• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The invention relates to an oligosaccharide which contains 4-8 monosaccharide units and which is characterized in that it contains at least one glucosamine unit which is 3-0-sulfated, and at least one additional glucosamine unit, whereby these units are linked via an intermediate monosaccharide unit, which is bound to the reducing end of the 3-0-sulfated glucosamine unit. The invention further relates to a pharmaceutical composition containing one or more such oligosaccharides, and to a process for the preparation of these oligosaccharides.
    公开号:SU1289392A3
    申请号:SU823440803
    申请日:1982-05-14
    公开日:1987-02-07
    发明作者:Пер Фредрик Линдаль Ульф；Элизабет Бекстрем Гудрун；Янгве Леннарт Тунберг Джон
    申请人:Каби Аб (Фирма)；
    IPC主号:
    专利说明:

    one
    The invention relates to a method for producing new heparin derivatives, namely an oligosaccharide fraction, in the key A-8 monosaccharide units, where said oligosaccharide contains at least one 3-0-sulfated glucosamine unit, which is also 2-0-sulfa tated and 2-l-sulfated, and where these links are connected by a transition monosaccharide link that is attached to the reduced end of the 3-0-sulfated link of glucosamine, which has a selective anticoagulant effect.
    The purpose of the invention is to obtain a new oligosaccharide fraction, which has an improved anticoagulant effect, in comparison with heparin.
    Example 1. Preparation of heparin fragments by depolymerization with nitrous acid of ordinary heparin.
    Heparin (0.5 g), isolated from the pig intestines and dissolved in 150 ml of water, is cooled to C and passed through :; a column filled with Dowex® 50 W-28 synthetic ion exchange resin (form), 200-400 mesh. The column is then washed with too MP of water and the washing liquid is combined with the sample. 250 ml of dimethoxyethane (g2yme) cooled to minus and 10 ml of isoamyl nitrite are added to the sample, after which the mixture having the schnus is left to stand for 35 minutes. Then the reaction is stopped by the addition of Na acetate. After the addition of 5.2 liters of ethanol, the precipitated carbohydrate (derived from heparin) was recovered by centrifugation. The product is dissolved in 500 ml of a 0.05 M HCE solution pH 7.4. This solution is divided into 100 ml portions and fractionated by affinity chromatography on a column containing 75 ml of antithrombin pharose (about 5 mg of protein per 1 mp gel). The adsorbent in the column is washed with a salt gradient (500 mp 3 M HaCr - 0.05 M Tris-NSG in the tank) I, with most of the material either passing through the column or washed out of the adsorbent with a low ionic strength (less than 0, 4 M NaCi),






    This material does not possess biological activity. Active ingredients (material in the form of purified oligosaccharide is washed as a broad head fraction with 0.5 M NaCJ and 3 M Nad, corresponding to approximately 3% of the starting material. These fractions are combined, concentrated and desalted by gel chromatography.
    Oligosaccharides prepared and purified by the indicated method have a predominant molecular weight of about 1200-2400 dalton, corresponding to that of the octasaccharide according to the invention.
    Investigation of anticoagulation activity.
    The oligosaccharides prepared according to Example 1 are examined, having in mind their ability: to act on the inhibition of the coagulation enzyme thrombin; impact on; inhibition of coagulation factor X; affect the inhibition of activated factor IX; affect the inhibition of activated factor X; affect the inhibition of activated factor XI; affect the inhibition of activated factor XII; prolong the coagulation duration during the screening test for coagulation of blood plasma by the APTT method (duration of activated partial formation of blood clots); to be neutralized by components of blood plasma and to influence platelet aggregation.
    Example 2. Inhibition of thrombin.
    The ability of oligosaccharides to enhance thrombin inhibition by antithrombin III is analyzed according to a modified method of Teien and co-workers. Oligosaccharides were found to have a specific activity of 8 U / mg compared to 120-170 U / mg for standard heparin.
    And p and M er 3. Inhibition
    activation factor X.
    The ability of oligosaccharides to enhance the inhibition of the activation factor X in plasma and in pure antithrombin III is investigated according to
    a modified version of Teie's way with the staff. Oligosaccharide has been shown to have a specific activity of 500 U / mg in the system of pure antithrombin III and 1900 U / mg in the plasma system compared to 120-170 U / mg for standard heparin.
    Example 4 Inhibition of the activation factor IX,
    The ability of oligosaccharides to enhance Inhibition of the activation factor IX in pure antithrombin III is investigated by Po2mer and co-workers. It has been shown that oligosac-RIDs have a specific activity of 18 U / mg compared to 120-180 U / mg for standard heparin.
    Example 5, Inhibition of the activation factor IX.
    The ability of oligosaccharides usi-. pour Inhibition of the activation factor IX in pure antithrombin III is investigated by Holmer and co-workers. Oligosaccharides have been shown to have a specific activity of 40 U / mg compared to 120-170 U / mg for standard heparin.
    Example 6. Inhibition of the activation factor XII
    The ability of oligo: BharidoB to enhance the inhibition of the activation factor XII in pure antithrombin III is investigated according to Holmer and his colleagues. It was found that the adagosagarides have a specific activity of 470 U / mg compared with standard heparin.
    Example 7, Prolongation of the coagulation duration.
    The ability of oligosaccharides to increase the length of time required for coagulation in the blood plasma is studied according to the APTT method (the time required for the formation of blood clots in partially activated thromboplatinin), the oligosaccharides show a specific (12 e / mg) activity
    compared with heparin in accordance with international standard 3, Standard Heparin shows specific activity in the range of 120-170 U / mg.
    Example 8, Neutralization
    oligosaccharides in the blood plasma.
    VNIIPI Order 7827/60 Circulation 369
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    0
    five
    five
    ABOUT
    five
    The neutralizing effect of plasma components on oligosaccharides is obtained by measuring the effects of heparin and oligosaccharides in the plasma and in the system of pure antithrombin. . It was shown that the activity of oligosaccharides is characterized by 18% neutralization of plasma components. It has been shown that the corresponding value for standard heparin is 75%,
    Example 9, Effect on platelets.
    The ability of oligosaccharides to aggregate platelets at critical concentrations of ADRs (adenosine diphosphate) is studied mainly according to Beck E. A. It was shown that the ability of oligosaccharides to aggregate platelets was 10 times less than that of standard heparin in terms of weight.
    thirty
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    A method of producing an oligosaccharide fraction comprising 4-8 saccharide units, wherein said oligosaccharide contains at least one 3-0 sulphated unit, glucosamine, which is also 2-0 sulphated and 2-N-sulphated , and where these links are connected by a transition monosaccharide link, which is attached to the reduced end of the 3–0-sulfated glucosamine link, which is also related to the fact that heparin is depolymerized by treatment with nitrous acid in dimethoxyethane at (-10) ° С followed by boarding Heath precipitates of heparin derivatives, after which it is dissolved in a 0.05 M solution of sodium chloride (0.05 M NSD at pH 7.4 and the resulting solution is subjected to separation using an affinity chromatograph (on a column containing antithrombin pharose.
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    同族专利:
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    引用文献:
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    法律状态:
    优先权:
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    SE8006459|1980-09-15|
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