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Sulfated heparin oligosaccharide as well as preparation method and application thereof

A technology of sulfated heparin oligosaccharides and heparin oligosaccharides, which is applied in the field of antitumor drugs, can solve the problems of difficult preparation and structure determination of N-acetylated heparin oligosaccharides, no significant improvement in heparanase activity, etc., and achieve good inhibition Tumor metastasis activity, good heparanase inhibitory activity, tumor metastasis inhibition effect

Active Publication Date: 2016-04-20
SHENZHEN HEPALINK PHARMA GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] This invention only solves the problem of difficult preparation and structure determination of heparin oligosaccharides containing N-acetylated structure, and there is no significant improvement in inhibiting the activity of heparanase in vivo

Method used

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  • Sulfated heparin oligosaccharide as well as preparation method and application thereof
  • Sulfated heparin oligosaccharide as well as preparation method and application thereof
  • Sulfated heparin oligosaccharide as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0091] The preparation of embodiment 1 heparin oligosaccharide

[0092] Take 24g of heparin, add 240mL of Tris-HCl buffer solution, stir to dissolve, add 480IU of heparinase I, stir evenly, and place it at 10°C for enzymolysis reaction for 16h. After the reaction was completed, the reaction mixture was heated to 95° C. for 6 min to inactivate, and ultrafiltered in a 10 KDa ultrafiltration centrifuge tube. The filtrate was separated with a Bio-GelP-10 (2.5×100cm) chromatographic column, and 0.2M NH 4 HCO 3 As the eluent, collect 1.75-2.15 times the column volume components to obtain the heparin tetrasaccharide mixture, collect 1.35-1.75 times the column volume components to obtain the heparin hexasaccharide mixture, collect 1.05-1.35 times the column volume components to obtain the heparin 8 Sugar-sugar mixture, collect 0.85-1.05 times column volume fraction to get heparin decasaccharide mixture, collect 0.75-0.85 times column volume fraction to get heparin dodecaose mixture,...

Embodiment 220

[0096] The preparation of the heparin octasaccharide of embodiment 220%~80% sulfate

[0097] Weigh 0.54g of heparin octasaccharide raw material, transfer it to a reaction bottle, add 25mL of anhydrous DMF, and stir to dissolve. Weigh 0.86g (CH 3 ) 3 N·SO 3 , gradually added to the above solution under stirring, and stirred for 10 min. The bottle cap was plugged, and the reaction bottle was placed in an oil bath at 80°C and stirred for 4 h. After stopping the reaction, it was naturally cooled to room temperature. The solid was dissolved in 90 mL of pure water, and the pH was adjusted to nearly neutral with 2M NaOH. The solution was transferred to a dialysis bag with a molecular weight cut-off of 100-500 Da, and after three days of dialysis, the solution was washed with BaCl 2 Check for the presence of large amounts of sulfate in the dialysate. If not, first adjust the pH to neutral with 2M NaOH, and concentrate to about 10 mL on a rotary evaporator. Put it on the P2 col...

Embodiment 340

[0106] Example 3 Heparanase Inhibitory Activity of 40% and 60% Sulfated Heparin Octaose

[0107] The method described in the literature [HammondE, LiCP, FerroV.Developmentofacolorimetricassayforheparanaseactivitysuitableforkineticanalysisandinhibitorscreening.Anal.Biochem.2011; 396:112-116] was used to measure the heparanase inhibitory activity of sulfated heparan oligosaccharides, that is, the specific experimental operations were as follows:

[0108]The reaction solution of the experimental group was composed of 40mM sodium acetate buffer (pH5.0) and 100mM fondaparinux sodium, and a specific concentration of heparin sulfated octaose, and the reaction solution of the control group was mixed with sulfated heparin octaose The SST0001 reference substance of the same concentration replaced sulfated heparin octasaccharide. Add 100 μL of the reaction solution of the experimental group or the control group to each well of the 96-well plate, and then add heparanase respectively to st...

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Abstract

The invention provides sulfated heparin oligosaccharide as well as a preparation method and application thereof. The sulfated heparin oligosaccharide is characterized in that a non-reducing end of a sulfated heparin oligosaccharide molecule contains an unsaturated double bond produced through enzymolysis of heparinase as well as uronic acid derivatives and glycosylamine derivatives; the sulfated heparin oligosaccharide has a structure represented by a formula I, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, Ra, Rb, Rc and Rd are independently SO3<-> or H; Rx', Ry' and Rz' are independently COCH3 or SO3<->, and n is 1-3. The sulfated heparin oligosaccharide with controllable sulfating degree prepared by virtue of the preparation method has very high activity for inhibiting heparanase in vitro, the activity of the sulfated heparin oligosaccharide for inhibiting cell adhesion and migration is 4-5 times higher than that of heparin, and the activity for inhibiting tumor metastasis in mice is 2-3 times higher than that of the heparin; the sulfated heparin oligosaccharide has relatively good tumor metastasis resistance and relatively high specificity.

Description

technical field [0001] The invention belongs to the field of antitumor drugs, and relates to a sulfated heparin oligosaccharide and a preparation method and application thereof. Background technique [0002] Tumor is a major threat to human health. More seriously, malignant tumors such as liver cancer and lung cancer are prone to metastasis. There is no drug to inhibit tumor metastasis, which makes the treatment of tumor metastasis difficult and makes tumor metastasis the most important cause of death in cancer patients. Two processes are critical for tumor cells to achieve invasion and migration: one is to break through the barrier composed of extracellular matrix (ECM) and basement membrane (BM) and the other is to form new blood vessels. ECM and BM are the barriers for tumor cell invasion and metastasis. To realize the spread and metastasis of malignant tumor cells, they must pass through the ECM and BM to enter the circulation. In this process, the degradation of variou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/10C12P19/26A61K31/727A61P35/00
CPCA61K31/727C08B37/0078C12P19/00C12P19/26
Inventor 李锂马小来田方方杜媛媛杜宏银刘亚晗
Owner SHENZHEN HEPALINK PHARMA GRP CO LTD
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