H) and ESI-TOF MS, as shown in Figures 2 to 4. The major fragments according to ESI-TOF MS vary between 1200 up to about 3300, with the major M.W. around 1710 (Compound 1) and 1350 and 2800 (Compound 2). Table 1 - Elementary analysis

Example 3: In vitro collagen fibrillogenesis

A previously-described method (Hedbom, E. and Heinegard, D. (1989) J. Biol. Chem. 264, 6898-6905) was adapted for the microtitre plate format. Briefly, water solutions of various natural (CS-E (Seikagaku, Code No: 400678), CS-C (Seikagaku, Code No: 400670), CS-A (Sigma-Aldrich, Cat. No. C9819:), Chondroitin-6-Sulphate (C6S; Sigma-Aldrich: Cat. No. C4384)) and synthetic p-CS^'s were transferred to microtitre plate (Corning, Cat. No. 3641) wells in appropriate volumes. A solution of bovine type I collagen monomers (INAMED BIOMATERIALS, PureCol, Code: 5409) was brought to neutral pH by addition of an appropriate volume of 0.012M NaOH and buffered by 20 mM HEPES, 150 mM NaCl at pH 7.4. The collagen solution was thereafter added to the microtitre plate wells to obtain a final collagen monomer level of 100 μg/ml and chondroitin sulphate levels as indicated in Figures 5 and 6. The microtitre plate was sealed with optic sealing tape (Nunc, Cat. No. 232701) and inserted into a pre-warmed (36°C) spectrophotometer (SpectraMAX^® 340pc, Molecular Devices), and the absorbance due to light scattering at 400 nm was monitored over time (700 minutes). An increased absorbance/turbidity is caused by collagen fiber formation.

Result of collagen fibrillogenesis experiment

Different types of natural and synthetic p-CS were tested for effect in the collagen fibrillogenesis assay and the result is shown in Figures 5 and 6. The synthetic p-CS facilitated collagen fibre formation, and affected both the time course and the maximal turbidity obtained (Figure 5). With respect to stimulatory activity in this assay, the most highly sulphated product was superior to the other synthetic p-CS's (Figure 5) and was also more active than natural CS-E at 1 and 0.06 μg/ml (Figure 6). Other naturally-occurring chondroitin sulphates tested, i.e., CS-A, CS-C, and C6S, were less efficacious (Figure 5).

Example 4: In vivo models

Healing of full-thickness, excisional skin wounds in healthy mice

CD-I (Crl.) derived male mice weighing 24 ± 2 g were used. During the study animals were housed in individual cages. Under hexobarbital (90 mg/kg, IP) anaesthesia, the shoulder and back region of each animal was shaved. A sharp punch (inner diameter 12 mm) was applied to remove the skin, including panniculus carnosus and adherent tissues. Polysulphated chondroitin sulphate (0.1, 0.01 and 0.001 μg/mouse) or vehicle (0.5% carboxymethylcellulose/phosphate-buffered saline (PBS), pH 7.4) were administered topically to the wound in volumes of 20 μΐ, immediately following cutaneous injury, thereafter once daily until the end of the experiment. The wound area, traced onto clear plastic sheets, was measured by use of an Image - ProPlus (Media Cybernetics, Version 4.5.0.29) on days 1, 3, 5, 7, 9 and 11. The percent reduction of the wound area was calculated, and wound half-closure time (CT50) was analyzed by linear regression using Graph-Prism (Graph Software USA).

Result of wound healing experiment in healthy mice

Topical treatment with Compound 1 promoted wound healing in healthy mice.

This was shown by its significant effect on wound area reduction (Figure 7) and on half-closure time, CT₅₀ (e.g., 5.9 ± 0.3 days and 7.0 ± 0.2 days for compound 1 (0.1 μg) and vehicle, respectively). Wound breaking strength of full-thickness, incisional wounds in healing-deficient, diabetic mice

Female ((C57 L/KsJ -Leprdb) db+/db+) mice (Charles River) aged 14 weeks and weighing about 48 g were used. During the experiment, the animals were housed one per cage, maintained under controlled environmental conditions (12-hr light/dark cycle, temperature approximately 23°C), and provided with standard laboratory food and water ad libitum. After general anaesthesia with thiopental sodium (80 mg/kg/i.p.), the hair on the back was shaved and the skin washed with povidone-iodine solution and wiped with sterile water. Two full-thickness longitudinal incisions (4 cm) were made on the dorsum of the mice, and the wound edges were closed with 4-0 silk surgical suture placed at 1-cm intervals. Compound 1 (0.1 , 1 and 10 μg/mouse) or vehicle (0.5% carboxymethylcellulose (Sigma-Aldrich, Cat. No. C-4888)/PBS (pH 7.4)) were applied topically to the wound in 200 μΐ volumes, starting immediately after wounding, thereafter once daily until sacrifice of the animal. All substances were prepared fresh daily from frozen aliquots. The animals were sacrificed after 14 days. The wounds were excised and used for breaking strength measurement. Two skin strips, 0.8 cm wide and 2 cm long, obtained from every animal were used for breaking strength measurement. The maximum load (breaking strength) tolerated by wounds was measured blindly on coded samples using a calibrated tensometer (Instron, Canton, MA) as previously described (Pierce, G. F., Mustoe, T. A., Senior, R. M. et al. (1988) J Exp Med 167, 974-987). The ends of each skin strip were pulled at a constant speed (20 cm/min) and breaking strength was expressed as the mean maximum level of tensile strength in Newton (N) before separation of wounds.

Result of wound healing experiment in healing-deficient, diabetic mice

Wound breaking strength was determined 14 days after full-thickness, incisional wounding of healing-deficient, diabetic mice. As shown in Figure 8, daily topical treatment with p-CS compound 1 enhanced breaking strength compared to vehicle treatment, and at the highest dose (10 μg) the difference was significant (PO.05).

Example 5: Formulations

Example of a preparation comprising a capsule

Per capsule

Active ingredient, optionally as salt 5 mg

Lactose 250 mg

Starch 120 mg

Magnesium stearate 5 mg

Total up to 380 mg

In case higher amounts of active ingredient are required, the amount of lactose used may be reduced. Example of a suitable tablet formulation.

Per tablet

Active ingredient, optionally as salt 5 mg

Potato starch 90 mg

Colloidal silica 10 mg

Talc 20 mg

Magnesium stearate 2 mg

5% aqueous solution of gelatine 25 mg

Total up to 152 mg

A solution for parenteral administration by injection can be prepared in aqueous solution of a water-soluble pharmaceutically acceptable acid addition salt of the active substance preferably in a concentration of 0.1% to about 10% by weight.

These solutions may also contain stabilising agents, buffering agents and/or gelating agents such as but not limited to hyaluronan, PEG, HPMC, EHEC, carboxymethyl cellulose, to obtain a controlled release and/or elimination.

Example of a topical formulation

A gel for topical administration can be prepared with active substance in a concentration of 0.1% to 10% by weight, optionally containing stabilising agents, buffering agents and/or additional gelating agents such as but not limited to hyaluronan, PEG, HMPC, EHEC, carboxymethyl cellulose to obtain controlled release and/or elimination.

Example 6: Collagen gel comprising pCS

An aqueous composition is made by combining 4 ml of sterile rat-tail type I collagen in 1 ml of 10 X concentration Eagle minimum essential medium and mixing in 5 mg polysulphated chondroitin sulphate (Compound 1). A collagen gel is made by neutralizing the aqueous composition with 0.5 ml 1M sodium hydroxide. The gel is cast into a mould and set/stabilized in a 37°C 0.5% C0₂ incubator for 30 min.

Claims

Claims

1. A polysulphated chondroitin sulphate (p-CS) with a sulphation degree of 2.8 to 3.8, or a pharmaceutically acceptable salt thereof, for the treatment of conditions or diseases related to collagen fibril formation.

2. Use of a polysulphated chondroitin sulphate (p-CS) with a sulphation degree of 2.8 to 3.8 or a pharmaceutically acceptable salt thereof, for the manufacture of medicament for the treatment of conditions or diseases related to collagen fibril formation.

3. A method of treating conditions or diseases related to collagen fibril formation comprising administering an effective amount of a polysulphated chondroitin sulphate (p-CS) with a sulphation degree of 2.8 to 3.8 or a pharmaceutically acceptable salt thereof, to a patient.

4. A p-CS, use or method as claimed in any one of claims 1 to 3, wherein the range of sizes of the p-CS is 5000-60000 Da, preferably 20000-59000 Da.

5. A p-CS, use or method as claimed in any one of claims 1 to 4, wherein the pCS has a degree of sulphation of 3.4 to 3.8 sulphate groups per disaccharide unit.

6. A p-CS, use or method as claimed in any one of the preceding claims, wherein the disease or condition to be treated is a wound or ulcer.

7. A p-CS, use or method as claimed in any one of claims 1 to 6, wherein the condition or disease is skeletal repair and/or reconstructive skeletal formation.

8. A p-CS, use or method as claimed in any one of claims 1 to 6, wherein the condition or disease is cartilage repair and/or reconstructive cartilage formation.

9. A p-CS, use or method as claimed in any one of claims 1 to 6, wherein the condition or disease is fibrosis.

10. A p-CS, use or method as claimed in any one of claims 1 to 9, wherein the structure or composition of the p-CS and/or the positions of the sulphate groups and/or the degree of sulphation is characterised by a spectrum or profile as shown in any one of Figures 2-4.

11. A modified collagen fibre comprising a polysulphated chondroitin sulphate (p- CS) as defined in any one of claims 1 and 4-5, or a pharmaceutically acceptable salt thereof.

12. A matrix scaffold or a hydrogel comprising a polysulphated chondroitin sulphate (p-CS) as defined in any one of claims 1 and 4-5, or a pharmaceutically acceptable salt thereof, wherein the p-CS is either embedded within the scaffold or hydrogel or chemically bonded thereto, the scaffold or hydrogel optionally additionally comprising cells entrapped therein.

13. A matrix scaffold or a hydrogel as claimed in claim 12, wherein the matrix scaffold or hydrogel is formed from collagen, alginic acid, an acrylic acid based polymer, a polymerizable cellulose derivative, a hydroxylether polymer or a natural gum.

14. A collagen fibre, a matrix scaffold or a hydrogel as claimed in any one of claims 11 to 13, for use as a medicament or for use in therapy or surgery.

15. A collagen fibre, a matrix scaffold or a hydrogel as claimed in any one of claims 11 to 13, for use in treating conditions and diseases related to collagen fibril formation.

16. Use of a collagen fibre, a matrix scaffold or a hydrogel as claimed in any one of claims 11 to 13, in the manufacture of medicament for treating conditions and diseases related to collagen fibril formation.

17. A method of treating conditions and diseases related to collagen fibril formation comprising contacting a patient with a collagen fibre, a matrix scaffold or a hydrogel as claimed in any one of claims 11 to 13.

18. A collagen fibre, a matrix or a hydrogel as claimed in any one of claims 11 to 15, a use as claimed in claim 16 or a method as claimed in claim 17, wherein the disease or condition to be treated is a wound or ulcer and the fibre, matrix or hydrogel is applied to the wound or ulcer.

19. A collagen fibre, a matrix or a hydrogel as claimed in any one of claims 11 to 15, a use as claimed in claim 16 or a method as claimed in claim 17, wherein the disease or condition to be treated is skeletal repair and/or reconstructive skeletal formation and the fibre, matrix or hydrogel is applied to the bones to be repaired or reconstructed.

20. A collagen fibre, a matrix or hydrogel as claimed in any one of claims 11 to 15, a use as claimed in claim 16 or a method as claimed in claim 17, wherein the disease or condition to be treated is cartilage repair and/or reconstructive cartilage formation and the fibre, matrix or hydrogel is applied to the cartilage to be repaired or reconstructed.

21. A collagen fibre, a matrix or a hydrogel as claimed in any one of claims 11 to 15, a use as claimed in claim 16 or a method as claimed in claim 17, wherein the disease or condition to be treated is a burn and the fibre, matrix or hydrogel is applied to the burn.

22. A process of producing a scaffold matrix or hydrogel, said process comprising precipitating a hydrogel-forming polymer from a hydro gel-forming polymer solution in the presence of a p-CS as defined in any one of claims 1 or 4-5, and optionally dehydrating or lyophilising the resultant precipitate.

23. A process as claimed in claim 22, which additionally comprises the step of cross-linking the hydrogel-forming polymer to the p-CS, preferably using

glutaraldehyde or a carbodiimide.

24. A process as claimed in claim 22 or claim 23, wherein the hydrogel-forming polymer is selected from the group consisting of collagen, alginic acid, an acrylic acid based polymer, a polymerizable cellulose derivative, a hydroxylether polymer and a natural gum.

25. A process for producing a modified collagen fibre, said process comprising cross-linking a polysulphated chondroitin sulphate (p-CS) as defined in any one of claims 1 and 4-5 to a collagen fibre, preferably using glutaraldehyde or a carbodiimide.