CN101531723A

CN101531723A - Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure

Info

Publication number: CN101531723A
Application number: CN200910024844A
Authority: CN
Inventors: 陈敬华; 金坚; 许正宏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2009-02-27
Filing date: 2009-02-27
Publication date: 2009-09-16

Abstract

The invention provides a method for selectively modifying a heparin structure by using biological enzyme, which can improve anticoagulation activity of heparin, reduce combination of heparin with blood protein such as platelet factors and the like, and reduce toxic and side effects. The invention belongs to the field of biological medicine. The antithrombus and anticoagulation activities of the heparin derivatives prepared by the method are 2 to 3 times higher than common heparin medicament, the 2-O-sulfate and 6-O-sulfate contents are about 20 percent of the common heparin, and the combination capability of the heparin derivatives with the platelet factors is about 20 percent of the common heparin. The novel heparin derivatives have high anticoagulation activity and low side effect.

Description

A kind ofly with biological enzyme heparin is carried out selective structure and modify the method prepare heparin derivatives

Technical field

The present invention relates to a kind ofly with biological enzyme heparin be carried out selective structure and modify the method prepare heparin derivatives, belong to biomedicine field.

Background technology

Heparin is the sulfated polysaccharides that is formed by height sulfated sugar aldehydic acid and glucosamine alternating copolymerization, mainly separates obtaining from mammiferous mastocyte.The advantage of heparin is that it has higher anti-bolt and anticoagulating active simultaneously, can produce blood coagulation resisting function rapidly after the subcutaneous injection, and price is relatively cheap, and can use in the protamine (Protamine) and the heparin [8] of excessive use in the body.But the clinical use of heparin has bigger limitation.Mainly show the following aspects: (1) heparin is to separate to obtain from pluck, and therefore, the heparin of different sources exists on composition, molecular weight, structure than big-difference, make very difficulty of quality control, therefore, lack unified quality standard, variant production can't be replaced use; (2) because the diversity of heparin structure and the polymolecularity of molecular weight, cause the albumen of heparin and plasma proteins, secretion of platelet and the non-specific interaction between the epithelial cell, and with AT-III competition active centre, therefore, dose-effect relationship varies with each individual, and needs before the use to determine dosage by the chemical examination test; (3) during the heparin subcutaneous injection, the treatment window is very narrow, and bioavailability is low, and the transformation period is short, and pharmacokinetics and pharmacodynamic properties are all very poor; (4) heparin may cause hemorrhage and side effects [9] such as induced platelet minimizing (HIT), osteoporosis in addition.These reasons have limited heparin application clinically.

The ideal anticoagulation medicine not only will have unified quality standard, activity is controlled, side effect is little, dosage is little and long half time.In order to develop this novel anti blood-clotting agent, the pharmaceuticals researcher attempts with the structure of modification of chemical method to heparin both at home and abroad.The acidifying of chemical method desulfuration can reduce the side effect of heparin on certain degree, but owing to lack selectivity, causes active centre quantity to reduce, and anticoagulant active significantly reduces; Chemical method makes heparin oversulfated, may improve the quantity at anticoagulant active center in theory, thereby improves its anticoagulant active.But, because degree is too high, cause its electric density excessive, itself and blood protein non-specific interaction are significantly increased.As a result, not only fail to increase its anticoagulant active, caused more serious toxic side effect on the contrary.

By the selectivity enzyme reaction, increase the quantity at anticoagulant active center, can increase substantially the anticoagulant active of low molecular weight heparin.Existing studies show that, the anticoagulant active of heparin depends on the quantity in its pentose active centre.N-in the pentose structure, 6-O-and 3-O-position sulfate participate in conjunction with AT-III, and be relevant with anticoagulant active, and the effect of 2-O-position sulfate and Ai Du aldehydic acid it be unclear that [13].Wherein 3-O-position sulfate is even more important: if lack 3-O-position sulfate, the anticoagulant active of heparin then can reduce more than 1000 times; On the contrary, increase a 3-O-position sulfate, then the anticoagulant active of heparin can be improved several times.Therefore, with 3-OST-1 and 3-OST-5 heparin is selected to modify, make 80% heparin that does not have the pentose active centre after reacting, have the pentose active centre, just can increase substantially the quantity in the pentose active centre of heparin, thereby improve its anticoagulant active (can improve anti-bolt and anticoagulating active 3-5 in theory simultaneously doubly).

By the desulfuration acidifying enzyme, the sulfate that selective removal and anticoagulant active are irrelevant reduces its electric density, can reduce its toxic side effect significantly.Heparin class polysaccharide is one of the most complicated molecule of the structure found up to now.It is made up of the disaccharides of 32 kinds of different structures.Corresponding with structural complexity is its functional complexity.Heparin can play an important role in physiological processs such as blood coagulation, wound healing, virus infection, fetal development and tumour [17] in conjunction with multiple protein.The function of heparin depends on that its sulfate replaces situation (sulfation pattern, sulfation pattern).The effect difference [18] of the sulfate performance of different positions.Such as, during anticoagulation, the N-position sulfate of glucosamine and the non-reduced terminal 6-O-that connects the glucosamine of glucuronic acid, 3-O-is essential, other sulfate is then inessential.And for example, binding growth factor, when promoting the cell growth, 2-O-, the sulfate of 6-O-position then are essential [19].Therefore, remove some unnecessary sulfates, just might the narrow spectrum heparin derivatives of complex functionality.This derivative will have two advantages: at first, structure is simple than heparin, homogeneous, has the function specificity; Secondly, the ability in other protein competition active centre reduces, and makes its pharmacological property more stable.Simultaneously, the variation of electric density can reduce the non-specific binding between heparin and blood plasma, blood platelet albumen matter, such as, reduce the binding ability with PF4, thereby side effect such as thrombocytopenia is reduced.

With the structure of biological enzyme selectivity transformation heparin, when improving the anticoagulant active centric quantity, reduce and the active 2-O-sulfate that has nothing to do and the content of 6-O-sulfate, do not see any relevant report so far both at home and abroad as yet.

Summary of the invention

The present invention has set up a kind of method for preparing heparin derivatives with biological enzyme selectivity transformation heparin structure.Distinctive feature of the present invention has 2 points: at first will carry out selective modification to existing heparin medicine with 3-OST-1 enzyme (or 3-OST-5), and improve the quantity at anticoagulant active center to greatest extent, thereby increase substantially the anticoagulant active of its heparin.Secondly remove and the irrelevant sulfate of anticoagulant active with 2-O-sulfatase and 6-O-sulfatase enzyme selectivity, reduce its electric density.The dosage and the toxic side effect of the clinical use of this heparin derivatives will reduce significantly, and the indication scope will be wider.The present invention will help to develop the anticoagulation medicine of new generation of independent intellectual property right.China is heparin big producing country, but the heparin series products of China mostly can only be as material outlet, the annual marketable value that only can create millions of dollar.China lacks the heparin class medicine with independent intellectual property right at present, and domestic market is also most of to be occupied by American-European company.At present, the annual heparin class anticoagulation medicine marketable value of selling in the whole world is about 5,000,000,000 dollars.Along with the arrival of aging population, the cardiovascular disease patient will progressively increase, and China's cardiovascular diseases sickness rate every year, the speed with nearly 30% increased [2].Therefore, this project not only has scientific research value, and will have bigger social value and economic worth.

Particular content of the present invention is divided into following five parts:

1. with Suleparoid 3-OH sulfate transferring enzyme, heparin 3-OH base is carried out selectively sulfated, increase active centre content, synthetic product I.

2. with Suleparoid 2-OH sulfate transferring enzyme, synthetic product I 2-OH sulfate is carried out the acidifying of selectivity desulfuration, reduce and active irrelevant 2-O-sulfate content, synthetic product II.

3. with Suleparoid 2-OH sulfate transferring enzyme, synthetic product II 2-OH sulfate is carried out the acidifying of selectivity desulfuration, reduce and active irrelevant 2-O-sulfate content synthetic product III.

4. use external anti-bolt and the anticoagulating active of colour developing luminous substrate assay heparin and synthetic product III.

5. measure the binding ability of heparin and synthetic product III and platelet factor PF4 with linear gradient elution method.

Description of drawings

Fig. 1. the HPLC collection of illustrative plates that the disaccharide of heparin and synthetic product I is formed

Fig. 2. the HPLC collection of illustrative plates that the disaccharide of heparin and product II is formed

Fig. 3. the HPLC collection of illustrative plates that the disaccharide of heparin and product III is formed

Fig. 4. the binding ability of heparin and product III and PF4

Embodiment

Following example will specify working method of the present invention, but can not be as limitation of the invention.

Example one

With Suleparoid 3-OH sulfate transferring enzyme, synthetic product I.Common sulfating reaction condition is that the 1mg heparin shakes (300rpm) reaction 6h in the 20ml reaction solution under room temperature.Reaction solution comprises 50mM Tris-HCl (pH7.2), 1%Triton X-100,1% BSA, 1mM MgCl ₂, 1mM MnCl ₂, 1mM PNPS, 40 μ M PAP, 8mg 3-OST-1 (or 3-OST-5) and 4mg AST-IV.Reaction product obtains synthetic product I through DEAE resin isolation, dialysis, freeze-drying.With mixing the depolymerized heparin enzyme heparin and synthetic product I are degraded into disaccharide fully, use C ₁₈Reversed-phase column HPLC method is analyzed their disaccharide and is formed and content.The results are shown in Fig. 1, the 3-O-sulfate disaccharide content that shows product I is about 3 times of heparin, discloses its anticoagulant active centric quantity and has improved 3 times.

Example two

With Suleparoid 2-O desulfuration acidic group enzyme, synthetic product II.Common desulfuration acidification reaction condition is that product I shakes (300rpm) reaction 6h in the 20ml reaction solution under room temperature.Reaction solution comprises 50mM Tris-HCl (pH7.2), 1%Triton X-100,1% BSA, 1mM MgCl ₂, 1mM MnCl ₂, 1mM CaCl ₂, 5mg 2-O-sulfatase.Reaction product obtains synthetic product II through DEAE resin isolation, dialysis, freeze-drying.With mixing the depolymerized heparin enzyme heparin and synthetic product II are degraded into disaccharide fully, use C ₁₈Reversed-phase column HPLC method is analyzed their disaccharide and is formed and content.The results are shown in Fig. 2, show that the 2-O-sulfate disaccharide content of product II is about 20% of heparin.

Example three

With Suleparoid 6-O desulfuration acidic group enzyme, synthetic product III.Common desulfuration acidification reaction condition is that product II shakes (300rpm) reaction 6h in the 20ml reaction solution under room temperature.Reaction solution comprises 50mM Tris-HCl (pH7.2), 1%Triton X-100,1% BSA, 1mM MgCl ₂, 1mM MnCl ₂, 1mM CaCl ₂, 5mg 6-O-sulfatase.Reaction product obtains synthetic product III through DEAE resin isolation, dialysis, freeze-drying.With mixing the depolymerized heparin enzyme heparin and synthetic product III are degraded into disaccharide fully, use C ₁₈Reversed-phase column HPLC method is analyzed their disaccharide and is formed and content.The results are shown in Fig. 3, show that the 6-O-sulfate disaccharide content of product III is about 20% of heparin.

Example four

External anti-bolt and anticoagulating active with substrate determination of color heparin and product III.At first, with the PBS that contains 1mg/ml BSA factor Xa, thrombin and antithrombin AT-III dilution being diluted respectively is that 1U/ml, 8U/ml and 27 μ M are standby.Chromogenic substrate S-2765 and S-2238 are made into the solution of 1mM respectively with PBS.Heparin and product III are with containing 50mM Tris-HCl (pH8.4), 7.5mM Na ₂EDTA, the damping fluid of 175mM NaCl is made into the solution of series concentration (1-1000ng/ml).During test, at first the polysaccharide soln of 25 μ l and the AT-III solution of 25 μ l are mixed, kept 2 minutes in 37 ℃.Add factor Xa or the thrombin solution of 25 μ l then, mix, kept 4 minutes in 37 ℃.At last, add chromogenic substrate S-2765 or the S-2238 of 25 μ l, mix, and the absorbancy at 405nm place changes in the METHOD FOR CONTINUOUS DETERMINATION 10 minutes, relatively and calculate factor Xa and thrombin half inhibiting value IC50.The results are shown in table 1, show, the external anti-thrombus activity of product III has improved 3 times, and anticoagulating active has improved 2.5 times.

External anti-bolt, the anticoagulating active of table 1. heparin and product III

Example five

Earlier PF4 is fixed on the CNBr-activated Sepharose 4B resin (Pharmacia).In the time of 4 ℃, measure the binding ability of PF4 and heparin and product III.PF4-Speharose 4B resin is filled in the chromatographic column, add heparin and product III respectively, carry out gradient elution then and collect fraction, then with the absorbancy of each fraction of ultraviolet determination at the 232nm place, and make corresponding elution curve, determine PF4 and different fractions binding ability by measuring wash-out concentration.The results are shown in Fig. 4, show that the amount of product III bonded PF4 greatly reduces, be about 20% of heparin binding capacity, and bonding force reduces.

Claims

1. one kind is carried out selective structure with biological enzyme to heparin and modifies the preparation heparin derivatives, improves its anticoagulant active, reduces its method in conjunction with the platelet factor ability.

2. the preparation method of heparin derivatives as claimed in claim 1, this method is made up of the following step:

(1) with Suleparoid 3-O-sulfate transferring enzyme,, the sulfate of PAPS is optionally transferred to heparin glucosamine 3-OH position, improve 3-O-sulfate content, synthetic product I by the enzyme sulfating reaction;

(2) handle synthetic product I with Suleparoid 6-O-desulfurization acidic group enzyme, by enzyme desulfuration acidification reaction, selectivity is taken off the glucosamine 6-O sulfate that non-reduced end is an iduronic acid, reduces 6-O-sulfate content, synthetic product II;

(3) handle synthetic product I with Suleparoid 2-O-desulfurization acidic group enzyme, by enzyme reaction, selectivity is taken off iduronic acid 2-O sulfate, reduces 2-O-sulfate content, synthetic product III;

(4) synthetic product III is carried out desalination, dialysis, drying, obtain white powder, very the heparin derivatives of present method preparation.

The heparin of being declared refers to the Calciparine/sodium salt that separation and purification goes out from the mastocyte of pluck.

The Suleparoid 3-O-sulfate transferring enzyme of being declared, 6-O-desulfurization acidic group enzyme, 2-O-desulfurization acidic group enzyme refer to the enzyme relevant with the Suleparoid biosynthesizing, can from people, animal or microorganism, separate obtaining, also can be by molecular biological technology preparation.

The enzyme reaction of being declared refers in certain pH, damping fluid, temperature, the reaction of carrying out with specific enzyme catalysis.

The heparin derivatives of being declared is a kind of sodium salt of sulfated polysaccharides, be white powder, molecular weight is at 5000-8000, uronic acid and glucosamine consist of 1:1, each sugar unit contains 1.5 sulfates approximately, and wherein 3-O-sulfate content accounts for the 5-10% of whole sulfate content, and N-sulfate content is 50-60%, 2-O-sulfate sulfate content is 10-15%, and 6-O-sulfate sulfate content is 10-15%.

3. the enzyme sulfating reaction of declaring in the claim 2 refers to by 50mM Tris-HCl (pH7.2) 1% Triton X-100,1%BSA, 1mMMgCl ₂, 1mM MnCl ₂, 1mM PNPS, 40 μ M PAP, the reaction of carrying out in the reaction solution that 8mg 3-OST-1 (or 3-OST-5) and 4mg AST-IV form is shaken reaction 2-6h down in 20-40 ℃.

4. the enzyme sulfating reaction of declaring in the claim 2 refers to by 50mM Tris-HCl (pH7.2), 1%Triton X-100,1%BSA, 1mMMgCl ₂, ImM MnCl ₂, 1mM CaCl ₂The reaction of carrying out in the reaction solution of forming is shaken reaction 2-6h down in 20-40 ℃.

5. the drying of declaring in the claim 2 can be vacuum-drying, spraying drying or lyophilize.