JPH0492673A - Antithrombotic hemo filter - Google Patents

Abstract

PURPOSE:To compatibly obtain an antithrombotic property and membrane performance by forming the above hemo filter of of a membrane base material consisting an acid group-contg. polyacrylonitrile copolymer, a porous chitosan layer clad on the inside surface of this membrane base material and heparin bonded to this layer. CONSTITUTION:This filter consists of the acid group-contg. PAN copolymer, the porous chitosan layer clad on the inside surface of this membrane base material and the heparin bonded to this layer. The membrane base film remains without being subjected to the oxidation reaction by a strong oxidizing reagent as the acid group is previously introduced into the base material. The acid group in the membrane base material forms the bond of the amino group of chitosan and ion and allows the stable lamination of the chitosan on the surface of the membrane base material. The antithrombotic property and the high membrane performance are both obtd. in this way.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a hemofilter with excellent antithrombotic properties.

(Prior Art) As one of the blood purification methods for patients with chronic renal failure, a treatment method using a hemofilter is performed. Hemofilter removes substances by filtration, so the hemofiltration rate (hereinafter referred to as
It must have a built-in membrane with excellent blood UFR (blood UFR). In recent years, not only low molecular weight uremic substances but also β2. -Microglobulin (approximately 11,800 molecules
It is also important to have a high sieving coefficient (hereinafter referred to as SC) for β2-MG so that low molecular weight proteins such as β2-MG (hereinafter referred to as β2-MG) can be efficiently removed.

A membrane that satisfies these conditions contains albumin (approximately 69
It is necessary to have as large a pore diameter as possible without allowing useful proteins such as ,000) to pass through. Synthetic polymer membranes are suitable from the viewpoint of pore size controllability and water permeability, and among these, polyacrylonitrile (hereinafter referred to as PAN) copolymer membranes are preferred because of their excellent biocompatibility.

On the other hand, anticoagulants are used during treatment to prevent thrombus formation in the hemofilter and circuit. Currently, heparin is the most commonly used drug, but long-term administration may cause side effects in some patients, and when used continuously for long periods of time, such as in continuous hemofiltration therapy, there is always a risk of bleeding. It was considered dangerous and improvements were desired.

In order to minimize the amount of heparin administered, several attempts have been made to immobilize heparin on blood purification membranes and improve its antithrombotic properties.

For example, in JP-A-57-162704, a PAN polymer is subjected to craft polymerization of a vinyl monomer containing an epoxy group and a vinyl monomer that does not contain an epoxy group, and the epoxy group in the craft chain is added to the amine in heparin. It is said that antithrombotic properties can be obtained by covalently bonding groups.

However, in this method, graft polymerization also proceeds inside the pores, so even if a PAN membrane, which originally has high water permeability, is used, the pores become smaller and the water permeability decreases. Is there no mention of blood 1UFR in this invention?In general, in PAN membranes, blood UFR is said to be about 1/10 of the water permeation rate, so the estimated plasma UFR is 10 mfl/m2-h.
r-mmHg or less, which is a practical level of about 20ml/l/
It is far below m2-hr-mmHg.

Also, E
PA method (End Point Attachmen
There is an example in which a method of covalently bonding heparin to the material surface by one-end bonding (T: one-end bond) was attempted on cellulose acetate hollow fiber membranes (P
roc, EDTA-ERA21.270, 84).

This invention involves oxidizing the surface with a permancanic acid-based oxidizing agent, coating it with polyethyleneimine, and then covalently bonding heparin with an aldehyde at one end to the polyethyleneimine layer, which is said to have excellent antithrombotic properties. I'm hitting. but,
The membrane material itself is a dialysis membrane, and β
High membrane performance such as removal of 2-MG and high blood UFR cannot be expected. Furthermore, during the oxidation treatment, there was a risk that the membrane material and pores would be eroded by the reagent, and since the coated polyethyleneimine was water-soluble, there was a risk that it would elute during use.

Furthermore, Japanese Patent Publication No. 1-45373 states that antithrombotic properties can be obtained by providing a chitosan coating layer on a polymer substrate and bonding an antithrombinogenic agent thereon. However, what is disclosed here is an application to non-permeable substrates and not membranes. In fact, even if the method shown in the Examples is directly applied to membranes, membrane performance will not be achieved.

As described above, when attempting to bind heparin to a membrane material to make it antithrombotic, membrane performance such as β2-MG removal and blood UFR deteriorates, making it impossible to achieve both antithrombotic and membrane performance. .

(9 Problems to be Solved by the Invention) An object of the present invention is to provide a hemofilter that has excellent antithrombotic properties and excellent membrane performance.

(Means for Solving the Problems) The antithrombotic hemofilter of the present invention is made of acidic group-containing PAN.
It consists of a membrane base material made of a copolymer, a porous chitosan layer coated on the surface of the membrane base material, and heparin bonded to the layer.

In the present invention, the presence of acidic groups in the PAN copolymer is
This is important for the following two points. ■Since acidic groups have been introduced into the membrane base material in advance, it is not subject to the violent oxidation reaction caused by the oxidizing agent that was a problem in the inventions of JP-A No. 58-147404 and JP-A No. 1-45373. It ends up being (2) The acidic groups in the membrane base form ionic bonds with the amino groups in chitosan, allowing chitosan to be stably laminated on the membrane base.

Furthermore, since the chitosan layer is porous, the pores of the membrane base material are not impaired, and high membrane performance as a blood filtration membrane, which has not been able to achieve a practical level in the past, can be achieved. At the same time, heparin can be bound via either ionic bonds, covalent bonds, or both.

The hemofilter having the above characteristics has both antithrombotic properties and high membrane performance.

(Structure of the Invention) The acidic group-containing PAN copolymer contains 50 wt% or more of acrylonitrile as a main component, and is copolymerized with at least one vinyl monomer having an acidic group. The acidic group referred to herein may be of any type as long as chitosan can form a coating layer through ionic bonding. However, from the viewpoint of stability during copolymerization, carboxyl groups and sulfonic acid groups are preferred, and either one or both may be present as a mixture. For example, examples of the vinyl monomer having a carboxyl group include acrylic acid, methacrylic acid, itaconic acid, and maleic acid, and those having a sulfonic acid group include vinylsulfonic acid, methallylsulfonic acid, and allylsulfonic acid.

The content of acidic groups should be sufficient for the porous PAN copolymer to stably exist and hang on the PAN copolymer; if it is contained in excess, the membrane will swell during blood flow, so it will not affect the blood flow. This can cause unevenness, thrombus formation, and performance deterioration. Therefore, 00
It is preferably 2 to 1.40 mmol/g (polymer).

In the present invention, the membrane base material has a pore size of 25 to 500 pores, and there is no particular limitation on the shape of the membrane base material;
Any form of film may be used.

The coating layer on the surface of the membrane base material is formed of porous chitosan. The chitosan layer G'j is used to form ionic bonds with the acidic groups in the membrane base material, and to form covalent bonds and ionic bonds with heparin by force. In the present invention, deacetylated chitin and its derivatives are defined as chitosan. Therefore, it also includes chitosan with modified hydroxyl groups at the 3- and 6-positions, such as glycol chitosan, carboxymethyl chitosan, and diethylaminoethyl chitosan. Furthermore, the molecular weight is not particularly limited, and includes chitooligosaccharides with a molecular weight of a few to several million polymers.

The coating amount of chitosan is preferably such that it can form ionic bonds with the acidic groups on the surface of the membrane substrate and at the same time bind heparin to the coating layer. If it is too small, the binding amount of heparin will be limited, and if it is too large, the membrane performance will be reduced.
μg/cm2 is good. Preferably 1 to 100 μg/cm
2, more preferably 5 to 50 μg/cm2.

The structure of the porous chitosan layer is porous with a pore size equal to but larger than that of the membrane substrate. For this reason, the coating layer does not become a resistance to the filtration performance of the membrane, and does not reduce the high membrane performance that the PAN membrane base material has.

Heparin is bonded to the amino groups of the porous chitosan layer by ionic bonds, covalent bonds, or a mixture of both. In the case of ionic bonding, an ion pair is formed between the acidic group in heparin and the amino group of the porous chitosan layer, and there are no limitations on the heparin used. When the ionically bonded heparin comes into contact with blood, the ionic bond is gradually broken off and it is slowly released into the blood, thereby exhibiting antithrombotic properties. In terms of covalent bonding, there are cases where heparin is covalently bonded to porous chitosan at multiple points and cases where it is covalently bonded at one end. In the case of multiple points, for example, the amino groups in heparin and the amino groups in the porous chitosan layer are linked by covalent bonds with dialdehyde or jepoxy compounds. In the case of one end, heparin which has been diazolyzed and has an aldehyde group at one end and the amino group of porous chitosan are separated by Schiff
ff) Bonded by a base. This double bond may be reduced with a borohydride reagent or the like.

Furthermore, both ionic bonds and covalent bonds may coexist. The activity of heparin is high when it is covalently bonded at one end, which has a large degree of freedom, and it is more effective when it is mixed with ionic bonds. During the initial phase of blood circulation, ionically bound heparin is released in a sustained manner.
After that, covalent heparin is most preferred because it has a long-lasting effect.

As described above, the antithrombotic hemofilter comprising the membrane base material, porous chitosan layer, and heparin is manufactured, for example, as follows. First, a membrane base material molded into a hollow fiber shape or a film shape is assembled to form a membrane base material module. The hollow fiber membrane substrate is prepared, for example, according to Example 1 in Japanese Patent Application No. 1-95223.

Next, water is passed through the module 5 to wash away the adhering chrycerin, and then the chitosan solution is passed through the liquid-passing material module for a certain period of time. The pH of the chitosan solution is not particularly limited as long as chitosan can be dissolved;
Number 3 is preferable since the amount of heparin binding continues to be the highest.

If water is passed immediately after passing through the solution, the chitosan will solidify rapidly and a porous chitosan layer will be formed. Further, after washing with water, a heparin solution is passed through it for a certain period of time, followed by washing with water. The heparin solution is not particularly limited; for example, in the case of ionic bonding, a normal aqueous heparin solution can be used. In the case of multi-point covalent bonding, after a normal aqueous heparin solution is poured, an aqueous solution containing glutaraldehyde is poured to effect covalent bonding. In the case of a covalent bond at one end, an aqueous solution of heparin after diazolysis can be used. After passing the heparin solution, it is washed with water, and a 50 wt % aqueous chrycerin solution is circulated, followed by vacuum drying to obtain an antithrombotic hemofilter.

Note that this series of operations can also be completed until heparin is fixed in the hollow fiber or film state, and then assembled into a module. Next, the present invention will be explained using Examples, but the present invention is not limited thereto.

The methods for measuring various values used in the examples are as follows.

1. Connect the circuit to the bovine blood UFR (mu/m2-hr-mmHg) module and add 200 ml of fresh blood at 37°C.
The liquid was passed at a flow rate of 1/min. Transmembrane pressure difference 200mmHg.

The calculation was based on the following formula.

UFR=i throughput (mJ2/hr)/(membrane area (m2)
X200 (mmHg))2, S of bovine plasma β2-MG
Hollow fibers were taken out from the C module and a mini module of 85 fibers x 15 cm was made. Add to this bovine plasma (TP6.5g/
du, 37° C.) at a flow rate of 2 ml/min. Transmembrane pressure difference: 50 mmHg.

SC=β2-MG concentration in filtrate/β2-MG concentration in bovine plasma The β2-MG concentration was measured by EIA method (Imusine, manufactured by Fujirebio Co., Ltd.).

3. Take out the hollow fibers from the rat blood residual rate module and prepare 100 fibers x 10 cm.
I made a mini module. Add 2 m of rat blood (heparin added 5000 U/ffi, 37°C) to this.
It was circulated for 4 hours at a flow rate of min. After washing with physiological saline, the mini module was filled with a still fluid (without heparin added) collected from another rat, and left to stand for 2 hours. Finally, it was washed with physiological saline, and blood or clotted threads were counted.

Remaining blood rate (%) = Number of hollow fibers with coagulated blood/10
0 × 100 4. Amount of bound heparin (total heparin amount) (μg/cm2) Hollow fiber taken out from the module (membrane surface M25Cm2)
was dissolved in 3 mJ2 of concentrated sulfuric acid. After heating at 100°C for 10 minutes, the mixture was cooled to room temperature, 0.1 mj2 of a carbazole reagent was added, and the mixture was heated again at IOOO°C for 15 minutes. After cooling to room temperature, absorbance at 530 mm was read using a spectrophotometer and calculated from a calibration curve.

5. Amount of bound heparin (amount of ionically bound heparin) (μg/
Cm2) The hollow fibers were taken out from the module and a mini module of 25 fibers x 16 cm was made. Add 2mn of 25% saline to this.
The liquid was passed for 30 minutes at a flow rate of /min. Hereinafter, it was calculated according to the procedure in 4 above, and the difference from the total heparin amount of the same sample was defined as the ionically bound heparin amount.

6. Amount of chitosan coated (μg/cm2) A hollow fiber (membrane area 500 cm2) taken out from a module coated with chitosan but not bound with heparin was dried under hot air at 105°C for 12 hours and weighed (defined as Amg). ). Similarly, a hollow fiber (membrane area: 500 cm2) taken out from a membrane base module not coated with chitosan was dried under hot air at 105° C. for 12 hours and weighed (denoted as Bmg).

7. Take out the hollow fiber from the pore diameter measurement module and measure the membrane area of 120 cm2.
I made a mini module. Using this, physiological saline containing 200 ppm of each labeled protein was filtered, and the rejection rate was measured. The protein used was cytochrome C (molecular weight 1
2400), trypsin (molecular weight 23000), egg albumin (molecular weight 47000), bovine albumin (molecular weight 6
7000) and γ-globulin (molecular weight 156000).

From the molecular fractionation curve thus obtained, the molecular weight corresponding to the 50% inhibition point was calculated using the following formula 0 (D).

M. areen et al., American Society of Artificial Internal Organs, pp. 627 et al., 1
976) to determine the molecular diameter. The obtained molecular diameter is 0 below.
The modified pore theory formula (Takezawa et al., Artificial Organs 13
@ No. 6, p. 1460 et seq., 1984) to determine the average pore diameter (dp).

Chids=1.32x (molecular weight) ・・・・・・・・・
■5C=0.5= (2N-q)2- (1-q)'
)(1-(2/3)q2-0.202q5)/(1-0
, 759CI5) -・・・・・・・・・
(here, q=ds/dp, ds: molecular diameter obtained by the formula 0, dP: average pore diameter, sc: sieving coefficient) (Example 1
) ■Acrylonitrile 18. 5mmo 17g (polymer), acrylic acid 0.17mmo Itg (polymer), sodium methallylsulfonate 0.07mmol
An acidic group-containing PAN copolymer consisting of /g (polymer) was obtained.

(2) A hollow fiber membrane substrate was prepared according to Example 1 in Japanese Patent Application No. 1-95223. That is, the copolymer obtained in step (1) above was dissolved in 70% nitric acid to obtain a spinning dope having a polymer concentration of 13%. The stock solution was discharged from the sheath of a sheath-core type hollow fiber nozzle, and 20 wt% of polyvinylpyrrolidone (K
-15) Aqueous solution was introduced. After running in the air, it was introduced into water at 50°C to form a hollow fiber shape. After passing through the water washing process, 4
After immersing in a 0wt% glycerin aqueous solution at 40°C for 6 hours, it was dried in a vacuum dryer at 40°C, and the inner diameter was 230.5.
m' membrane base material module.

■After dissolving 5 g of chitosan (manufactured by Katakura Chikkarin; CTA-10, molecular weight approximately 160,000) in 500 ml of 2% acetic acid aqueous solution, 4N sodium hydroxide was added to adjust the pH of the solution to 6.10.
Adjusted to. Water was passed through the above module (2) to wash away the adhering glycerin, and then this chitosan solution was circulated through the above module (2) for 30 minutes, and then water was immediately passed through it to coagulate it, followed by further washing with water.

(1) 1 g of commercially available heparin (VGF, 178 U/mg) was dissolved in 300 m of distilled water, and the solution was water-cooled to 0°C. After adjusting the pH of the liquid to 3.21 by adding acetic acid, 10 mg of sodium nitrite was added and stirred for 2 hours. Next, neutralize with 4N-sodium hydroxide and transfer to phosphate buffer (pH 6, 9).
) 200 m It, 250 mg of sodium cyanoborohydride were added.

(2) The solution (2) was circulated through the module (2) for 24 hours, and then washed with water.

(2) A 50 wt% aqueous glycerin solution was circulated at 40° C. for 2 hours in the module (2) above, and then vacuum dried to obtain an antithrombotic hemofilter. Various evaluation results for the obtained hemofilter are shown in Tables 1 and 2. When briming,
The amount of physiological saline required to completely remove air was 0.72 for the module with only the membrane base material, whereas it was 0.3 Il for the Honhemo filter. The obtained hemofilter had excellent membrane performance and antithrombotic properties, and had good air release.

(Example 2) The following operation was carried out in the same manner as in Example 1, except that the chitosan solution was adjusted to pH 6.30. The evaluation results are shown in Table-1 and Table-2.

(Example 3) The same operation as in Example 1 was performed except that the chitosan solution was adjusted to pH 3.50. The evaluation results are shown in Table-1 and Table-2.

(Example 4) (1) According to (1) to (4) of Example 1, a porous chitosan-coated module was obtained.

■ 1g of commercially available heparin in phosphate buffer (pH 6,9) 5
00ml and transfer this solution to the module 2 above.
After 4 hours of circulation, the procedure of Example 1 was followed. Table of evaluation results
1. Shown in Table-2.

(Comparative Example 1) Example 3 in the prior art (Japanese Unexamined Patent Publication No. 58-147404) was followed. However, since a hollow membrane base material made of an acidic group-containing PAN copolymer was used instead of the polyethylene tube, the introduction of a negative charge due to an oxidation reaction was omitted. Commercially available polyethyleneimine (manufactured by Tokyo Kasei Co., Ltd.) was added to boric acid buffer (PH8,9) 500mf to a concentration of 0.1%.
The base material was coated with a solution dissolved in fi, and the following operations were carried out in the same manner as in steps (1) to (2) of Example 1.

The evaluation results are shown in Table-1. It can be seen that the membrane performance deteriorates and does not reach a practical level.

(Comparative Example 2) Example 1 in the prior art (Japanese Unexamined Patent Publication No. 1-45373)
．． 4 and 6 were followed. However, since a hollow fiber membrane base material made of an acidic group-containing PAN copolymer was used as the polymer article, the introduction of a negative charge through surface treatment was omitted. The substrate was coated with a 0.6 wt% chitosan solution in a 1% aqueous acetic acid solution, dried, and washed sequentially with IM ammonium hydroxide and distilled water. A 0.2M phosphate buffer having a pH of 7.0 containing 1% heparin and 1% sodium cyanoborohydride was passed through this for 3 hours, and after washing with water, the same operations as 1 to 2 in Example 1 were performed.

The evaluation results are shown in Table-1. It can be seen that the membrane performance deteriorates and does not reach a practical level.

(Reference) The bovine blood UFR using only membrane material is 201, bovine Il [L
SC of plasma β2 MG is O (Effects of the Invention) The antithrombotic hemofilter of the present invention has antithrombotic properties and high membrane performance. Also, air escape during brimming is good.

The Hesen filter of the present invention is used in normal hemofiltration therapy, but because of its good antithrombotic properties, it is particularly useful in continuous hemofiltration therapy where it is used continuously for a long time. It can also be used for hemofiltration and dialysis therapy.

Claims (1)

[Claims] An antithrombotic hemoglobin comprising a membrane base material made of an acidic group-containing polyacrylonitrile copolymer, a porous chitosan layer coated on the surface of the membrane base material, and heparin bonded to the layer. filter.