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WO2013006906A1 - Sulfate de dermatane, compositions pharmaceutiques et leur procédé de fabrication - Google Patents

Sulfate de dermatane, compositions pharmaceutiques et leur procédé de fabrication Download PDF

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Publication number
WO2013006906A1
WO2013006906A1 PCT/AU2012/000828 AU2012000828W WO2013006906A1 WO 2013006906 A1 WO2013006906 A1 WO 2013006906A1 AU 2012000828 W AU2012000828 W AU 2012000828W WO 2013006906 A1 WO2013006906 A1 WO 2013006906A1
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WIPO (PCT)
Prior art keywords
dermatan sulphate
molecular weight
low molecular
isolated
fraction
Prior art date
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PCT/AU2012/000828
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English (en)
Inventor
Paul Pantaleone Masci
Kirthi De Silva
Simone OSBORNE
Robert Bruce SEYMOUR
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Commonwealth Scientific and Industrial Research Organization CSIRO
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Commonwealth Scientific and Industrial Research Organization CSIRO
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Priority claimed from AU2011902761A external-priority patent/AU2011902761A0/en
Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Publication of WO2013006906A1 publication Critical patent/WO2013006906A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

  • the present application relates to a method or process for the enrichment, isolation and/or modification of the dermatan sulphate (DS) from hide processing waste streams.
  • the present application also provides a use of the isolated dermatan sulphate and/or low molecular weight dermatan sulphate in the treatment of hematological disorders and in the preparation of medicaments, supplements, nutraceuticals, functional food ingredients and functional food.
  • Dermatan sulphate also known as chondroitin sulphate B (CSB)
  • CSB chondroitin sulphate B
  • GAG glycosaminoglycan
  • Dermatan sulphate forms a complex with heparin cofactor II (HOI), which binds to thrombin, inactivating the protease and preventing completion of the coagulation cascade. It does not bind to anti-thrombin III, consequently it is not as potent as heparin as an anti-coagulant. It is also effective at preventing thrombosis because it enhances the rate of formation of thrombin-HCII complexes while at the same time posing less hemorrhagic risk.
  • HOI heparin cofactor II
  • WO 1990/04607 entitled “Novel dermatan sulfate and heparin oligosaccharides having antiatherosclerotic activity” discloses a process for the preparation of low molecular weight dermatan sulphate based on peroxide free radical reaction of dermatans (or fractions thereof) with an oxidizing agent, such as a periodate ion followed by reduction of the aldehyde groups at the C(2)-C-(3) positions to hydroxymethyl groups and fractionation of the products by anion exchange to separate the fragments of different charge density, and subsequent acid hydrolysis to produce the oligosaccharides having the desired molecule weight.
  • an oxidizing agent such as a periodate ion followed by reduction of the aldehyde groups at the C(2)-C-(3) positions to hydroxymethyl groups and fractionation of the products by anion exchange to separate the fragments of different charge density, and subsequent acid hydrolysis to produce the oligosaccharides having the desired molecule weight.
  • WO 199855514 entitled "Oligosaccharide mixtures having antithrombotic activity” discloses depolymerisation of dermatan sulphate by periodate oxidation, followed by borohydride reduction and acid hydrolysis and then ion exchange fractionation. These prior art processes are time consuming and involve multiple steps often resulting in problems with scale-up and reproducibility. The prior art process also tend to use alcoholic solvents such as methanol, ethanol, propenol or isopropanol. There is therefore a need for improved methods and processes for preparing dermatan sulphate.
  • dermatan sulphate can be isolated from hide processing waste using a method that does not involve the use of such alcohol solvents. This dermatan sulphate is able to inhibit thrombin activity and inhibit clot formation in a rabbit model of deep vein thrombosis.
  • One aspect relates to a method for enriching or extracting dermatan sulphate (DS) from an aqueous composition of hide waste processing waste that is able to inhibit thrombin activity and prevent clot formation, the method comprising the following steps:
  • LMW low molecular weight
  • Another aspect also provides an isolated dermatan sulphate (DS) or low molecular (LMW) dermatan sulphate fraction thereof, wherein said isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof comprises at least one of the disaccharides selected from the group comprising ⁇ -di-non S; ⁇ -di-mono 6S; ⁇ -di-mono 4S; ⁇ -di-di 2,6S and ⁇ -di-di 2,4S and is derived from animal hide disaccharides.
  • DS dermatan sulphate
  • LMW low molecular
  • One aspect provides an isolated dermatan sulphate (DS) or low molecular weight (LMW) dermatan sulphate fraction thereof, wherein said isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof comprises at least one of the following disaccharides: diOs [UAl,3GalNAc)], di6s [UAl,3GalNAc 6(S0 4 )], di4s [UAl,3GalNAc 4(S0 4 )], di2s [UA 2(S0 4 )l,3GalNAc], di2,6dis [UA 2(S0 4 )l,3GalNAc 6(S0 4 )], di4,6dis' [UAl,3GalNAc 4(S0 4 ) 6(S0 4 )], di2,4dis [UA 2(S0 )l,3GalNAc 4(S0 )], di2,4,6tris [UA 2(S0 4 )l,3GalNA
  • the dermatan sulphate may be from any suitable source.
  • the dermatan sulphate is derived from an animal source.
  • the animal source may be, for example, animal hides, skins, organs, tissues or the like.
  • the primary animal source is animal hide processing waste.
  • [10] in another aspect relates to a method or process for isolating a low molecular weight (LMW) dermatan sulphate that includes the step of at least partially depolymerising the dermatan sulphate to obtain a product with similar or enhanced activity compared to the original dermatan sulphate product.
  • LMW low molecular weight
  • Another aspect relates to least one LMW dermatan sulphate produced by the method or process of the invention.
  • the optionally isolated dermatan sulphate or LMW dermatan sulphate may be a liquid concentrate or a dried solid.
  • the dried solid may be a powder.
  • Another aspect relates to a dermatan sulphate or LMW dermatan sulphate that is able to inhibit thrombin activity and delay clot formation.
  • This dermatan sulphate has no effect on platelets allowing the delayed clot to be of normal strength.
  • FIGURE 1 illustrates an agarose gel electrophoresis of glycosaminoglycans extracted from bovine hide processing waste stream compared to commercial glycosaminoglycan standards, before and after digestion with chondroitin ABC lyase (Chase ABC) and chondroitin ACII lyase (Chase ACII).
  • glycosaminoglycans were fixed using 0.1% (w/v) cetyl-trimethyl-ammonium bromide solution and stained using 0.1% toluidine blue (w/v) / 50% (v/v) ethanol/1% acetic acid (v/v) solution.
  • FIGURE 2 illustrates an anion exchange chromatography of depolymerised dermatan sulphate using Q Sepharose Big Beads. Elution profile of depolymerised dermatan sulphate fractionated using a 1.5M NaCl gradient at pH 7.0. Fractions were collected every 5 minutes (for 150 minutes) using a 2mL/minute flow rate. Interaction with DMMB dye (525nm) confirmed the presence of dermatan sulphate. Fractions 5 to 16 were collected for activity and structural analysis.
  • FIGURE 3 illustrates a heparin cofactor II mediated thrombin inhibition of bovine hide derived dermatan sulphate prepared using a pilot scale process (prototype DS) and depolymerised prototype DS (LMW prototype DS) compared to a commercial dermatan sulphate (commercial DS) preparation, as measured in vitro.
  • prototype DS pilot scale process
  • LMW prototype DS depolymerised prototype DS
  • commercial DS commercial dermatan sulphate
  • FIGURE 4 illustrates the effect of bovine hide derived dermatan sulphate prepared using a pilot scale process (DS) compared to commercial heparin, on time to clot formation and clot strength in vitro;
  • DS pilot scale process
  • A Thromboelastographs show that the application of DS inhibits time to clotting but has no effect on clot strength when compared to normal blood.
  • Table 1 illustrates (B) thromboelastography (TEG), which is a measure of the efficiency of coagulation in the blood, and anti-thrombotic parameters show that DS significantly inhibits time to clotting (R time, PT and aPTT) without significantly affecting clot strength (MA value) when compared to negative (citrate and saline) controls in vitro.
  • TAG thromboelastography
  • FIGURE 5 illustrates in vivo antithrombotic activity of bovine hide derived dermatan sulphate prepared using a pilot scale process (DS), saline and commercial heparin in rabbit blood following intravenous (IV) administration in a deep vein thrombosis model;
  • DS pilot scale process
  • IV intravenous
  • A Representative thromboelastograph traces of animals IV administrated with: (1) Saline; (2) DS dose 1; (3) DS dose 2; (4) DS dose 3; and (5) heparin with blood samples (A), (B) and (C) as marked.
  • Table 2 illustrates (B) three IV doses of DS reduced clot formation and extended aPTT and R times, without significantly affecting clot formation (MA value) or PT.
  • FIGURE 6 illustrates in vivo antithrombotic activity of bovine hide derived dermatan sulphate prepared using a pilot scale process (DS), saline and commercial heparin in rabbit blood following oral administration in a deep vein thrombosis model;
  • A Representative thromboelastograph traces of animals orally administrated with: (1) saline; (2) DS; and (3) heparin with blood samples (A), (B), (C) and (D) as marked.
  • Table 3 illustrates (B) oral administration of DS significantly reduced clot formation; extended R time and increased GAG levels in serum without significantly affecting clot strength (MA value).
  • FIGURE 7 illustrates total glycosaminoglycans (GAG) in serum, following intravenous administration (IV) of 120 mg/kg dermatan sulphate (DS) to one animal, were measured using the Blyscan Sulphated Glycosaminoglycan Assay.
  • a one-phase exponential decay model (R 2 0.9930) revealed an average half life of 9.2 minutes (with a range of 8.8 - 9.7 minutes) and an average plateau concentration of 189 g/mL (with a range of 177 - 201 g/mL).
  • the model also predicted that it would take 255 minutes for the amount of GAG in serum to return to basal levels (40 ⁇ g/mL).
  • the R time from the saline control was 342 ⁇ 37 seconds and is denoted by «— .
  • FIGURE 8 illustrates a pilot-scale process for isolation of dermatan sulphate from a bovine hide processing waste stream.
  • FIGURE 9 illustrates a pilot-scale process for depolymerising and isolating depolymerised dermatan sulphate from a bovine hide processing waste stream.
  • the aqueous composition containing dermatan sulphate may be derived from a waste stream.
  • the waste stream may be that resulting from enzymatic or chemical treatment of an animal hide, skin, or the like, with for example a protease or an alkaline or acid solution as known in the art.
  • the method or process of the invention may include a heat hold step to precipitate such matter and improve the performance of subsequent filtration steps.
  • this optional step may extend over a period of about 1 to 8 hours and is at a temperature between about 35°C and 75°C (inclusive).
  • a person skilled in the art would be able to determine empirically the appropriate time and temperature depending on the nature of the undesired matter.
  • the coarse filtration may occur across a filter of about 5 - 10 ⁇ .
  • the filter may be selected from the group consisting of a liquid bag filter; a static screen, a centrifugal screen or vibratory screen; or any other device which permits separation of insoluble and/or precipitated matter from soluble material.
  • a person skilled in the art would be able to determine empirically the appropriate filter depending on the nature of insoluble and/or precipitated matter.
  • the clarification step may be effected by, for example, a centrifugal clarifier such as continuous solids discharging horizontal centrifuge or the like.
  • a centrifugal clarifier such as continuous solids discharging horizontal centrifuge or the like.
  • a person skilled in the art would be able to determine empirically the appropriate clarifier depending on the nature of insoluble and/or precipitated matter.
  • the method/process of the invention may include at least one microfiltration step.
  • the microfiltration may include diafiltration. Diafiltration may serve the purpose of recovering the maximum amount of dermatan sulphate from the MF retentate.
  • the method or process of the invention may include an ultrafiltration and/or diafiltration step to remove, for example, sulphide ions.
  • the step may be continued until the level of hydrogen sulphide is acceptable following pH adjustment. pH adjustment may be effected by addition of any suitable acid, for example, HC1 or H2SO 4 . This step may also be used to change the pH to 5.5 - 7.5, preferably to the pH may be adjusted to 6.3.
  • the aqueous composition is subjected to ion exchange.
  • Ion exchange chromatography involves the interaction of charged functional groups in the sample with ionic functional groups of opposite charge on an adsorbent surface. Two general types of interaction are known. Anionic exchange chromatography is mediated by negatively charged compounds interacting with positively charged surfaces and cationic exchange chromatography is mediated by positively charged compounds interacting with negatively charged surfaces.
  • strongly basic anion exchange resins may be used to fractionate the depolymerised dermatan sulphate are typically in HSO3 " form.
  • the strongly basic anion exchange resin may have a styrene or acrylic skeleton.
  • the resin may be crosslinked with divinylbenzene.
  • Other alkenylaromatic polymer resins such as those based on monomers like alkyl-substituted styrene or mixtures thereof, can also be applied.
  • a weakly basic anion exchange resin may be used to fractionate the depolymerised dermatan sulphate.
  • the weakly basic anion exchange resin has an acrylic skeleton derived from acrylic esters such as methyl acrylate, ethyl acrylate, butyl acrylate, methyl methacrylate, acrylonitrile or acrylic acids or a mixture thereof.
  • the weakly basic anion exchange resin structure is epichlorohydrin-based polycondensation anion exchangers.
  • the chloromethyl and epoxy groups of epichlorohydrin react with polyamines forming crosslinked gel type anion exchangers.
  • the ion exchange resin may be a gel-type resin.
  • the ion exchange step may be achieved using column or batch ion exchange chromatography, simulated moving bed chromatography, continuous ion exchange (e.g. CSEP technology), or any combination thereof.
  • the isolated dermatan sulphate may be achieved by methods known to those skilled in the art.
  • the dermatan sulphate may be isolated by evaporation, freeze drying, or any combination thereof.
  • One aspect relates to an isolated dermatan sulphate (DS) or low molecular weight (LMW) dermatan sulphate fraction thereof, wherein said isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof comprises at least one of the ⁇ - disaccharides selected from the group comprising:
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof may comprise at least two of the ⁇ -disaccharides.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof may comprise at least three of the ⁇ - disaccharides. It may also comprise at least four of the ⁇ -disaccharides.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof may comprise at least five of the ⁇ -disaccharides.
  • the isolated dermatan sulphate will compromise any number and all possible combinations of all the disaccharides as identified above.
  • the low molecular weight dermatan sulphate will compromise any number and all possible combinations of all the disaccharides as identified above but is likely that enhanced anti-thrombin activity will come from those fragments enriched for the more highly sulphated disaccharides.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof is obtained from hide processing waste.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof may be in a sterile form.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof contains no more than 10% by weight of non-dermatan sulphate biological materials.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof contains no more than 2% by weight of non-dermatan sulphate biological materials.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof has a molecular weight in the range about 5 to about 30 kDa.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof has a molecular weight in the range about 5 to 15 kDa.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof has a molecular weight in the range about 5 to 8 kDa.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, together with a physiologically tolerable carrier or excipient, and optionally also a therapeutic or prophylactic drug substance.
  • Another aspect relates to a pharmaceutical that may be formulated for oral administration in the form of tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions, functional beverages or functional foods.
  • Another aspect relates to a method of reducing blood coagulation in a human or mammalian body which method comprising administering an anticoagulant effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof to a human or mammal in need thereof.
  • Another aspect relates to a cosmetic use of dermatan sulphate. It may be formulated for use as a cosmeceutical using various carrier creams and lotions.
  • Another aspect relates to a method of treating thrombosis comprising: administering to a human or mammal in need thereof an amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, said amount being effective to treat thrombosis in the subject.
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction may be administered orally.
  • the dermatan sulphate may be delivered orally as a tablet or syrup formulated to provide delivery to the stomach.
  • These products may be a pharmaceutical product or a nutraceutical product.
  • the dermatan sulphate may also be delivered via a food product such as a muffin to provide a therapeutic dose.
  • One aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of orally administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof.
  • One aspect relates to a method for enriching or extracting dermatan sulphate (DS) from an aqueous composition of hide waste processing waste, the method comprising the following steps:
  • LMW low molecular weight
  • the method includes the step of at least one heat hold step.
  • the heat hold step may involve heating the aqueous composition to about 35 to about 75°C for a period of about 1 to about 8 hours. Preferable heating to 53°C and holding for two hours for the non-soluble solids to precipitate and separate from the soluble solids.
  • the step of course filtration may occur across a filter of about 5 - 10 ⁇ .
  • the filter may be selected from the group comprising a liquid bag filter; a static screen, a centrifugal screen or vibratory screen; or any other device which permits separation of insoluble and/or precipitated matter from soluble material.
  • the step of clarification is performed on a continuous solid discharging horizontal centrifuge or the like.
  • the step of microfiltration may include diafiltration.
  • the pH of the aqueous solution is maintained at pH of about 6.3.
  • the ion exchange step may be performed on an exchange resin selected from the group comprising a strongly basic anion exchange resin, or a weakly basic anion exchange resin.
  • Step (h) may involve isolating the dermatan sulphate by evaporation, freeze drying, and /or a combination thereof.
  • the methods may further comprises a step j) fractionating the depolymerised dermatan sulphate.
  • the fractionation of the depolymerised dermatan sulphate may be performed by gel filtration or gel permeation chromatography, hydrophobic interaction chromatography, fractionation based on charge differences, affinity chromatography or any combination thereof.
  • One aspect relates to a method or process for producing a low molecular weight dermatan sulphate from native dermatan sulphate, the method or process comprising depolymerising the dermatan sulphate to yield a low molecular weight dermatan sulphate with similar or enhanced biological activity compared to the native dermatan sulphate.
  • Depolymerisation may be achieved by, for example, periodate depolymerisation, free radical depolymerisation, ⁇ -elimination, and nitrous acid reaction.
  • the depolymerisation step may be an oxidative process.
  • the oxidative process may comprise generation of free radicals in solution; for example, the generation of free radicals results in HO " free radicals.
  • the HO ' free radicals are generated by contacting the aqueous composition with metal ions and hydrogen peroxide.
  • the metal ions are copper ions.
  • the depolymerisation step may produce a mixture of low molecular weight (LMW) dermatan sulphate molecules.
  • the mixture of LMW dermatan sulphate molecules may have an average molecular weight in the range of 5 kDa - 30 kDa.
  • the mixture of LMW dermatan sulphate molecules has an average molecular weight of 5 kDa - 8 kDa.
  • the depolymerisation step precedes a subsequent fractionation step.
  • the depolymerisation step may further comprise fractionation of the depolymerised dermatan sulphate by gel filtration or gel permeation chromatography (also known as size exclusion chromatography), hydrophobic interaction chromatography, fractionation based on charge differences, affinity chromatography, hydrophobic interaction chromatography, or any combination of the above-mentioned forms of chromatography.
  • the fractionation based on charge differences is ion exchange chromatography.
  • the ion exchange chromatography may be anion exchange.
  • a particularly suitable ion exchange resin is a gel-type resin.
  • compositions comprising the dermatan sulphate and/or at least one LMW dermatan sulphate produced by the method of the present invention.
  • the composition may be a pharmaceutical composition, a nutraceutical composition, or a functional food.
  • the international food information council (IFIC) defines functional food as foods that provide health benefit in addition to basic nutrition. Functional foods may play a role in reducing the risks of disease and promote good health. Functional foods can include whole, enhanced, fortified or enriched foods.
  • compositions may be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or times release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions, functional beverages or functional foods. They may also be administered in an intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, and all using dosage forms well known to those of ordinary skill in the art of pharmacy.
  • the present compositions may be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, for example using conventional transdermal skin patches. The dosage administration in a transdermal delivery system will be continuous rather than intermittent throughout the dosage regime.
  • the dermatan sulphate and/or LMW dermatan sulphate prepared by the methods described herein is effective in the treatment of a haematological disorder.
  • one aspect relates to a method of treating a haematological disorder in a subject comprising administering to a subject in need thereof a therapeutically effective amount of dermatan sulphate and/or LMW dermatan sulphate according to the invention.
  • the haematological disorder is a blood coagulation disorder.
  • Subjects that may receive a treatment or be administered a composition of dermatan sulphate and/or LMW dermatan sulphate include animals, including mammalians, and particularly humans. Animals also include domestic animals, such as horses, cows, sheep, swine, cats, dogs, and zoo animals.
  • One aspect also relates to a use of dermatan sulphate and/or LMW dermatan sulphate in the manufacture of a medicament for the treatment of a haematological disorder.
  • the haematological disorder may be a blood coagulation disorder.
  • the blood coagulation disorder may be an inappropriate or pathological formation of a solid mass from the constituents of blood within a blood vessel or an organ, such as a cerebral thrombosis and/or a coronary thrombosis.
  • dermatan sulphate and/or LMW dermatan sulphate of the invention in cell or tissue culture.
  • the substrate may be an artificial blood vessel, or equipment for clinical application used in regions directly contacted with blood, for example, a blood vessel catheter, a tube for an artificial kidney, artificial heart and lung, a blood vessel bypass tube, an artificial heart pumping chamber, bone implants etc.
  • the process is free of volatile solvents.
  • the normal process for the production of glycosaminoglycans from a range of different source materials is to use ethanol precipitation.
  • This invention utilises a commercially efficient ion exchange process for the production of the dermatan sulphate free of volatile solvents such as alcoholic solvents. It has surprisingly been found that despite the use of alcoholic solvents in prior art methods, the present invention provides superior results without using alcoholic solvents.
  • one aspect provides a novel source of dermatan sulphate.
  • Previously hide processing waste has not been considered as a viable commercial source of this product.
  • the process developed makes it commercially viable to purify the dermatan sulphate from this source material.
  • the hide processing waste may be either from the production of leather or from the production of collagen from selected hides as both processes require alkaline treatment to remove hair and condition the hide.
  • the only GAG detected in the hide processing waste is dermatan sulphate thus the process results in the production of a pure dermatan sulphate product uncontaminated by other glycosaminoglycans.
  • Embodiments as described herein are the first time that it has been demonstrated that oral delivery of dermatan sulphate results in the reduction of clot formation in vivo. Whilst dermatan has been used intravenously to reduce clotting in human clinical situations it has not been shown to be orally bioavailable and active. lt has also been demonstrated that following oral delivery of the dermatan sulphate to rabbits there is a significant increase in blood levels of glycosaminoglycans from 6.7 - 8.3 ⁇ / ⁇ , in the control animals, to 13.2 - 15.9 ⁇ g/mL in the animals that received oral doses of dermatan sulphate (Figure 6). This was an indication that the dermatan sulphate had been absorbed across the gut wall.
  • the dermatan sulphate and/or low molecular weight dermataii sulphate fraction thereof may be useful as an anti-thrombotic with two potential applications.
  • the first is as a pharmaceutical replacement for heparin being an intravenous therapy for blood clotting diseases that include deep vein thrombosis.
  • the compositions may be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or times release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions, functional beverages or functional foods.
  • compositions, of the invention may be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, for example using conventional transdermal skin patches.
  • the dosage administration in a transdermal delivery system will be continuous rather than intermittent throughout the dosage regime.
  • the second potential anti thrombotic application is as an oral therapy for the prevention of deep vein thrombosis during long haul air flights.
  • One aspect relates to a method of reducing blood coagulation in a subject, the method comprising administration to a subject in need thereof, an anticoagulant effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof prepared by a method comprising the following steps:
  • LMW low molecular weight
  • One aspect relates to a method of treating thrombosis comprising:
  • LMW low molecular weight
  • One aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of orally administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof,
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof prepared by a method comprising the following steps:
  • LMW low molecular weight
  • Another aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of intravenously administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, that increases the time until clot formation by up to 300% (compared to normal) 40 - 70 minutes post intravenous administration, and remains elevated in serum for more than four hours following intravenous administration,
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof prepared by a method comprising the following steps:
  • LMW low molecular weight
  • Another aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of intravenously administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, that increases the time until clot formation by up to 300% (compared to normal) 40 - 70 minutes post intravenous administration, and remains elevated in serum for more than four hours following intravenous administration,
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof prepared by a method comprising the following steps: a) subjecting the aqueous composition to a heat hold step;
  • LMW low molecular weight
  • Another aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of orally administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, that increases the time until clot formation by up to 300% (compared to normal) 40 - 70 minutes post intravenous administration, and remains elevated in serum for more than four hours following intravenous administration,
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof prepared by a method comprising the following steps:
  • One aspect relates to a method of reducing blood coagulation in a subject, the method comprising administration to a subject in need thereof, an anticoagulant effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof prepared by a method comprising the following steps:
  • LMW low molecular weight
  • One aspect relates to a method of treating thrombosis comprising:
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof prepared by a method comprising the following steps:
  • LMW low molecular weight
  • One aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of orally administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof,
  • the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof prepared by a method comprising the following steps:
  • LMW low molecular weight
  • One aspect relates to a method of reducing blood coagulation in a subject, the method comprising administration to a subject in need thereof, an anticoagulant effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, wherein said isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof comprises at least one of the following disaccharides; diOs [UAl,3GalNAc)], di6s [UAl,3GalNAc 6(S0 4 )], di4s [UAl,3GalNAc 4(S0 4 )], di2s [UA 2(S0 4 )l,3GalNAc], di2,6dis [UA 2(S0 4 )l,3GalNAc 6(S0 4 )], di4,6dis [UAl,3GalNAc 4(S0 ) 6(S0 4 )], di2,4dis [UA 2O 2
  • One aspect relates to a method of treating thrombosis comprising: administering to a human or mammal in need thereof an amount of the isolated dermatan sulphate or low molecular weight- dermatan sulphate fraction thereof, or a salt or solvate thereof,
  • said isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof comprises at least one of the following disaccharides; diOs [UAl,3GalNAc)], di6s [UAl,3GaINAc 6(S0 4 )], di4s [UAl,3GalNAc 4(S0 4 )], di2s [UA 2(S0 4 )l,3GalNAc], di2,6dis [UA 2(S0 4 )l,3GalNAc 6(S0 4 )], di4,6dis [UAl,3GalNAc 4(S0 4 ) 6(S0 4 )], di2,4dis [UA 2(S0 4 )l,3GalNAc 4(S0 4 )], di2,4,6tris [UA 2(S0 4 )l,3GalNAc 4(S0 4 ) 6(S0 4 )].
  • One aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of orally administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof,
  • said isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof comprises at least one of the following disaccharides; diOs [UAl,3GalNAc)], di6s [UAl,3GalNAc 6(S0 4 )], di4s [UAl,3GalNAc 4(S0 4 )], di2s [UA 2(S0 4 )l,3GalNAc], di2,6dis [UA 2(S0 4 )l,3GalNAc 6(S0 4 )], di4,6dis [UAl,3GalNAc 4(S0 ) 6(S0 4 )], di2,4dis [UA 2(S0 )l,3GalNAc 4(S0 4 )], di2,4,6tris [UA 2(S0 4 )l,3GalNAc 4(S0 4 ) 6(S0 )].
  • Another aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of intravenously administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, that increases the time until clot formation by up to 300% (compared to normal) 40 - 70 minutes post intravenous administration, and remains elevated in serum for more than four hours following intravenous administration,
  • said isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof comprises at least one of the following disaccharides; diOs [UAl,3GalNAc)], di6s [UAl,3GalNAc 6(S0 4 )], di4s [UAl,3GalNAc 4(S0 4 )], di2s [UA 2(S0 4 )l,3GalNAc], di2,6dis [UA 2(S0 4 )l,3GalNAc 6(S0 )], di4,6dis [UAl,3GalNAc 4(S0 ) 6(S0 4 )], di2,4dis [UA 2(S0 4 )l,3GalNAc 4(S0 4 )], di2,4,6tris [UA 2(S0 4 )l,3GalNAc 4(S0 4 ) 6(S0 )].
  • Another aspect relates to a method of treating and/or preventing deep vein thrombosis in a subject, the method involves the step of intravenously administering to the subject an antithrombotically effective amount of the isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof, or a salt or solvate thereof, that increases the time until clot formation by up to 300% (compared to normal) 40 - 70 minutes post intravenous administration, and remains elevated in serum for more than four hours following intravenous administration,
  • said isolated dermatan sulphate or low molecular weight dermatan sulphate fraction thereof comprises at least one of the following disaccharides; diOs [UAl,3GalNAc)], di6s [UAl,3GalNAc 6(S0 4 )], di4s [UAl,3GalNAc 4(S0 4 )], di2s [UA 2(S0 4 )l,3GalNAc], di2,6dis [UA 2(S0 4 )l,3GalNAc 6(S0 4 )], di4,6dis [UAl,3GalNAc 4(S0 4 ) 6(S0 4 )], di2,4dis [UA 2(S0 )l,3GalNAc 4(S0 4 )], di2,4,6tris [UA 2(S0 4 )l,3GalNAc 4(S0 4 ) 6(S0 4 )].
  • chondroitin sulphate a glycosaminoglycan similar to dermatan sulphate
  • Dermatan sulphate may also have application for the treatment of inflammation as it is known that thrombin plays a role in enhancing inflammation.
  • dermatan sulphate that is able to inhibit thrombin is orally available and thus may be able to reduce inflammation as an oral therapy.
  • Hyaluronan is another glycosaminoglycan molecule that is currently used in cosmeceutical preparations for the enhancement of skin health. Predominantly it is used based on the theory that hyaluronan promotes skin health through its ability to retain water. Dermatan sulphate is likely to also have application in cosmeceuticals as it has been shown to promote the growth of fibroblasts through its interaction with growth factors. Thus a rational explanation can be provided as to why dermatan sulphate may be beneficial in a cosmeceutical application.
  • both the native and the low molecular weight dermatan sulphate molecules have confirmed in vitro anti-thrombin activity when complexed with the protein heparin cofactor II (HCII).
  • HCII protein heparin cofactor II
  • the dermatan sulphate (and low molecular weight dermatan sulphate) and the heparin cofactor II are combined and then incubated with the thrombin substrate chromozym and then with thrombin itself. Cleavage of the substrate by thrombin releases a chromophore that can be measured by spectrophotometer.
  • the hide derived dermatan sulphate produced as part of the novel process has been shown to inhibit thrombin in this in vitro assay, as shown by a reduced production of the chromophore.
  • the HCII-mediated thrombin inhibition by DS is measured using a commercial heparin standard curve and expressed as USP units/mg DS required to inhibit thrombin activity by 50% ( Figure 3).
  • the dermatan sulphate product that is produced from the process described has been shown to increase the time to clot formation (R time) in an in vitro assay using whole blood, as well as impact on several other parameters of blood clotting ( Figure 4). Included in the analysis of the effect of dermatan in vitro on whole blood is the measurement of clot strength upon clot formation (MA value), prothrombin time (PT) and activated partial thromboplastin time (aPTT) (Table 2).
  • the level of glycosaminoglycans in serum was measured to determine the half-life of intravenous DS (Figure 7).
  • the amount of GAG in serum was determined in one animal every 5 minutes over a 100 minute time-course post intravenous administration of DS dose 3.
  • the concentration of DS in serum was estimated using the BlyscanTM Sulphated Glycosaminoglycan Assay.
  • Clot formation following DS treatment was reduced by approximately 90%, however unlike heparin, DS did not significantly affect clot strength (MA value).
  • MA value the level of glycosaminoglycans in serum was measured to determine the uptake of the product (Table 3). The results from this work demonstrate that oral dermatan sulphate is able to cross the gut wall and significantly increase the levels of glycosaminoglycan present in serum, and having done so is able to reduce clot formation in the rabbit model of deep vein thrombosis and delay time until clot formation for more than three hours after oral administration.
  • red blood cell count ranged from 5.3 - 5.5 xl0 6 /mm 3
  • white blood cell count ranged from 3.8 - 5.1 xloVmm 3
  • platelets ranged from 153 - 164 xl0 9 /L.
  • the hide processing waste stream was initially heated and held at 53°C for the non- soluble solids to precipitate and separate from the soluble solids.
  • the supernatant was decanted and passed through a GAF filter and processed in a microfiltration plant to remove any fine particles and fat.
  • the final retentate was diafiltered to minimise the dermatan in the retentate waste stream.
  • the MF permeate was concentrated in an ultrafiltration plant to reduce the volume of liquid and reduce the diafiltration volume
  • the UF retentate was diafiltered using water to reduce the concentration of sodium sulphite in the UF retentate and reduce the pH of the UF retentate at the same time.
  • the UF retentate was diafiltered until there was ⁇ lppm of hydrogen sulphide produced when the retentate was pH adjusted to pH 6.3. Diafiltration eliminated the requirement to add large volumes of acid and produce poisonous hydrogen sulphide gas.
  • the pH of the UF retentate was adjusted to about pH 6.3 and passed through CSEP chromatographic columns.
  • the columns with the bound proteins are washed and proteins and peptides bound on to the column are pre-eluted using 150 mM sodium citrate or 0.4 M sodium chloride solution followed by eluting with 1-2 M and preferably with 2 M sodium chloride solution.
  • the eluted dermatan sulphate solution can be desalted using ultrafiltration membranes of 5 - 10 kDa molecular weight cut-off membranes and preferably 10 kDa membrane and includes diafiltration.
  • the concentrated dermatan sulphate solution can be provided either as a concentrated liquid or as a powder by optionally evaporating followed by spray or freeze or any other form of drying technology.
  • Depolymerisation can be carried out according to the methods described in the literature for preparing low molecular weight dermatan sulphate, for example, by periodate depolymerisation (see, for example, application of this approach in EP-0,287,477), or by free radical depolymerisation (see, for example, application of this approach in EP-0, 121,067. This approach is based on the generation of HO " free radicals by means of metal ions and hydrogen peroxide. Depolymerisation can also be carried out by ⁇ -elimination (see, for example, EP- 0,040,144). Another approach to depolymerising is by reacting large molecular weight dermatan sulphate with nitrous acid (see, for example, EP-0,037,319).
  • the depolymerisation step produces a mixture of LMW dermatan sulphates.
  • the mixture of LMW dermatan sulphates has an average molecular weight in the range of 5 kDa - 10 kDa.
  • the mixture of LMW dermatan sulphates has an average molecular weight of 5 - 8 kDa.
  • the depolymerisation step precedes a subsequent fractionation step.
  • Fractionation of the depolymerised dermatan sulphates can be achieved by a number of methods including, for example, gel filtration or gel permeation chromatography, also known as size exclusion chromatography, ion exchange chromatography, fractionation based on charge differences, and hydrophobic interaction chromatography.
  • the depolymerised dermatan sulphates can be fractionated by gel filtration or gel permeation chromatography.
  • Hydrophobic interaction chromatography is a useful tool for separating molecules based on their hydrophobicity.
  • sample molecules in a high salt buffer are loaded on the HIC column.
  • the salt in the buffer interacts with water molecules to reduce the solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently adsorbed by the HIC column.
  • the more hydrophobic the molecule the less salt needed to promote binding.
  • a decreasing salt gradient is used to elute samples from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases and molecules elute from the column in order of increasing hydrophobicity.
  • Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer.
  • the depolymerised dermatan sulphates can be fractionated by hydrophobic interaction chromatography.
  • Affinity chromatography relies oh the specific interaction of a compound with an immobilized ligand.
  • the ligand can be designed to be specific for the compound of interest. Alternatively, the ligand may be able to react with a number of related compounds. A person skilled in the art would understand the nature of this technique and its utility in fractionating depolymerised dermatan sulphates.
  • the process includes a fractionation step based on affinity chromatography.
  • dermatan sulphates or LMW dermatan sulphates of the invention would, therefore, in addition to the application to artificial blood vessels, be extremely valuable as materials for a variety of equipment for clinical application used in regions directly contacted with blood, for example, a blood vessel catheter, a tube for an artificial kidney, artificial heart and lung, a blood vessel bypass tube, an artificial heart pumping chamber, bone implants etc.
  • the haematological disorder is a blood coagulation disorder.
  • One aspect also relates to a use of the dermatan sulphates and/or LMW dermatan sulphates in the manufacture of a medicament for the treatment of a haematological disorder.
  • the haematological disorder is a blood coagulation disorder.
  • the blood coagulation disorder is an inappropriate or pathological formation of a solid mass from the constituents of blood within a blood vessel or an organ, such as a cerebral thrombosis and/or a coronary thrombosis.
  • stromal cells are to be used for transplantation or implantation in vivo it is preferable to obtain the stromal cells from the patient's own tissues.
  • the growth of cells in the presence of the three-dimensional stromal support matrix may be further enhanced by adding to the matrix, or coating the matrix support with proteins (e.g., collagens, elastic fibres, reticular fibres) glycoproteins, glycosaminoglycans (e.g., dermatan sulphate, etc.), a cellular matrix, and/or other materials.
  • proteins e.g., collagens, elastic fibres, reticular fibres
  • glycoproteins e.g., glycoproteins, glycosaminoglycans (e.g., dermatan sulphate, etc.)
  • glycosaminoglycans e.g., dermatan sulphate, etc.
  • stromal cells may be added to form a three-dimensional stromal matrix required to support long term growth in culture.
  • other cells found in loose connective tissue may be inoculated onto the three-dimensional support along with fibroblasts.
  • Such cells include but are not limited to endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, etc.
  • These stromal cells may readily be derived from appropriate organs such as skin, liver, etc., using methods known in the art such as those discussed above.
  • stromal cells which are specialized for the particular tissue to be cultured may be added to the fibroblast stroma.
  • stromal cells of hematopoietic tissue including but not limited to fibroblasts, endothelial cells, macrophages/monocytes, adipocytes and reticular cells, could be used to form the three-dimensional subconfluent stroma for the long term culture of bone marrow in vitro.
  • Hematopoietic stromal cells may be readily obtained from the "buffy coat" formed in bone marrow suspensions by centrifugation at low forces, e.g., 3000 x g.
  • Stromal cells of liver may include fibroblasts, Kupffer cells, and vascular and bile duct endothelial cells.
  • glial cells could be used as the stroma to support the proliferation of neurological cells and tissues; glial cells for this purpose can be obtained by trypsinization or collagenase digestion of embryonic or adult brain.
  • the dermatan sulphate of the invention can be used in cell - or tissue culture. In a preferred embodiment, the dermatan sulphate of the invention is used in cell - or tissue culture prior to transplant the cell or tissue into a recipient.
  • the major proteoglycans found in cartilage are chondroitin sulphate, dermatan sulphate, keratan sulphate and hyaluronan. Chondrocytes are active cells within the cartilage matrix, which manufacture new collagen and proteoglycan molecules while excreting enzymes, which aid in removal of damaged cartilage and proteoglycans.
  • the dermatan sulphates and/or LMW dermatan sulphates is dermatan sulphate and LMW dermatan sulphate, respectively.
  • alcoholic solvent is meant that the solvent is based on an alcohol.
  • alcoholic solvents include methanol, ethanol, propenol and isopropanol
  • Unsaturated chondro/dermato/hyaluro-disaccharides (ADi-0S, ADi-4S, ADi-6S, ADi- UA2S, ADi-diSB, ADi-diSD, ADi-diSE and ADi-triS) and chondroitin ACII lyase (Arthrobacter aurescens) were from Seikagaku Corporation (Tokyo City, Japan).
  • Chondroitin sulphate A (bovine trachea), chondroitin sulphate B (porcine intestinal mucosa), chondroitin sulphate C (shark cartilage), dextran sulphates (8kDa and lOkDa), 2-Amino-9(10H)- acridinone (AMAC), sodium cyanoborohydride, 1 ,3-diaminopropane, toluidine blue O, acetic acid (glacial), dimethyl sulfoxide and chondroitin ABC lyase (Proteus vulgaris) were from Sigma-Aldrich (St Louis, MO, USA).
  • a hide waste stream from the alkaline extraction of collagen from bovine hides was collected and stored at 4°C for no longer than 48 hours.
  • the waste liquid was heated as described, clarified and the pH adjusted to pH 7.0.
  • Dermatan sulphate was extracted using Q Sepharose Big Beads.
  • Anion exchange chromatography was performed at pH 7.0 using 2M NaCl to elute the bound dermatan sulphate.
  • the eluant was de-salted using ultrafiltration prior to freeze drying of the final material.
  • the dermatan sulphate concentration was estimated in the freeze dried material using the BlyscanTM Sulphated Glycosaminoglycan Assay (according to the manufacturer's instructions).
  • the dermatan sulphate samples were digested with ABC lyase and then assayed for antithrombin activity. No significant antithrombin activity was observed (data not shown).
  • Example 4 Preparation of depolymerised dermatan sulphate
  • Depolymerised dermatan sulphate was prepared from the freeze dried bovine dermatan sulphate as described by Volpi et al. in IV Convegno su Recenti Sviluppi ed Applicazioni nell'Analisi Farmaceutica, Vol. 47 841-853 (II Farmaco, Bologna; 1992) with modifications. This controlled method of depolymerisation is based on the generation of hydroxyl radicals by means of metal ions and hydrogen peroxide.
  • dermatan sulphate (as determined using the BlyscanTM Sulphated Glycosaminoglycan Assay) was dissolved in 10ml sterile deionised water. 40mg copper acetate' was added with stirring at 55°C. The pH was adjusted to 7.5 and maintained using 1M sodium hydroxide. Depolymerisation was facilitated by adding 9% v/v hydrogen peroxide at a rate of 2mL/hour. Various incubation times produced depolymerised material with a variety of molecular masses. Final incubation time was 50 minutes producing a depolymerised dermatan sulphate.
  • Example 5 Separation ofdepolymerise.d DS using anion exchange chromatography
  • the depolymerised material was fractionated using anion exchange chromatography facilitated by Q Sepharose Big Beads, see Figure 2. lg of depolymerised material was fractioned at pH 7 using a 0-1.5M NaCl gradient in 50mM sodium citrate. Fractions were collected every- 5 minutes, for a total 150 minutes, using a 2ml/minute flow rate. Fractions containing dermatan sulphate were confirmed by interaction with dimethymethylene blue dye measured at 525nm. The glycosaminoglycan containing fractions were desalted using Chemicon Tube-O-DIALYZERs with a 1 kDa MWCO.
  • Molecular weight was determined via HPLC analysis using a TS -GEL G4000SW (7.5mmx30cm) and a G2000SW (7.5mmx30cm) connected in series.
  • the HPLC system from Shimadsu consisted of a Model SIL-lOAi auto injector, a SCL-IOA vp System Controller and a SPD-M10A diode array detector.
  • the mobile phase was composed of 125mM sodium sulphate (Na 2 S0 4 ) and 2mM sodium dihydrogen orthophosphate (NaH 2 P0 4 ) adjusted to pH 6.0 with 0.1M NaOH. Flow rate was l.Oml/min.
  • Glycosaminoglycan elution was detected at 206 nm and confirmed via fraction interaction with dimethylmethylene blue dye. 200 g to 400 ⁇ g of each sample was solubilised in water with the molecular mass calculated as an average of three determinations. The peak molecular mass was calculated using a calibration curve plotted with commercial chondroitin sulphate A, chondroitin sulphate B (DS), chondroitin sulphate C and dextran sulphates (8 kDa and 10 kDa molecular mass).
  • DS required for HCII mediated thrombin inhibition are repeating disulphated disaccharides of either iduronic acid-2-O-sulphate— >N-acetyl-D-galactosamine 4- O-sulphate or uronic acid ⁇ N-acetyl-D-galactosamine 4,6-0-sulphate7,15, 16, 18,24,27,28.
  • oligosaccharides containing the high affinity HCII binding site iduronic acid-2-0-sulphate ⁇ N-acetyl-D-galactosamine 4-O-sulphate, were isolated. . v ⁇
  • Example 7 Heparin cof actor II mediated thrombin inhibition by DS preparations
  • HCII Heparin cof actor II
  • the assay was incubated at 37°C for 40 minutes with the absorbance measured at 405 nm (Spectra max PLUS 384) at 2 minute intervals. All DS samples with and without digestion with chondroitin ABC lyase were assayed twice in triplicate.
  • the HCII mediated thrombin inhibition by the DS samples was calculated using a heparin standard curve (0.15625 ⁇ g/mL-2.5 g/mL or 0.028125 USP units/mL-0.45 USP units/ml) and expressed as USP units/mg DS required to inhibit thrombin activity by 50%. Each sample was assayed twice in triplicate and expressed as the mean ⁇ the standard error.
  • Figure 3 represents the results of a prototype dermatan sulphate sample from one of the pilot scale trails. Results from analysis of the dermatan sulphate produced from the bench and pilot scale separation trials were qualitatively indistinguishable.
  • the gel was run using a Bio-Rad Mini-Protean II Cell. To load the samples glycerol and bromophenol blue was added to the labelling mix. Approximately 180pM of labelled disaccharide was run per well, with cooling to maintain running at 0°C. Once the gel run was completed (25mA for 40 minutes) the gel was removed and placed under a UV transilluminator for visualisation of the bands. The digested samples were compared to standard unsaturated disaccharide controls as obtained from Seikagaku (Seikagaku catalogue numbers 400571-1 and 400572-1).
  • the PT assay involved adding 50 of plasma, containing either DS or heparin, to 150 Thromborel S (containing calcium). Clotting was determined as defined in the aPTT assay (above). Thromboelastography studies were performed using a TEG ® Haemostasis Analyser 5000 series (Haemoscope Corporation, Niles, USA) according to manufacturer's instructions. The TEG ® Haemostasis Analyser measures the shear elasticity of clot formation (or lysis). The TEG measurements included reaction time (R) in seconds until the first indication that a clot has formed and maximum amplitude (MA) of the developed clot measured in mm.
  • R reaction time
  • MA maximum amplitude
  • Example 10 In vivo anti-thrombotic activity ofDS in a rabbit model of deep vein thrombosis following intravenous and oral administration
  • the tubing was attached to a No.20 needle to enable blood sampling and was also connected to a Gilson pump to flush 0.5 mL/min saline to keep the artery clear between sample collections and for administration of intravenous saline, DS or Clexane.
  • a vertical incision in the neck area was made to surgically isolate the jugular veins from the fascia. The dissection was performed carefully using a scalpel, forceps and cautery so as not to damage or traumatize the vessels. Collateral vessels were cauterized and 1 cm of each jugular vein (including the bifurcation) was isolated.
  • Mersilk sutures were loosely tied around each branch of the jugular vein without interfering with blood circulation.
  • the exposed neck area was kept moist with saline-soaked gauze patches.
  • IV intravenous
  • three animals were challenged for each treatment and dose.
  • the first 3.5 mL blood sample (A) was taken from the femoral artery before any IV administration, and then flushed with 1 mL of sterile saline.
  • Intravenous administration included three doses of DS: Dose 1: 1 mg/mL in plasma (30 mg kg); Dose 2: 2 mg/mL in plasma, (60 mg/kg); and Dose 3: 4 mg/mL in plasma, (120 mg/kg).
  • the third and final 3.5 mL blood sample (C) was taken 30 minutes after thrombogenic challenge (70 minutes post-intravenous administration).
  • the animal was euthanized using 1 mL/kg of Lethabarb.
  • the third 3.5 mL blood sample (C) was taken 30 minutes after thrombogenic challenge (3 hours post-gavage).
  • the fourth and final 3.5 mL blood sample (D) was taken 60 minutes after thrombogenic challenge (3.5 hours post-gavage). At completion of the experiment the animal was euthanized using 1 mL/kg of Lethabarb.
  • the samples were vortexed, centrifuged at 9000 x g for 10 minutes with the aqueous (top) layer retained for GAG analysis.
  • the Blyscan Sulphated Glycosaminoglycan Assay was then used according to manufacturer's instructions to estimate total GAGs in the aqueous layer with an internal positive DS control (from porcine intestinal mucosa) used to ensure recovery of GAG from serum.
  • Example 13 Pilot scale production of LMW dermatan sulphate (Low Molecular Weight dermatan sulphate)

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Abstract

L'invention concerne des procédés d'enrichissement, d'isolement et/ou de modification du sulfate de dermatane (DS) dérivé de sources comprenant les courants de déchets issus du traitement de peaux. L'invention concerne également des utilisations de sulfate de dermatane et/ou de sulfate de dermatane de poids moléculaire faible dans le traitement de troubles hématologiques; la préparation de médicaments; de compléments; de produits nutraceutiques; d'ingrédients alimentaires fonctionnels et de produits alimentaires fonctionnels.
PCT/AU2012/000828 2011-07-11 2012-07-11 Sulfate de dermatane, compositions pharmaceutiques et leur procédé de fabrication Ceased WO2013006906A1 (fr)

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RU2693262C2 (ru) * 2014-03-21 2019-07-01 Альтергон С.А. Способ очистки хондроитин сульфата

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WO1998034959A1 (fr) * 1997-02-06 1998-08-13 Dermatan Products Limited Le bisulfate de dermatan, inhibiteur de la production de thrombine, et de l'activation du complement
US5801162A (en) * 1993-09-30 1998-09-01 Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) Dermatan sulfate compositions and antithrombotic compositions containing same
WO1998055514A1 (fr) * 1997-06-03 1998-12-10 Leo Pharmaceutical Products Ltd. A/S (Løvens Kemiske Fabrik Produktionsaktieselskab) Melanges d'oligosaccharides presentant une activite antithrombotique

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US5801162A (en) * 1993-09-30 1998-09-01 Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation) Dermatan sulfate compositions and antithrombotic compositions containing same
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