Preparation method of low-anticoagulant heparin with anti-tumor activity

A technology with anti-tumor activity and low anti-coagulation, applied in the field of biomedicine, can solve the problems of lack of selectivity, insignificant anti-tumor activity, and low degree of sulfation, achieve great social and economic value, and broaden the scope of clinical applications. Effect

Inactive Publication Date: 2013-02-13
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Drug researchers at home and abroad try to use chemical methods to modify the structure of heparin. Chemical desulfation can reduce the anticoagulant activity of heparin to a certain extent, but due to the lack of selectivity and the low degree of sulfation, the antitumor activity is not good. obvious

Method used

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  • Preparation method of low-anticoagulant heparin with anti-tumor activity
  • Preparation method of low-anticoagulant heparin with anti-tumor activity

Examples

Experimental program
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Effect test

Embodiment 1

[0011] Example 1: Preparation of Completely Desulfated Heparin

[0012] Dissolve 1 g of heparin sodium in 20 mL of deionized water, pass through a hydrogen ion exchange column to obtain a heparin acid solution, adjust the pH of the solution to about 5.5 with tributylamine (TBA), and freeze-dry to obtain heparin tributylamine salt. The freeze-dried powder was dissolved in 25 mL of dimethyl sulfoxide solution containing 10% methanol, and reacted at 105° C. for 24 h. After the reaction was completed, the pH of the reaction liquid was adjusted to 9 after being diluted with an equal volume of water, and then alcohol precipitation with 4 times the volume of saturated sodium acetate ethanol solution, dialyzed, desalted, and freeze-dried to obtain completely desulfated heparin. The sulfur content is determined by elemental analysis to determine that the desulfurization rate is above 95%.

Embodiment 2

[0013] Example 2: Preparation of N-position sulfated heparin

[0014] Completely desulfated heparin was dissolved in water, and trimethylammonium sulfur trioxide copolymer (trimethylamine sulfurtrioxide complex, TMA·SO3) was gradually added at a mass ratio of 1:2, and reacted at 55°C and pH 9 for 6 hours. After the reaction, adjust the pH to neutral, dialyze and freeze-dry to obtain N-position sulfated heparin.

Embodiment 3

[0015] Example 3: Preparation of N-position, 6-O-position sulfated heparin

[0016] Usually, the enzymatic sulfation reaction condition is 1 mg of N-sulfated heparin in 20 ml of reaction liquid and shaken (300 rpm) at room temperature for 6 h. The reaction solution contains 50mM Tris-HCl (pH 7.2), 1% Triton X-100, 1% BSA, 1mM MgCl 2 , 1mM MnCl 2 , 1 mM PNPS, 40 μM PAP, 8 mg 6-OST-1 and 4 mg AST-IV. Heating the reaction solution at 100°C for ten minutes to stop the reaction, centrifuging to obtain the supernatant, mixing the supernatant with twice the volume of 2.0% NaCl aqueous solution, and loading the DEAE column; sequentially with 2.0% NaCl aqueous solution and 3.0 % NaCl aqueous solution was used for gradient elution, and the eluate obtained from the second elution was collected. The obtained solution was dialyzed with deionized water for 24 hours, and freeze-dried to obtain freeze-dried powder of N-position, 6-O-position sulfated heparin.

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Abstract

The invention belongs to the field of biomedicine technique, and particularly relates to a preparation method of low-anticoagulant heparin with anti-tumor activity. The method comprises the steps of using heparin as a starting material to prepare completely desulfated heparin, carrying out N- site resulfation, carrying out enzymatic synthesis to obtain specific-site sulfated heparin through combination of Arylsulfotransferase-IV (AST-IV), 6-OST-1 and 2-OST by a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration system based on the substances obtained in the previous steps, and carrying out N- site sulfation on the specific-site sulfated heparin to obtain the final low-anticoagulant activity heparin derivative with specific anti-tumor function.The heparin derivative avoids the side effects such as bleeding, decrease of blood platelet and osteoporosis when common heparin is used for the tumor patients, and is beneficial to expanding the clinical application ranges of the heparin drugs.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a preparation method of low anticoagulant heparin with antitumor activity. Background technique [0002] Heparin has been widely used clinically as an anticoagulant drug since 1935, but in addition to its anticoagulant effect, it also has the functions of promoting the release of lipoprotein lipase and hepatic lipase, inhibiting complement activation, inhibiting angiogenesis and tumor growth, antiviral activity. Recently it was found that heparin also plays an important role in anti-inflammation and anti-tumor metastasis. However, due to the strong anticoagulant activity of heparin, its clinical application is limited. Chemical or enzymatic modification of heparin can reduce its anticoagulant activity but enhance its antitumor effect. [0003] The anticoagulant activity of heparin comes from its special pentose sugar structure sequence, and the combination of this pentose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/10A61P35/00
Inventor 滕丽萍陈敬华
Owner JIANGNAN UNIV
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