JPH0686808A - Bound body of oligosaccharide or polysaccharide and base material - Google Patents

Abstract

PURPOSE:To provide a bound body of an oligosaccharide or a polysaccharide and a base material by reducing the oligosaccharide or polysaccharide under the presence of ammonium salt to form terminal amino radical and react the amino radical with the functional group of the base material through. CONSTITUTION:Oligosaccharide or polysaccharide is reduced under the presence of ammonium salt to form terminal amino group. A bound body of the oligosaccharide or polysaccharide and a base material is obtained by allowing the reaction with a functional group of the base material through the amino group. Also, diazotation, hydrolysis by acid and alkali and decomposition by end gulcosidase, etc., are enumerated as processes for forming chemically or enzy-matically terminal reduction group from the oligosccharide or polysaccharide. Heparin or the like is preferable for the oligosaccharide or polysaccharide bonded covalently with the base material. The base material is of one having, on the surface, carboxyl group, amino group, isocyanate group or isothiocyanate group, or plastic or gel or the like capable of inducing these functional groups.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a conjugate of an oligosaccharide or a polysaccharide with a substrate having a functional group. The present invention also relates to a base material comprising a conjugate of an oligosaccharide or a polysaccharide and a base material having a functional group and having excellent antithrombotic property and biocompatibility.

[0002]

2. Description of the Related Art It has been conventionally practiced to coat the surface of a substrate with an oligosaccharide or a polysaccharide in order to impart physical, chemical or physiological properties to the surface of the substrate. In particular, for medical devices and the like, various methods have been tried in order to improve biocompatibility and antithrombotic property. For example, as a technique for imparting antithrombotic properties by coating a medical device with heparin, there is a method of ion-complexing heparin, which is an anion, with a cation on the surface of a base material. When brought into contact, it is unstable and the effect lasts only for a short time because heparin is easily eluted. In addition, attempts have been made to coat heparin on the surface of the substrate covalently in order to sustain its effect, most of which result in a large reduction in the biological activity of heparin. This is because an unspecified functional group of the sugar chain including the active sequence is provided for covalent bonding. As a method of avoiding this decrease in activity, there is a method of covalently bonding a free terminal aldehyde group generated by decomposing a polysaccharide containing glucosamine or galactosamine by diazotization and a primary amino group on the surface of the substrate. Japanese Examined Patent Publication 3-70722
No. However, in this method, the activity of heparin itself is lowered due to decomposition by diazotization to generate a free terminal aldehyde group. Moreover, oligosaccharides or polysaccharides that decompose by diazotization are limited to substances containing glucosamine or galactosamine residues as constituent sugar components. Furthermore, the presence of primary amino groups was required on the surface of the substrate to be coated.

[0003]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art, and the effect of elution of oligosaccharides or polysaccharides coated on a substrate is not lowered, Further, the activity of the oligosaccharide or polysaccharide itself does not decrease due to the binding reaction with the base material, and the oligosaccharide is not limited to the constituent sugar component of the oligosaccharide or polysaccharide and the functional group existing on the surface of the base material. It is an object to provide a conjugate of a sugar or a polysaccharide and a substrate.

[0004]

The present inventors have found that the above object can be achieved by the present invention having the following constitution. That is, the object is a conjugate of an oligosaccharide or a polysaccharide having a reducing end and a substrate having a functional group on the surface, and the oligosaccharide or the polysaccharide is subjected to reduction in the presence of an ammonium salt. To produce a terminal amino group, and reacting the oligosaccharide or polysaccharide with a functional group of the base material via the amino group, and comprising a conjugate of the oligosaccharide or polysaccharide and the base material. Achieved by the invention.

[0005] The present invention also aims at binding an oligosaccharide or a polysaccharide having a reducing end to a substrate having a functional group on the surface by a covalent bond so that the reducing end of the oligosaccharide or the polysaccharide is converted to an ammonium salt. According to the present invention, which comprises a method for producing a covalent bond between an oligosaccharide or a polysaccharide and a substrate, which is reduced in the presence of the amino acid to generate a terminal amino group, and the amino group is bonded to a functional group on the surface of the substrate. To be achieved. The present invention also produces a terminal amino group by reducing a terminal reducing group formed by chemically or enzymatically treating an oligosaccharide or a polysaccharide in the presence of an ammonium salt, and generating the terminal amino group through the amino group. And a method for producing the same, which is a covalent bond of an oligosaccharide or polysaccharide bound to a functional group on the surface of a substrate and the substrate.

The chemical or enzymatic treatment of oligosaccharides or polysaccharides in the present invention includes diazotization, hydrolysis with acid or alkali, and decomposition with endoglucosidase.

The oligosaccharides or polysaccharides covalently bound to the substrate according to the present invention include heparin, heparan sulfate,
Dermatan sulfate, chondroitin sulfate, hyaluronic acid,
Keratan sulfate, chitin, dextran, dextran sulfate, amylose or sialic acid and the like and derivatives thereof are preferred.

Examples of the substrate include plastics or gels having carboxyl groups, amino groups, isocyanate groups or isothiocyanate groups on the surface thereof or capable of inducing these functional groups.
In the present invention, the oligosaccharide or polysaccharide may be directly reacted with the functional group of the base material via its amino group to obtain a conjugate of the oligosaccharide or polysaccharide and the base material, but via a spacer or the like. It is also possible to react to obtain a conjugate of an oligosaccharide or a polysaccharide and a substrate. For example, as the spacer used for binding to the amino group on the surface of the substrate, a substance such as dialdehyde, dicarboxylic acid, diisocyanate or diisothiocyanate that crosslinks the amino group and the amino group can be used, and glutardialdehyde is preferable. 1,6
-Hexamethylene diisocyanate or cyanuric chloride is used.

The conjugate of the oligosaccharide or polysaccharide of the present invention and the substrate can be used as a medical material such as various antithrombogenic materials and biocompatible materials, and particularly, the portion that comes into contact with blood can be used. It can be used as a medical device. Examples of medical materials include tubes, sheets, fibers and hollow fibers. Examples of the medical device include artificial lung, artificial kidney, artificial blood vessel, artificial skin, catheter and the like. The present invention will be described in detail below with reference to examples.

[0010]

【Example】

Example 1 5 g of heparin (manufactured by sigma), 1
61 U / mg) is dissolved in 50 ml of 10% aqueous ammonium acetate solution. To this, 3 g of sodium cyanoborohydride was added, reacted at 40 ° C. for 4 to 5 days, and then desalted by dialysis against purified water to obtain Dowex 50W (N
a ⁺ ) (manufactured by Dow Chemical Co.) to freeze-dry as a sodium salt to prepare aminated heparin.

Separately, MDI (OCN-C ₆ H ₄ -CH ₂ -C ₆ H ₄ -NCO)
Or Millionate MTL-C (OCN-C ₆ H ₄ -CH ₂ -C ₆ H ₄ -NC
NC ₆ H ₄ -CH ₂ -C ₆ H ₄ -NCO) tetrahydrofuran (TH
F) A polyvinyl chloride (PVC) tube having an isocyanate group introduced on the surface was prepared by immersing the solution in the tube.

Then, an aminated heparin aqueous solution (0.2%) adjusted to pH 9 to 10 with sodium hydrogen carbonate was placed in the tube and immersed at 60 ° C. for 17 hours to immobilize heparin. After washing with water, it was confirmed by staining with toluidine blue that heparin was immobilized on the inner surface of the tube.

(Example 2) 300 mg of heparin (Sigma)
(sigma), 161U / mg) and sodium nitrite 30mg
Was dissolved in 3 ml of water, adjusted to pH 3 to 3.5 with dilute hydrochloric acid, allowed to stand at 4 ° C. for 1 hour, and then adjusted to pH 7 to 8 with sodium hydroxide. An ethanol solution containing 0.2% sodium acetate is added to this in an amount of 4 times to precipitate the heparin derivative. The precipitate was dissolved in 50 ml of 10% aqueous ammonium acetate solution, added with 3 g of sodium cyanoborohydride, reacted at 40 ° C. for 4-5 days, and then desalted by dialysis against purified water. 50W (N
a ⁺ ) and lyophilized as the sodium salt to give aminated heparin. Thereafter, the same operation as in Example 1 was performed to immobilize the heparin derivative on the PVC tube.

Example 3 An aminated heparin aqueous solution prepared in the same manner as in Example 1 was passed through Dowex 50W (H ⁺ ) at 4 ° C., and the eluate was neutralized with a tri-n-butylamine ethanol solution and freeze-dried. To give a tri-n-butylammonium salt, and 3-5% dimethylformamide (D
It was dissolved in a THF solution containing MF). Apply on the inner surface of PVC tube with isocyanate group introduced,
Heparin was immobilized by drying for 7 hours.

Example 4 Chondroitin sulfate aminated in the same manner as in Example 1 containing 5% DMF in THF.
The solution was treated as in Example 3 and immobilized on the PVC tube inner surface.

Example 5 The inner surface of a soft PVC tube was ozone-oxidized and then reacted with polyethyleneimine to introduce an amino group into the inner surface. The aminated heparin prepared in the same manner as in Example 1 was ionically bonded to the inner surface of the PVC tube by glutardialdehyde to covalently bond the amino group in heparin.

Example 6 The inner surface of the thermoplastic polyurethane tube was ozone-oxidized and then reacted with polyethyleneimine to introduce an amino group into the surface. This was immersed in 50 ml of 0.1 M borate buffer (pH 10) containing 5 times the molar amount of cyanuric chloride with respect to the amino group at 4 ° C. for 2 hours and then washed with water to remove the residual cyanuric chloride. . Next, 0.1 containing aminated heparin prepared as in Example 1
50 ml of M borate buffer (pH 10) was introduced into the tube and immersed at 37 ° C. for 2 hours to immobilize heparin.

(Comparative Example) A 10% tridecylmethylammonium chloride (TDMAC) solution was introduced into a PVC tube and immersed therein, and then heparin (manufactured by sigma, 161 U / mg) was introduced to the inner surface. Heparin was ionically bound.

The tubes of the respective examples and comparative examples were cut into a certain length, and the heparin titer was measured with a test team heparin quantification kit (made by KABI) to obtain the same strength. Those showing the value were used in the following test examples.

Test Example 1 By using the Chandler's loop method, the conjugate of the present invention obtained in Example 1, the ionic conjugate obtained in Comparative Example and an untreated soft PVC tube were used to obtain anticoagulant properties. Persistence levels were measured. As a result, the soft PVC could be confirmed to coagulate in less than 10 minutes, and in the comparative example, coagulation could be confirmed in 3 to 10 hours, but the conjugate of the present invention of Example 1 did not coagulate for 25 hours or more. It showed excellent anticoagulant properties. Further, the present invention other than Example 1 also showed the same excellent effect.

(Test Example 2) The conjugate tube of the present invention prepared in Example 1 was cut to a length of 10 cm, immersed in 10 ml of physiological saline and incubated at 37 ° C.
The amount of eluted heparin after 2, 4, 6 and 8 hours was measured by quantifying uronic acid by the carbazole sulfate method. The same operation was performed for the comparative example. The result is shown in FIG. The conjugate of the present invention obtained in Example 1 was
Compared with the ionic binder of the comparative example, excellent heparin elution resistance was exhibited.

[0022]

As described above in detail, according to the present invention, the reducing end originally possessed by the oligosaccharide or polysaccharide and the base material or the reducing end generated by the chemical or enzymatic treatment is treated with a reducing amino acid. Is converted into a terminal amino group and covalently bonded to a functional group on the surface of the base material via the amino group, the oligosaccharide or polysaccharide is not eluted, and the biological property of the oligosaccharide or polysaccharide is not increased. A conjugate of the oligosaccharide or polysaccharide and the base material can be obtained without any decrease in activity. Furthermore, according to the present invention, conjugates of various base materials and oligosaccharides or polysaccharides can be obtained without being limited by the type of the base material.

[Brief description of drawings]

FIG. 1 shows the measurement results of the amount of eluted heparin of the conjugate of the present invention and the ion conjugate of Comparative Example.

Claims (1)

[Claims] 1. A conjugate of an oligosaccharide or polysaccharide having a reducing end and a substrate having a functional group on the surface, the oligosaccharide or polysaccharide being subjected to reduction in the presence of an ammonium salt. A conjugate of the base material and the oligosaccharide or polysaccharide obtained by producing a terminal amino group and reacting the oligosaccharide or polysaccharide with a functional group of the base material via the amino group.