NO157580B - PROCEDURE FOR THE PREPARATION OF OLIGOSACCARIDES WITH SELECTIVE ANTICOAGULATION ACTIVITY. - Google Patents
PROCEDURE FOR THE PREPARATION OF OLIGOSACCARIDES WITH SELECTIVE ANTICOAGULATION ACTIVITY. Download PDFInfo
- Publication number
- NO157580B NO157580B NO82821631A NO821631A NO157580B NO 157580 B NO157580 B NO 157580B NO 82821631 A NO82821631 A NO 82821631A NO 821631 A NO821631 A NO 821631A NO 157580 B NO157580 B NO 157580B
- Authority
- NO
- Norway
- Prior art keywords
- sulfated
- unit
- oligosaccharides
- heparin
- glucosamine
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title claims abstract description 4
- 230000000694 effects Effects 0.000 title claims description 15
- 230000010100 anticoagulation Effects 0.000 title 1
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 35
- 150000002482 oligosaccharides Chemical class 0.000 claims abstract description 35
- MSWZFWKMSRAUBD-IVMDWMLBSA-N glucosamine group Chemical group OC1[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims abstract description 15
- 230000008569 process Effects 0.000 claims abstract description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 24
- 229960002897 heparin Drugs 0.000 claims description 24
- 229920000669 heparin Polymers 0.000 claims description 24
- 150000002772 monosaccharides Chemical group 0.000 claims description 15
- 102000004411 Antithrombin III Human genes 0.000 claims description 8
- 108090000935 Antithrombin III Proteins 0.000 claims description 8
- 229960005348 antithrombin iii Drugs 0.000 claims description 8
- 238000005345 coagulation Methods 0.000 claims description 8
- 230000015271 coagulation Effects 0.000 claims description 8
- 239000004019 antithrombin Substances 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- XTHFKEDIFFGKHM-UHFFFAOYSA-N Dimethoxyethane Chemical compound COCCOC XTHFKEDIFFGKHM-UHFFFAOYSA-N 0.000 claims description 3
- 108010014173 Factor X Proteins 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 238000001042 affinity chromatography Methods 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- LKAPTZKZHMOIRE-KVTDHHQDSA-N (2s,3s,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carbaldehyde Chemical group OC[C@H]1O[C@H](C=O)[C@@H](O)[C@@H]1O LKAPTZKZHMOIRE-KVTDHHQDSA-N 0.000 claims description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims description 2
- 230000009257 reactivity Effects 0.000 claims description 2
- AEMOLEFTQBMNLQ-HNFCZKTMSA-N L-idopyranuronic acid Chemical group OC1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-HNFCZKTMSA-N 0.000 claims 2
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical group OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- GXYKBDAKQXPXFV-SLPGGIOYSA-N [(2r,3r,4r,5r)-2-amino-4,5,6-trihydroxy-1-oxohexan-3-yl] hydrogen sulfate Chemical group O=C[C@H](N)[C@@H](OS(O)(=O)=O)[C@H](O)[C@H](O)CO GXYKBDAKQXPXFV-SLPGGIOYSA-N 0.000 abstract description 2
- 125000001483 monosaccharide substituent group Chemical group 0.000 abstract 2
- 239000008194 pharmaceutical composition Substances 0.000 abstract 1
- 230000005764 inhibitory process Effects 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 210000002381 plasma Anatomy 0.000 description 9
- 208000007536 Thrombosis Diseases 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 108010048049 Factor IXa Proteins 0.000 description 3
- 108010071241 Factor XIIa Proteins 0.000 description 3
- 108010080805 Factor XIa Proteins 0.000 description 3
- 108010074860 Factor Xa Proteins 0.000 description 3
- 108090000190 Thrombin Proteins 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 2
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 2
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 2
- 102100029117 Coagulation factor X Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108010000499 Thromboplastin Proteins 0.000 description 2
- 102000002262 Thromboplastin Human genes 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 229940105756 coagulation factor x Drugs 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- IAJILQKETJEXLJ-LECHCGJUSA-N iduronic acid Chemical group O=C[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-LECHCGJUSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000014508 negative regulation of coagulation Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 206010003178 Arterial thrombosis Diseases 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 206010047249 Venous thrombosis Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical group O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 239000002628 heparin derivative Substances 0.000 description 1
- 239000002565 heparin fraction Substances 0.000 description 1
- 239000002634 heparin fragment Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- OWFXIOWLTKNBAP-UHFFFAOYSA-N isoamyl nitrite Chemical compound CC(C)CCON=O OWFXIOWLTKNBAP-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Søknaden vedrrer et oligosakkarid som inneholder 4-8 monosakkaridenheter og som erat de inneholder minst en glukosaminenhet som er 3-0-sulfatert, og minst en ytterligere glukosaminenhet, idet disse enheter er bundet gjennom en mellomliggende monosakkaridenhet, som er bundet til den reduserende ende av den 3-0-sulfaterte glukosaminenhet. Videre vedrører søknaden en farmasøytisk blanding som inneholder en eller flere slike oligosakkarid, og en fremgangsmåte for fremstilling av disse oligosakkarider.The application relates to an oligosaccharide which contains 4-8 monosaccharide units and which contains at least one glucosamine unit which is 3-0-sulphated, and at least one further glucosamine unit, these units being bound through an intermediate monosaccharide unit, which is bound to the reducing end of the 3-O-sulfated glucosamine unit. The application further relates to a pharmaceutical composition containing one or more such oligosaccharides, and to a process for the preparation of these oligosaccharides.
Description
Foreliggende oppfinnelse vedrører en fremgangsmåte av den The present invention relates to a method thereof
art som er angitt i kravets ingress. species specified in the claim's preamble.
Heparin er et sulfatholdig polysakkarid som kan isoleres Heparin is a sulfated polysaccharide that can be isolated
fra tarmslim fra svin eller lunger fra storfe. Det brukes klinisk som et middel for behandling og forebygning av trombose. Imidlertid er bruken -.v heparin forbundet med prob-lemer såsom risk for blødningskomplikasjoner og stor indi-viduell variasjon mellom forskjellige pasienter. Et annet problem med nåværende type av heparinterapi er den svake virkning på arteriell trombose. Ved denne type trombose er trombosittaggregeringen et mer dominerende trekk enn ved venøs trombose hvor heparin gir en god virkning. Standard heparin stimulerer i en viss grad trombosyttaggregering og gir følgelig en negativ virkning i denne henseende. Det er vist at heparinfraksjoner med forskjellige molekylvekter på-virker koaguleringsforløpet på forskjellige måter [L.-O. Andersson et al, Thromb. Res. 9_, 575 (1976).]. Koaguleringsfaktor X inntar en sentral stilling i midten av koaguleringstrinnene og inhiberingen derav betraktes av mange som spesielt viktig for å oppnå en effektiv trombose-forebyggende virkning [S. Wessler, Thromb. Diath. Haemorrh. 33, 81 (1974)]. from intestinal mucus from pigs or lungs from cattle. It is used clinically as an agent for the treatment and prevention of thrombosis. However, the use of heparin is associated with problems such as the risk of bleeding complications and great individual variation between different patients. Another problem with the current type of heparin therapy is the weak effect on arterial thrombosis. In this type of thrombosis, platelet aggregation is a more dominant feature than in venous thrombosis, where heparin provides a good effect. Standard heparin stimulates platelet aggregation to a certain extent and consequently has a negative effect in this respect. It has been shown that heparin fractions with different molecular weights affect the coagulation process in different ways [L.-O. Andersson et al, Thromb. Res. 9_, 575 (1976).]. Coagulation factor X occupies a central position in the middle of the coagulation steps and its inhibition is considered by many to be particularly important for achieving an effective thrombosis-preventing effect [S. Wessler, Thromb. Diath. Haemorrh. 33, 81 (1974)].
Ifølge foreliggende oppfinnelse har det vist seg å være mulig å oppnå oligosakkarider med god selektiv antikoaguleringsaktivitet in vivo og in vitro, spesielt affinitet til antitrombin og evne til å forbedre reaksjonsevnen for antitrombin med koaguleringsfaktorer X og XII. Dyreforsøk har vist at oligosakkarider av denne type gir effektiv forebyggende virkning mot trombose uten noen tilsvarende øket virkning til tendens til blødning. I oligosakkaridene, som er fremstilt ifølge forelig<g>ende oppfinnelse, består sekvensen av mono-sakkarider som oppbygger polysakkaridene av bestemte monosakkaridenheter i en bestemt intern rekkefølge som skal defi- According to the present invention, it has been shown to be possible to obtain oligosaccharides with good selective anticoagulant activity in vivo and in vitro, especially affinity to antithrombin and ability to improve the reactivity of antithrombin with coagulation factors X and XII. Animal experiments have shown that oligosaccharides of this type provide an effective preventive effect against thrombosis without any corresponding increased effect on the tendency to bleed. In the oligosaccharides, which have been produced according to the present invention, the sequence of monosaccharides that make up the polysaccharides consists of specific monosaccharide units in a specific internal order that must be defined
neres i det følgende. nered in the following.
Oppfinnelsen vedrører fremstilling av slike The invention relates to the production of such
oligosakkarider som kan avledes fra heparin som er særpreget ved at de har en glukosaminenhet som er 3-O-sulfatert, og en ytterligere glukosaminenhet, hvorunder disse enheter er forbundet med en mellomliggende monosakkaridenhet som er bundet til de reduserende ender av den 3-0-sulfaterte glukosaminenhet. Ifølge en utførelsesform er den 3-0-sulfaterte glukosaminenhet også 2-N-sulfatert. Videre kan den ytterligere glukosaminenhet som gjennom en mellomliggende monosakkaridenhet er bundet til den reduserende ende av den 3~-0-sulfaterte glukosaminenhet være 2-N-sulfatert. Den mellomliggende monosakkaridenhet er fortrinnsvis en sulfatert iduronsyreenhet, hvor sulfateringen normalt består av en 2-0-sulfatgruppe. Oppfinnelsen vedrører spesielt oligosakkarider hvor den 3-0-sulfaterte glukosaminenhet er en enhet i en sekvens av monosakkaridrester med 8 enheter Eionosakkaridrester. oligosaccharides derivable from heparin that are characterized by having a glucosamine unit that is 3-O-sulfated, and a further glucosamine unit, wherein these units are linked by an intervening monosaccharide unit that is attached to the reducing ends of the 3-0- sulfated glucosamine unit. According to one embodiment, the 3-O-sulfated glucosamine unit is also 2-N-sulfated. Furthermore, the further glucosamine unit which is bound through an intermediate monosaccharide unit to the reducing end of the 3~-0-sulfated glucosamine unit can be 2-N-sulfated. The intermediate monosaccharide unit is preferably a sulphated iduronic acid unit, where the sulphation normally consists of a 2-0-sulphate group. The invention relates in particular to oligosaccharides where the 3-0-sulphated glucosamine unit is one unit in a sequence of monosaccharide residues with 8 units of Eionosaccharide residues.
De fremstilte oligosakkarider kan ifølge de foregå- The produced oligosaccharides can, according to the preceding
ende definisjoner også kan inneholde en ikke-sulfatert iduronsyreenhet bundet i C 4-stilling av den 3-0-sulfaterte glukosaminenhet mellom 2 mellomliggende monosakkaridenheter, idet fortrinnsvis en av disse mellomliggende monosakkaridenheter er en acetylert glukosaminenhet som kan være 6-0-sulfatert og den andre monosakkaridenhet er fortrinnsvis en glukuronsyreenhet. end definitions can also contain a non-sulfated iduronic acid unit bound in the C 4 position of the 3-0-sulfated glucosamine unit between 2 intermediate monosaccharide units, preferably one of these intermediate monosaccharide units is an acetylated glucosamine unit which can be 6-0-sulfated and the second monosaccharide unit is preferably a glucuronic acid unit.
En eller flere ytterligere monosakkaridenheter kan gå forut for disse enheter, fortrinnsvis en glukosaminenhet, og foran denne kan igjen sitte en uronsyreenhet. One or more additional monosaccharide units may precede these units, preferably a glucosamine unit, and this may again be preceded by a uronic acid unit.
2,5-anhydro-D-mannoseenheten innføres fortrinnsvis ved fremstillingen av oligosakkarider ved spaltning (deamine-rende spaltning) av et utgangsmateriale med høyere molekyl-vekt. The 2,5-anhydro-D-mannose unit is preferably introduced during the production of oligosaccharides by cleavage (deaminating cleavage) of a starting material with a higher molecular weight.
01igosakkaridene kan fremstilles fra naturlig heparin eller eventuelt fremstilles syntetisk. Når naturlig heparin anvendes som utgangsmateriale kan de følgende metoder anvendes: (a) behandle heparin med salpetersyrling i dimetoksyetan og The oligosaccharides can be produced from natural heparin or possibly synthetically. When natural heparin is used as starting material, the following methods can be used: (a) treat heparin with nitric acid in dimethoxyethane and
rense det erholdte materiale, eller purify the obtained material, or
(b) behandle heparin med nitrit, fortrinnsvis natriumnitrit (b) treating the heparin with nitrite, preferably sodium nitrite
i vandig løsning og rense det erholdte materiale, in aqueous solution and purify the material obtained,
og fremgangsmåten er særpreget ved det som er angitt i kravets karakteriserende del. and the method is characterized by what is stated in the characterizing part of the claim.
Separasjonsmetoden for isolering av de sterkt aktive forbin-delser og separere de svakt aktive eller inaktive bestanddeler kan utføres ved affiniteteskromatografi på matrisebundet antitrombin III [H55k et al,. FEBS Lett. 66, 90 (1976); Hopwood et al,. FEBS Lett. 69, 51 (1976); L.-0. Andersson et al,. Thromb. Res. 9, 575 (1976)]. The separation method for isolating the highly active compounds and separating the weakly active or inactive components can be carried out by affinity chromatography on matrix-bound antithrombin III [H55k et al,. FEBS Easy. 66, 90 (1976); Hopwood et al. FEBS Easy. 69, 51 (1976); L.-0. Andersson et al. Thromb. Res. 9, 575 (1976)].
Oppfinnelsen illustreres videre gjennom fLe/ følgende eksempler. The invention is further illustrated through the following examples.
Eksempel 1 Example 1
Fremstilling av heparinfragmenter ved depolymerisering av standard heparin med salpetersyrling Preparation of heparin fragments by depolymerization of standard heparin with nitric acid
Heparin (0,5 g) isolert fra svinetarmer og oppløst i 150 ml vann avkjøles til +4°C og føres gjennom en 3x7 cm kolonne av Dowetf*%0 W-28 (H+ forml, 200-400 mesh. Kolonnen vaskes så med 100 ml vann og vaskevæsken kombineres med prøven. Prøven tilsettes 250 ml dimetoksyetan (glyml avkjølt til ;-20°C og 10 ml isoamylnitrit og blandingen får ved en temperatur på nøyaktig -10°C stå i 35 minutter. Reaksjonen av-brytes så ved en tilsetning av 10 ml 10% Na<+->acetat. Etter tilsetning av 5,2 liter etanol utvinnes utfelt karbohydrat (heparinderivater) ved sentrifugering. Produktet oppløses ;i 500 ml 0,05 M NaCl - 0,05 M Tris-HCl, pH 7,4. Denne løs-ning oppdeles i 100 ml porsjoner og fraksjoneres ved affinitetskromatografi på en kolonne som inneholder 75 ml antitrombin - Sepharose<4*> (Pharmacia Fine Chemicals, Uppsala, Sverige) (ca. 5 mg protein pr. ml gel).. Kolonnen elueres med en saltgradient (500 ml 0,05 NaCl - 0,05 M Tris-HCl i Heparin (0.5 g) isolated from pig intestines and dissolved in 150 ml of water is cooled to +4°C and passed through a 3x7 cm column of Dowetf*%0 W-28 (H+ formula, 200-400 mesh. The column is then washed with 100 ml of water and the washing liquid are combined with the sample. 250 ml of dimethoxyethane (glyml cooled to -20°C and 10 ml of isoamyl nitrite are added to the sample and the mixture is allowed to stand at a temperature of exactly -10°C for 35 minutes. The reaction is then stopped by an addition of 10 ml of 10% Na<+->acetate. After the addition of 5.2 liters of ethanol, precipitated carbohydrate (heparin derivatives) is recovered by centrifugation. The product is dissolved in 500 ml of 0.05 M NaCl - 0.05 M Tris-HCl , pH 7.4. This solution is divided into 100 ml portions and fractionated by affinity chromatography on a column containing 75 ml of antithrombin - Sepharose<4*> (Pharmacia Fine Chemicals, Uppsala, Sweden) (approx. 5 mg protein per ml gel).. The column is eluted with a salt gradient (500 ml 0.05 NaCl - 0.05 M Tris-HCl in
blandebeholderen; 500 ml 3 M NaCl - 0,05 M Tris-HCl i reser-voiret), idet hoveddelen av det påsatte materialet enten går uretardert gjennom kolonnen eller elueres ved lav ionestyrke (mindre enn 0,4 M NaCl); dette materialet har ingen biologisk aktivitet. De aktive bestanddeler (renset oligosakkaridmate-rialeX elueres i en bred topp 0,5 M NaCl og 3 M Na Cl tilsvarende ca. 3% av utgangsmaterialet. Disse fraksjoner slås sammen, konsentreres og avsaltes ved gelkromatografi. the mixing container; 500 ml 3 M NaCl - 0.05 M Tris-HCl in the reservoir), with the main part of the applied material either going unretarded through the column or eluted at low ionic strength (less than 0.4 M NaCl); this material has no biological activity. The active ingredients (purified oligosaccharide material X are eluted in a broad peak of 0.5 M NaCl and 3 M NaCl corresponding to approx. 3% of the starting material. These fractions are combined, concentrated and desalted by gel chromatography.
Oligosakkarider fremstilt og renset på denne måten har en dominerende molekylstørrelse som svarer til den for et okta-sakkarid ifølge oppfinnelsen. Oligosaccharides produced and purified in this way have a predominant molecular size corresponding to that of an octa-saccharide according to the invention.
Studier av antikoaguleringsaktivitet Studies of anticoagulant activity
Oligosakkaridene fremstilt ifølge eksempel 1 ble undersøkt med hensyn til deres evne til å: A), påvirke inhiber ingen av koaguler ingsenzymet trombin; B). påvirke inhiberingen av aktivert koaguleringsf aktor X; Cl påvirke inhiberingen av aktivert faktor IX; D). påvirke inhiberingen av aktivert faktor XI; The oligosaccharides prepared according to Example 1 were examined with regard to their ability to: A), inhibit the coagulation enzyme thrombin; B). affect the inhibition of activated coagulation factor X; Cl affect the inhibition of activated factor IX; D). affect the inhibition of activated factor XI;
El påvirke inhiberingen av aktivert faktor XII; El affect the inhibition of activated factor XII;
Fl forlenge koaguleringstiden i blodplasmakoaguleringsfor-søket APTT (Aktivert Partiell Tromboplastin Tidl; Fl prolong the coagulation time in the blood plasma coagulation test APTT (Activated Partial Thromboplastin Time;
Gl nøytraliseres av blodplasraabestanddeler; Gl is neutralized by blood plasma components;
H) påvirke aggregeringen av trombocytter. H) affect the aggregation of platelets.
Eksempel A. Inhibering av trombin Example A. Inhibition of thrombin
Oligosakkaridenes evne til å forsterke inhiberingen av trombin med antitrombin III ble analysert ifølge en modifikasjon av metoden til Teien et al. (Thrombosis Research 11, s. 107-117, 1977).. Oligosakkaridene ble funnet å ha en spesifikk aktivitet på 8 E/mg sammenlignet med 120-170 E/mg for standard heparin. The ability of the oligosaccharides to enhance the inhibition of thrombin by antithrombin III was analyzed according to a modification of the method of Teien et al. (Thrombosis Research 11, pp. 107-117, 1977).. The oligosaccharides were found to have a specific activity of 8 U/mg compared to 120-170 U/mg for standard heparin.
Eksempel B. Inhibering av aktivert faktor X Example B. Inhibition of activated factor X
Oligosakkaridenes evne til å forsterke inhiberingen av aktivert faktor X i plasma og i rent antitrombin III ble under-søkt ifølge en modifisert versjon av metoden til Teien et al. The ability of the oligosaccharides to enhance the inhibition of activated factor X in plasma and in pure antithrombin III was investigated according to a modified version of the method of Teien et al.
(Thrombosis Research 8, 413, 1976L. Oligosakkaridene ble vist å ha en spesifikk aktivitet på 500 E/mg i et rent antitrombin III system og 1900 E/mg i et plasmasystem sammenlignet med 120-17 0 E/mg for standard heparin. (Thrombosis Research 8, 413, 1976L. The oligosaccharides were shown to have a specific activity of 500 U/mg in a pure antithrombin III system and 1900 U/mg in a plasma system compared to 120-170 U/mg for standard heparin.
Eksempel C. Inhibering av aktivert faktor IX Example C. Inhibition of activated factor IX
Oligosakkaridenes evne til å forsterke inhiberingen av aktivert faktor IX i rent antitrombin III ble undersøkt ifølge Holmer et al. (Biochem. J. 1981, Volume 193 s. 395-4001. Oligosakkaridene ble vist å ha en spesifikk aktivitet på 18 E/mg sammenlignet med 120-180 E/mg for standard heparin. The ability of the oligosaccharides to enhance the inhibition of activated factor IX in pure antithrombin III was investigated according to Holmer et al. (Biochem. J. 1981, Volume 193 pp. 395-4001. The oligosaccharides were shown to have a specific activity of 18 U/mg compared to 120-180 U/mg for standard heparin.
Eksempel D. Inhibering av aktivert faktor XI Example D. Inhibition of Activated Factor XI
Oligosakkaridenes evne til å forsterke inhiberingen av aktivert faktor XI i rent antitrombin III ble undersøkt ifølge Holmer et al. (Biochem. J. 1981. Volume 193 s. 395-400). Oligosakkaridene ble vist å ha en spesifikk aktivitet på 40 E/mg sammenlignet med 120-170 E/mg for standard heparin. The ability of the oligosaccharides to enhance the inhibition of activated factor XI in pure antithrombin III was investigated according to Holmer et al. (Biochem. J. 1981. Volume 193 pp. 395-400). The oligosaccharides were shown to have a specific activity of 40 U/mg compared to 120-170 U/mg for standard heparin.
Eksempel E. Inhibering av aktivert faktor XII Example E. Inhibition of Activated Factor XII
Oligosakkaridenes evne til å forsterke inhiberingen av aktivert faktor XII i rent antitrombin III ble studert ifølge Holmer et al. (Biochem. J. 1981. Volume 193 s. 395-400). Oligosakkaridene ble funnet å ha en spesifikk aktivitet på 470 E/mg sammenlignet med standard heparin 120-170E/mg. The ability of the oligosaccharides to enhance the inhibition of activated factor XII in pure antithrombin III was studied according to Holmer et al. (Biochem. J. 1981. Volume 193 pp. 395-400). The oligosaccharides were found to have a specific activity of 470 U/mg compared to standard heparin 120-170U/mg.
Eksempel F. Forlengelse av koaguleringstiden Example F. Extension of the coagulation time
Oligosakkaridenes evne til å forlenge koaguleringstiden for blodplasma ble undersøkt ifølge APTT (Aktivert Partiell Tromboplastin Tid), metoden [Andersson et al,. Thromb. Res. The ability of the oligosaccharides to prolong the coagulation time of blood plasma was examined according to APTT (Activated Partial Thromboplastin Time), the method [Andersson et al,. Thromb. Res.
9_ 575 (1976)]. Oligosakkaridene viste en spesifikk aktivitet på 12 E/mg sammenlignet med tredje Internasjonale Heparin Standard. Standard heparin viste en spesifikk aktivitet i området 120-170 E/mg. 9_ 575 (1976)]. The oligosaccharides showed a specific activity of 12 U/mg compared to the third International Heparin Standard. Standard heparin showed a specific activity in the range of 120-170 U/mg.
Eksempel G. Nøytralisering av oligosakkaridene i blodplasma Example G. Neutralization of the oligosaccharides in blood plasma
Den nøytraliserende virkning på oligosakkaridene av plasma-komponenter ble studert ved å måle virkningen til heparin og til oligosakkaridene i- plasma og i et rent antitrombin-system. Dette ble utført ved å måle den mengde aktivert faktor X som var inhibert i de to systemer i nærvær av en viss mengde heparin eller oligosakkarid. Oligosakkaridenes aktivitet viste en 18% nøytralisering av plasmabéstanddeler, mens det tilsvarende tall for standard heparin hie vist å være 7 5%. The neutralizing action on the oligosaccharides of plasma components was studied by measuring the action of heparin and of the oligosaccharides in plasma and in a pure antithrombin system. This was done by measuring the amount of activated factor X that was inhibited in the two systems in the presence of a certain amount of heparin or oligosaccharide. The activity of the oligosaccharides showed an 18% neutralization of plasma constituents, while the corresponding figure for standard heparin was shown to be 75%.
Eksempel H. TrombocyttLnnflytelse Example H. Platelet influx
Oligosakkaridenes evne til å aggregere trombocytter ved kri-tisk ADP (Adenosine DiPhosphate).-konsentrasjoner ble studert hovedsakelig ifølge E.A. (Thromb Haem Stuttg. 1977, 38_, 578). Det ble vist at den trombocyttaggregerende evnen til oligosakkaridene var ti ganger lavere enn den for standard heparin, beregnet ut fra vekt. The ability of the oligosaccharides to aggregate platelets at critical ADP (Adenosine DiPhosphate) concentrations was studied mainly according to E.A. (Thromb Haem Stuttg. 1977, 38_, 578). It was shown that the platelet-aggregating ability of the oligosaccharides was ten times lower than that of standard heparin, calculated on the basis of weight.
Claims (1)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE8006459 | 1980-09-15 | ||
| PCT/SE1981/000252 WO1982001005A1 (en) | 1980-09-15 | 1981-09-10 | Oligosaccharides having selective anticoagulation activity |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| NO821631L NO821631L (en) | 1982-05-14 |
| NO157580B true NO157580B (en) | 1988-01-04 |
| NO157580C NO157580C (en) | 1988-04-13 |
Family
ID=26657673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO821631A NO157580C (en) | 1980-09-15 | 1982-05-14 | PROCEDURE FOR THE PREPARATION OF OLIGOSACCARIDES WITH SELECTIVE ANTICOAGULATION ACTIVITY. |
Country Status (1)
| Country | Link |
|---|---|
| NO (1) | NO157580C (en) |
-
1982
- 1982-05-14 NO NO821631A patent/NO157580C/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NO821631L (en) | 1982-05-14 |
| NO157580C (en) | 1988-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4496550A (en) | Oligosaccharides having selective anticoagulation activity | |
| EP0014184B1 (en) | Heparin fragments having a selective anticoagulation activity and process for their preparation | |
| US4981955A (en) | Depolymerization method of heparin | |
| US5013724A (en) | Process for the sulfation of glycosaminoglycans, the sulfated glycosaminoglycans and their biological applications | |
| US4933326A (en) | Oligosaccharides obtained by heparin depolymerization having antiatherosclerotic activity | |
| Wu et al. | Anticoagulant and antithrombotic evaluation of native fucosylated chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor Xase | |
| AU643531B2 (en) | Mixtures of low molecular weight polysaccharides, and their preparation and use | |
| AU601566B2 (en) | Low molecular weight heparins of regular structure, their preparation and their biological uses | |
| US4401662A (en) | Oligosaccharides having anti-Xa activity and pharmaceutical compositions containing them | |
| US4916219A (en) | Oligosaccharide heparin fragments as inhibitors of complement cascade | |
| EP0337327A1 (en) | Process for the preparation of new oligosaccharide fractions by controlled chemical depolimerization of heparin | |
| US4533549A (en) | Antithrombotic agent | |
| JPH06506685A (en) | Novel non-anticoagulant heparin derivative | |
| DE68921633T2 (en) | OLIGOSACCHARIDES WITH ANTIATHEROSCLEROTIC EFFECT. | |
| WO1992002232A1 (en) | Heparin fragment showing complement inhibition activity | |
| EP0101141A2 (en) | Process for manufacturing low molecular weight heparins by depolymerization of normal heparin | |
| USRE35770E (en) | Oligosaccharides having anti-Xa activity and pharmaceutical compositions containing them | |
| EP1513880B1 (en) | Epimerized derivatives of k5 polysaccharide with a very high degree of sulfation | |
| LINDAHL et al. | The antithrombin-binding sequence of heparin | |
| NO157580B (en) | PROCEDURE FOR THE PREPARATION OF OLIGOSACCARIDES WITH SELECTIVE ANTICOAGULATION ACTIVITY. | |
| Vijayabaskar et al. | Low-molecular weight molluscan glycosaminoglycan from bivalve Katelysia opima (Gmelin) | |
| Mascellani et al. | Relative influence of different disulphate disaccharide clusters on the HCII-mediated inhibition of thrombin by dermatan sulphates of different origins | |
| RU2333222C2 (en) | Epimerised k5 polysaccharide derivatives with high sulfation degree | |
| JP2000080102A (en) | O-persulfated glycosaminoglycan and anticoagulant containing same as effective ingredient | |
| JPH02124902A (en) | Blood coagulation inhibitor |