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WO1994029352A1 - Polysaccharides a forte activite antithrombotique et aticoagulante - Google Patents

Polysaccharides a forte activite antithrombotique et aticoagulante Download PDF

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Publication number
WO1994029352A1
WO1994029352A1 PCT/EP1994/001660 EP9401660W WO9429352A1 WO 1994029352 A1 WO1994029352 A1 WO 1994029352A1 EP 9401660 W EP9401660 W EP 9401660W WO 9429352 A1 WO9429352 A1 WO 9429352A1
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WO
WIPO (PCT)
Prior art keywords
polysaccharides
sulfate group
disaccharide units
disaccharide
hydrogen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1994/001660
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English (en)
Inventor
Benito Casu
Giordana Grazioli
Annamaria Naggi
Giangiacomo Torri
Ulf Lindahl
Nahid Razi
Pasqua Oreste
Maria Luisa Bossi
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Italfarmaco SpA
Original Assignee
Italfarmaco SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Italfarmaco SpA filed Critical Italfarmaco SpA
Priority to AU68448/94A priority Critical patent/AU6844894A/en
Publication of WO1994029352A1 publication Critical patent/WO1994029352A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof

Definitions

  • the present invention relates to novel polysaccharides consisting of chains or mixtures of chains having a molecular weight ranging from about 1,000 to about 100,000 Da, or more, said polysaccharides being characterized in that they have a repeating disaccharide structure of formula I
  • A is a D-glucuronic acid moiety
  • B is a D- glucosamine moiety
  • R can be hydrogen, acetyl or a sulfate group
  • R ⁇ , R2, 3 and R 4 are independently hydrogen or a sulfate group
  • R is a sulfate group and in the remaining disaccharide units R is mainly the acetyl group
  • R, R ⁇ and R2 are at the same time a sulfate group and R3 and R 4 are hydrogen.
  • the present invention also relates to the salts of these polysaccharides, for example the salts with alkali or alkaline-earth metal cations. Said polysaccharides have remarkable
  • Preferred polysaccharides according to the present invention are those in which, in at least 40% of the in disaccharide units, R is a sulfate group and in the remaining disaccharide units R is mainly the acetyl group, and in at least one of the disaccharide units R, RH and R2 are at the same time sulfate and R3 and R 4 are hydrogen; and the salts thereof with alkali or alkaline-earth metal cations.
  • polysaccharides according to the present invention are those in which in nearly all or in all of the disaccharide units R is a sulfate group and, if that is the case, in the remaining units R is ⁇ > mainly acetyl, and in at least one of the disaccharide units R, R 1 and R 2 are at the same time sulfate and R and R 4 are hydrogen; and the salts thereof with alkali or alkaline-earth metal cations.
  • polysaccharides object of the present invention can be prepared using separation methods known to those skilled in the art, such as suitable ion-exchange, or preferably affinity, systems, for example subjecting to affinity chromatography particular compounds obtained from N-desacetylation, N- sulfation and subsequent O-sulfation of N- acetylheparosanes extracted from fermentation broth of E. coli strains (hereinafter named polysaccharides K5).
  • sulfaminoheparosansulfates are, for example, those disclosed in the above cited Patent application publications, or those described by way of example in the present invention and more generally all those compounds deriving from the above mentioned transformations of the various polysaccharides K5 obtained from fermentation broth of E. coli strains. i" Examples of said trasformations are described in the above mentioned Patent application publications, or they will be illustrated hereinafter.
  • E . coli strains which can produce polysaccharides K5 are those showing the presence of the capsular ⁇ ⁇ antigene K5 and they are available from different sources, such as American Type Culture Collection, Institut Pasteur (Collection Nationale de Cultures de Microorganismes) , International Escherichia Centre, Statens Serum Institut, Copenhagen, Denmark, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, and the like.
  • E . coli strains capable of producing polysaccharides K5 are also available from different strains collections or can be isolated clinically, mainly from infections of the urinary tract and from pyelonephritis. They can be characterized by means of API SYSTEM 20 ⁇ as strains which show the presence of the antigene of capsular polysaccharide K5 according to W Nimmich et al., Z. Automate Hyg., 35(10), 583, 1989,
  • the obtained polysaccharides K5 are then subjected to N-desacetylation, by treatment with a suitable agent such as sodium or potassium hydroxide from about IN to about 3N, or with anhydrous hydrazine containing about i'. 10% by weight of hydrazine sulfate.
  • a suitable agent such as sodium or potassium hydroxide from about IN to about 3N
  • anhydrous hydrazine containing about i'. 10% by weight of hydrazine sulfate In this case, the reaction is carried out in a sealed tube, for a time ranging from about 30 minutes to about 6 hours, at a temperature from about 80 to about 100 * C.
  • the subsequent N-sulfation reaction is carried out , ⁇ according to known techniques, using as sulfating agents complexes of sulfur trioxide and nitrogen organic bases such as trimethylamine.SO3, pyridine.S0 3 and analogues, at a temperature ranging from about 45 to about 65 ⁇ C, for a time ranging from about 6 to about 24 hours.
  • the O-sulfation reaction is finally carried out substantially according to the procedure described by A. Ogamo et. al., Carbohydrate Research, 193, 165, ⁇ 1989, or as shown in the following.
  • the products deriving from N-sulfation are converted into the corresponding salts of nitrogen organic bases, such as trimethylamine, triethyla ine or tributylamine, which are then treated with suitable sulfating agents, for example those used in the N- sulfation procedure, in anhydrous inert organic ,” solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, or mixtures thereof.
  • the reaction temperature is advantageously from about -5°C to room temperature, for a time ranging from about 1 to about 24 hours.
  • Sulfaminoheparosansulfates are obtained which, according to a preferred aspect of the present invention, are subjected to affinity chromatography, by passing them through a column containing a suitable carrier, on which antithrombin iv. Ill (AT III) is immobilized.
  • Carriers which can be used are those known to those skilled in the art. Conveniently, SEPHAROSE R 4-B (PHARMACIA) on which AT III is immobilized according to the procedure described by Hook et al., FEBS Lett., 66,
  • Suitable solvent systems are, for example, buffer solutions at neutral pH, such as 0.05M Tris HC1 (pH 7.4).
  • the solution of the selected sulfaminoheparosansulfate is preferably added with an alkali or alkaline-earth metal salt soluble in the used solvent system, and the whole is applied at the head of the column.
  • the column is eluted applying a linear saline gradient in the same solvent system used, and eluates are collected in distinct fractions.
  • a suitable linear saline gradient consists of 0.05M sodium chloride and 3M sodium chloride in 0.05M Tris HC1 (pH
  • the eluates are collected in distinct fractions containing constant volumes, and the chromatography elution profile is characterized indirectly, by evaluating the content in uronic acid present in each single fraction (colorimetric reaction with carbazole) (A530).
  • An elution profile is obtained, characterized by the presence of two main peaks, as it is evident from Fig. 1, which shows the elution profile of the illustrative example of the present invention.
  • the second peak obtained in the area of higher molarity of the applied gradient is i ' . ⁇ considered, which corresponds to fractions of increasing affinity to AT III.
  • the fractions progressively collected which caused the profile of this second peak can be pooled in further fractions of equal volume, depending on the progressive increasing molarity of the applied gradient.
  • the single distinct fractions or the pooled fractions are subsequently salted off and freeze-dried, according to procedures known to those skilled in the art.
  • the polysaccharides of the present invention were ' «( ' characterized structurally by deaminative degradation with nitrous acid (G. Thunberg et al. , Carbohydr. Res., 100, 393, 1982), followed by reduction with NaB 3 H 4 , according to the procedure described by H.E. Conrad, Biochem. J. 191, 355, 1980.
  • hexuronosyl[l- 3 H]-2,5-anidromannitol disaccharides marked are then chromatographed by HPLC on a ion-exchange column, for example according to the procedure described by M. Kusche et al. in Journ. Biol. Chem. , Vol. 265, 13, 7292, 1990, e by high voltage •" paper electrophoresis, thereby revealing quali-quanti- tatively the characteristic peaks corresponding to characteristic disaccharide in the integer sulfated polysaccharide.
  • the polysaccharides object of the present invention are characterized in that, in at least one of the disaccharide units of formula I forming the chains or the mixtures of chains constituting them, R, R 1 and R 2 are at the same time a sulfate group and R3 and R 4 are hydrogen.
  • the polysaccharide obtained in lb) (100 mg) is dissolved in distilled water (20 ml), and the solution is eluted through an Amberlite IR-120 H+ column at room temperature. The column is washed with a further 20 ml of distilled water and the eluates are collected, pH is adjusted to 5.5 with 10% tributylamine in ethanol (w/v) (3 ml). The tributylamine excess is removed with ether ethyl (40 ml) and the product is freeze-dried.
  • the resulting product (188.2 mg) is dissolved in anhydrous dimethylformamide (33 ml), the pyridine.sulfur trioxide adduct (765 mg) dissolved in anhydrous dimethylformamide (15 ml) is added and the reaction mixture is kept at 0 ⁇ C for 1 hour. The reaction mixture is then added with an equal volume of distilled water and pH is adjusted to 9 by means of 2N NaOH. The sulfaminoheparosansulfate is recovered as described above.
  • 500 mg of a sulfaminoheparosansulfate prepared according to the procedures described above were dissolved in 500 ml of 0.05M Tris.HCl buffer (pH 7.4) containing 0.05M NaCl, and the resulting solution was placed in a cm 5x15 column, packed with 300 ml of SEPHAR0SE R -4B on which AT III had been immobilized according to the procedure described by Hook et al. , FEBS Lett., 66, 90-93, 1976, previously equilibrated with the same buffer, at a temperature of about 4 ⁇ C.
  • the column was eluted with a NaCl linear gradient consisting of 1500 ml of 0.05M NaCl and 1500 ml of 3M NaCl in 0.05M Tris.HCl buffer (pH 7.4).
  • the eluates were collected in distinct fractions of about 30 ml each, and the uronic acid content was determined for each fraction by means of the colorimetric reaction with carbazole (A530) (T. Bitter and H.M. Muir, Anal. Biochem, 4, 330, 1962).
  • the elution profile of Fig. 1 is obtained, i. characterized by the presence of two peaks.
  • the distinct fractions which cause the profile of the peak obtained in the area of higher molarity of the applied gradient were in their turn pooled in 4 fractions, of about the same volume, depending on the progressive i- increasing molarity of the applied saline gradient.
  • the fractions were then salted off by passing them through a Sephadex R G-15 chromatographic column of suitable size, and the eluates were collected and freeze-dried.
  • B) Characterization r
  • freeze-dried fractions 2 and 4 obtained as described in A) were characterized structurally by deaminative degradation with HNO2 at pH 1.5, followed by reduction with tritiated sodium borohydride, thereby obtaining the labelled hexuronosyl(l- 3 H)-2,5- ' ⁇ . ' anhydromannitol disaccharides which were chromatographed by HPLC on a Whatman Partisil-10 SAX ion-exchange column.
  • the electrophoretic pattern of the disaccharide after enzyme digestion corresponded to that of the two selected standards, i.e. the disaccharide itself and the 2,5-anhydromannitol sulfated at the oxygens at the 3- and 6- positions.
  • the fraction 4 had a molecular weight of about 35,000 Da, determined by gel-chro atography on Sephacryl R -S300. From the evaluation of the biological properties of the polysaccharide of the invention recovered from fraction 4, this proved to be the one having a higher affinity to Antithro bin III. In particular, the inhibition of the activity of factor Xa by AT III, in the presence of standard heparin and of
  • the polysaccharides of the present invention are anticoagulant and antithrombotic agents. They can be administered one or more times a day, for example in injectable unitary doses, ranging from about 5 to about 400 mg, as such or in form of an alkali or alkaline-earth metal salt.
  • Said injectable dosage forms are prepared according to the conventional technique related to polysaccharide formulations.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

Polysaccharides composés de chaînes ou de mélanges de chaînes d'un poids moléculaire compris entre au moins 1000 et 10000 Da et caractérisés en ce qu'ils présentent une structure de disaccharides à répétition de la formule (I), dans laquelle A, B, R, R1, R2, R3 et R4 sont tels que définis dans le descriptif. Ces polysaccharides, et leurs sels à cations de métal alcalin ou alcalino-terreux, présentent des activités anticoagulante et antithrombotique remarquables.
PCT/EP1994/001660 1993-06-04 1994-05-24 Polysaccharides a forte activite antithrombotique et aticoagulante Ceased WO1994029352A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU68448/94A AU6844894A (en) 1993-06-04 1994-05-24 Polysaccharides having high antithrombotic and anticoagulant activity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITMI93A001175 1993-06-04
ITMI931175A IT1270823B (it) 1993-06-04 1993-06-04 Polisaccaridi ad elevata attivita' antitrombotica e anticoagulante

Publications (1)

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WO1994029352A1 true WO1994029352A1 (fr) 1994-12-22

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AU (1) AU6844894A (fr)
IL (1) IL109881A0 (fr)
IT (1) IT1270823B (fr)
WO (1) WO1994029352A1 (fr)
ZA (1) ZA943868B (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998009636A1 (fr) * 1996-09-06 1998-03-12 Istituto Scientifico Di Chimica E Biochimica 'g. Ronzoni' Sulfaminoheparosansulfates semi-synthetiques possedant une activite anti-metastatique elevee et presentant un risque d'hemorragies reduit
RU2167163C2 (ru) * 1996-07-19 2001-05-20 Санофи Синтетические полисахариды, способ их получения и содержащая их фармацевтическая композиция
US6329351B1 (en) 1997-08-28 2001-12-11 Istituto Scientifico Di Chimica E Biochimica “G. Ronzoni” Semi-synthetic sulphaminoheparosansulphates having high anti-metastatic activity and reduced haemorrhagic risk
US6608042B2 (en) * 2000-03-28 2003-08-19 Aventis Pharma, S.A. Pharmaceutical compositions containing oligosaccharides, the novel oligosaccharides and preparation thereof
US6670339B1 (en) 1997-10-14 2003-12-30 The Australian National University Use of sulfated oligosaccharides in lowering blood triglyceride levels
US8071570B2 (en) 2002-10-10 2011-12-06 Aventis Pharma S.A. Mixtures of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AUPO556297A0 (en) * 1997-03-11 1997-04-10 Australian National University, The Sulfated oligosaccharides having anticoagulant/ antithrombotic activity

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0209924A1 (fr) * 1985-07-12 1987-01-28 Akzo N.V. Agent antithrombotique à base de glycosaminoglycanes, procédé de préparation et composition pharmaceutique
WO1992017507A1 (fr) * 1991-03-28 1992-10-15 Italfarmaco S.P.A. Anticoagulants et leurs procedes de preparation
EP0544592A2 (fr) * 1991-11-28 1993-06-02 Sanofi Héparosanes-N,O-sulfates de haute masse moléculaire, leur procédé de préparation et les compositions pharmaceutiques qui les contiennent

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0209924A1 (fr) * 1985-07-12 1987-01-28 Akzo N.V. Agent antithrombotique à base de glycosaminoglycanes, procédé de préparation et composition pharmaceutique
WO1992017507A1 (fr) * 1991-03-28 1992-10-15 Italfarmaco S.P.A. Anticoagulants et leurs procedes de preparation
EP0544592A2 (fr) * 1991-11-28 1993-06-02 Sanofi Héparosanes-N,O-sulfates de haute masse moléculaire, leur procédé de préparation et les compositions pharmaceutiques qui les contiennent

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
M. KUSCHE ET AL.: "Biosynthesis of heparin", THE BIOCHEMICAL JOURNAL, vol. 275, no. 1, 1991, LONDON, pages 151 - 158 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2167163C2 (ru) * 1996-07-19 2001-05-20 Санофи Синтетические полисахариды, способ их получения и содержащая их фармацевтическая композиция
WO1998009636A1 (fr) * 1996-09-06 1998-03-12 Istituto Scientifico Di Chimica E Biochimica 'g. Ronzoni' Sulfaminoheparosansulfates semi-synthetiques possedant une activite anti-metastatique elevee et presentant un risque d'hemorragies reduit
RU2176915C2 (ru) * 1996-09-06 2001-12-20 Иституто Скьентифико Ди Кимика Е Биокимика "Г. Ронцони" Полусинтетические сульфаминогепаросансульфаты, имеющие высокую антиметастатическую активность и пониженный геморрагический риск
US6329351B1 (en) 1997-08-28 2001-12-11 Istituto Scientifico Di Chimica E Biochimica “G. Ronzoni” Semi-synthetic sulphaminoheparosansulphates having high anti-metastatic activity and reduced haemorrhagic risk
US6670339B1 (en) 1997-10-14 2003-12-30 The Australian National University Use of sulfated oligosaccharides in lowering blood triglyceride levels
US6608042B2 (en) * 2000-03-28 2003-08-19 Aventis Pharma, S.A. Pharmaceutical compositions containing oligosaccharides, the novel oligosaccharides and preparation thereof
US8071570B2 (en) 2002-10-10 2011-12-06 Aventis Pharma S.A. Mixtures of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them

Also Published As

Publication number Publication date
ITMI931175A0 (it) 1993-06-04
ZA943868B (en) 1995-02-02
IT1270823B (it) 1997-05-13
AU6844894A (en) 1995-01-03
IL109881A0 (en) 1994-10-07
ITMI931175A1 (it) 1994-12-04

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