WO2011089446A1 - Utilisation de composés pharmaceutiques actifs - Google Patents

Utilisation de composés pharmaceutiques actifs Download PDF

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Publication number
WO2011089446A1
WO2011089446A1 PCT/GB2011/050112 GB2011050112W WO2011089446A1 WO 2011089446 A1 WO2011089446 A1 WO 2011089446A1 GB 2011050112 W GB2011050112 W GB 2011050112W WO 2011089446 A1 WO2011089446 A1 WO 2011089446A1
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hydrogel
collagen
matrix
fibre
scaffold
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PCT/GB2011/050112
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English (en)
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Torbjorn Lundstedt
Arne Boman
Niklas Palmqvist
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Anamar Ab
Webber, Philip
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Publication of WO2011089446A1 publication Critical patent/WO2011089446A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate

Definitions

  • 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.
  • ECM extracellular matrix
  • 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).
  • GAGs glycosaminoglycans
  • 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 are named to indicate that one of the amino acids
  • 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.
  • glycosaminoglycans There are 4 types of glycosaminoglycans: hyaluronic acid, chondroitin sulphate/ dermatan sulphate (CS), heparan sulphate/heparin (HS) and keratan sulphate.
  • CS chondroitin sulphate/ dermatan sulphate
  • HS heparan sulphate/heparin
  • 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.
  • CS glucuronic acid-N-acetyl- galactosamine
  • GlcA-GalNAc glucuronic acid-N-acetyl- galactosamine
  • 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.
  • 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).
  • 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.
  • 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.
  • the p-CS facilitates the formation of collagen fibril formation in a wound or ulcer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • p-CS of the invention includes 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.
  • p-CS 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)).
  • amine-S03 complex 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 S0 3 -trimethylamine and S0 3 -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);
  • 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.
  • alkali metal salts sodium, potassium, caesium
  • alkaline earth salts e.g. magnesium, zinc, calcium, strontium
  • ammonium e.g. magnesium, zinc, calcium, strontium
  • 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.
  • 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.
  • parenteral administration by injections (e.g., intravenous, intramuscular and the like), suppositories, percutaneous preparations, transnasal preparations, inhalations and the like or by topical administration.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pCS composition may be administered at a dose of 50 ng/mL - 200 ⁇ g/mL vehicle. In some embodiments, the pCS composition may be administered at a dose of 50 ng/mL - 1 ⁇ g/mL, 1 ⁇ g/mL - 25 ⁇ g/mL, 10 ⁇ g/mL - 100 ⁇ g/mL, 50 ⁇ g/mL - 100 ⁇ g/mL or 100 ⁇ g/mL to 200 ⁇ g/mL. Such dosages are particularly preferred for wound area reduction.
  • the pCS composition may be administered at a dose of 25 ng/mL - 50 ⁇ g/mL vehicle. In some embodiments, the pCS composition may be administered at a dose of 25 ng/mL - 1 ⁇ g/mL, 1 ⁇ g/mL - 25 ⁇ g/mL, 10 ⁇ g/mL - 100 ⁇ g/mL, 50 ⁇ g/mL - 100 ⁇ g/mL or 100 ⁇ g/mL to 200 ⁇ g/mL. Such dosages are particularly preferred for improving wound tissue tensile strength.
  • 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.
  • aqueous solvent for example, distilled water for injection and physiological saline may be included.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • stabilising agents such as but not limited to hyaluronan, PEG, HMPC, EHEC and carboxymethyl cellulose to obtain controlled release and/or elimination.
  • 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).
  • the invention relates to topical
  • 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).
  • CFF collagen fibril formation
  • 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.
  • 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.
  • the dermal extracellular matrix supports the processes of re-epithelialisation and angiogenesis by acting as a scaffold for migrating cells.
  • these processes are severely disturbed due to a deficient composition and reorganization of the extracellular matrix.
  • the facilitation of collagen fibril formation provides a means to promote healing of wounds in general, and chronic ulcers in particular.
  • 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.
  • fibrotic disorders e.g. pulmonary fibrosis, fibrosis caused by toxins and fibrosis as a result of ischemic conditions
  • skeletal reconstruction e.g. pulmonary fibrosis, fibrosis caused by toxins and fibrosis as a result of ischemic conditions
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the hydrogel-forming polymer is collagen.
  • the collagen hydrogel comprises a matrix of collagen fibrils which form a continuous scaffold around an interstitial liquid and optionally the cells.
  • 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
  • the collagen which is used in the modified fibres or gel may be any fibril- forming collagen.
  • 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.
  • the fibres or gel comprise or consist of Type I collagen.
  • 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.
  • the hydrogel-forming polymer is alginic acid or a alginate salt of a metal ion.
  • 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).
  • the polymer is sodium alginate or calcium alginate.
  • the hydrogel-forming polymer is a cross-linked acrylic acid-based (e.g. polyacrylamide) polymer, e.g. Carbopol ®
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the hydrogel forming polymer is collagen.
  • 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.
  • GlcA ⁇ 1-3 GalNAc All hydroxy positions may be sulphated or/and epimerised. The various positions open for sulphation are numbered.
  • 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
  • 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 ⁇ g dose, day 7-11 for 0.01 ⁇ g dose, and on day 3, 7-11 for 0.1 ⁇ g dose.
  • Figure 8 Effect of treatment with p-CS compound 1 on wound breaking strength in diabetic mice, 14 days after wounding.
  • 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.
  • 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
  • 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 ⁇ g/ml and chondroitin sulphate levels as indicated in Figures 5 and 6.
  • the microtitre plate was sealed with optic sealing tape (Nunc, Cat. No.
  • Example 4 In vivo models
  • 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 ⁇ g/mouse) or vehicle (0.5% carboxymethylcellulose/phosphate-buffered saline (PBS), pH 7.4) were administered topically to the wound in volumes of 20 ⁇ , immediately following cutaneous injury, thereafter once daily until the end of the experiment.
  • PBS carboxymethylcellulose/phosphate-buffered saline
  • Topical treatment with Compound 1 promoted wound healing in healthy mice.
  • 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.
  • 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.
  • 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 ⁇ g) the difference was significant (PO.05).
  • Example of a preparation comprising a capsule
  • Active ingredient optionally as salt 5 mg
  • Example of a suitable tablet formulation In case higher amounts of active ingredient are required, the amount of lactose used may be reduced.
  • Active ingredient optionally as salt 5 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.
  • stabilising agents such as but not limited to hyaluronan, PEG, HPMC, EHEC, carboxymethyl cellulose, to obtain a controlled release and/or elimination.
  • 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.
  • stabilising 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% C0 2 incubator for 30 min.

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  • Pharmacology & Pharmacy (AREA)
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  • Medicinal Chemistry (AREA)
  • Dermatology (AREA)
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  • Veterinary Medicine (AREA)
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Abstract

La présente invention concerne l'utilisation de sulfates de chondroïtine polysulfatés (p-CS) pour le traitement de maladies ou d'affections liées à la formation de fibrilles de collagène. L'invention porte en particulier sur un sulfate de chondroïtine polysulfaté (p-CS) qui présente un degré de sulfatation de 2,8 à 3,8, ou un sel de qualité pharmaceutique de celui-ci, pour le traitement d'affections ou de maladies liées à la formation de fibrilles de collagène.
PCT/GB2011/050112 2010-01-25 2011-01-24 Utilisation de composés pharmaceutiques actifs WO2011089446A1 (fr)

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JP2018514641A (ja) * 2015-06-10 2018-06-07 ジェジァン サンメン ヘンカン ファーマスーティカル カンパニー リミテッド ヘパリノイドの製造方法
CN111334543A (zh) * 2020-04-28 2020-06-26 山东冰文生物技术有限公司 一种新的提取鱼软骨中硫酸软骨素的方法

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CN111334543A (zh) * 2020-04-28 2020-06-26 山东冰文生物技术有限公司 一种新的提取鱼软骨中硫酸软骨素的方法

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