JPH07816A - Endotoxin adsorbent - Google Patents

Abstract

PURPOSE:To enable extensive applications to the therapeutic and prophylactic treatment of endotoxima as well as the refining and purification of water and an aqueous solution by adsorbing and removing endotoxin selectively. CONSTITUTION:The subject endotoxin adsorbent comprises cellulose with a particle diameter of 20 to 2000mum and an average pore diameter of 200 to 3000Angstrom , crosslinked polyacrylic acid, polyvinyl alcohol, polystyrene or their derivatives, the which chitosan having a molecular weight of 1000 to 20000 is immobilized within a range of 0.05 to 3.00meq/g of amino group content.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to the treatment and prevention of endotoxemia caused by Gram-negative bacterial infection by selectively adsorbing and removing endotoxin, which is a cell wall component of Gram-negative bacteria, or water contaminated with Gram-negative bacteria. The present invention relates to an adsorbent for the purpose of purifying and purifying an aqueous solution.

[0002]

2. Description of the Related Art Many gram-negative bacteria such as Escherichia coli, Salmonella, Shigella, Pseudomonas aeruginosa and Serratia marcescens are well known. The endotoxin corresponds to lipopolysaccharide, which is a cell wall component of these Gram-negative bacteria, and forms a macromolecule having a molecular weight of several hundred thousand to several million. Lipid A is its active center and has various biological activities such as lethal toxicity, pyrogenicity, immunoadjuvant activity, Schwartzman reaction, horseshoe crab blood cell component coagulation reaction, interferon inducing activity, and antitumor activity. It has a very important effect in the body.

The needs for selective removal of endotoxin have a wide range of fields including medical science and biochemistry. In the medical field, there is a cure and prevention of endotoxemia caused by Gram-negative bacteria, especially intestinal bacterial infection. During the course of Gram-negative bacterial infection, chills, fever, etc. are associated with rapid hypotension and peripheral circulatory insufficiency, which may cause so-called endotoxin shock. Large-scale administration of steroids or other anti-shock therapy has been attempted as a treatment method for such endotoxemia, but its success rate is extremely low.

In addition to blood purification, in the medical field, water for injection, physiological saline, glucose, which is directly administered into the living body,
Infusions or dialysis fluids containing nutrients such as vitamins are required to undergo not only sterilization but also strict removal of endotoxin. Also in the biochemical field, the same applies to, for example, water and nutrient solutions used for raising sterile animals.

Furthermore, the rapid progress of biotechnology technology in recent years has enabled mass production of many pharmaceuticals and various useful proteins by gene recombination technology. As a host for gene recombination, Escherichia coli is widely used because it is easy to culture, and in this field, strict removal of endotoxin is required for purification.

[0006] The production of sterile water and removal of bacterial cells from protein solutions and the like are usually carried out by filtration using a porous membrane, but endotoxin is not completely removed and other useful components are also removed at the same time. . Therefore, many attempts have been made for selective removal by adsorption using various carriers.

As an endotoxin adsorbent, in the area of blood purification, PMX-F, in which polymyxin B, which is a kind of antibiotic, is immobilized on polystyrene fiber which is a water-insoluble carrier
(Artificial organ 17 (2) 583-586 (1988)),
An adsorbent in which an organic low molecular weight compound containing an aromatic ring is immobilized on a cross-linked polymer containing polyvinyl alcohol as a main component (JP-A-59-22557) has been reported.
However, when polymyxin B is immobilized, there is a problem in safety when polymyxin B is desorbed in blood or in the treatment solution, and when an organic low-molecular compound containing an aromatic ring is immobilized, endotoxin adsorption There is a problem of low capacity. Further, in any case, when used for removing endotoxin in blood, there is a problem with blood compatibility, plasma separation operation and administration of a large amount of anticoagulant are necessary, and side effects such as bleeding tendency are caused. There are drawbacks. As another method for treating endotoxemia, for example, the effect of administration of an anti-endotoxin human monoclonal antibody has been reported, but safety and cost issues remain (Pharmacia 27 (9) 935-936 (1991). )).

As an endotoxin adsorbent for purification in water, an aqueous solution, etc., Pyro Sep (Bioscience and Industry 47 (2) 222-223 (198) manufactured by Daicel Chemical Industries, in which histidine is immobilized on sepharose with hexamethylenediamine as a spacer.
9)), Kurita Kogyo's Klimsorber II (JP-A-3-109940) in which porous chitosan is crosslinked, and many others. However, these are not sufficient in terms of adsorption specificity and versatility.

[0009]

On the basis of the above background, the present invention is excellent in adsorption performance and specificity or blood compatibility, and is applicable to blood purification, water, aqueous solution or pharmaceutical purification. It is intended to provide a wide range of adsorbents for endotoxin.

[0010]

The endotoxin adsorbent of the present invention, which has achieved the above object, is characterized in that chitosan is immobilized on a water-insoluble carrier. Further, the endotoxin adsorbent of the present invention has an amino group content depending on the purpose,
It can be applied in a wide range by controlling the particle size, the pore size or the filling amount.

Examples of the water-insoluble carrier used in the present invention include cellulose, crosslinked polyacrylic acid, polyvinyl alcohol, polystyrene and derivatives thereof. Of these, cellulose is particularly preferable because of its adsorption specificity and ease of immobilization reaction. The shape may be a fibrous shape, a membrane shape, a hollow fiber shape or the like, but a particle shape is preferable from the viewpoints of securing a surface area and easiness of handling during preparation.

Chitosan, which has a high affinity for endotoxin as a ligand, is introduced into the above water-insoluble carrier.
Chitosan is obtained by deacetylating chitin, which is a constituent polysaccharide of outer shell of shellfish such as shrimp and crab, by heating with alkali. The molecular weight of chitosan is preferably 1000 to 20000 from the viewpoint of handling such as solubility when preparing the adsorbent, and 2000 to 10
000 is more preferable.

In addition, in order to improve the adsorption efficiency of endotoxin, the influence of the introduction rate of chitosan is also important. If the chitosan content of the adsorbent is low, the adsorption efficiency will not be sufficient,
On the other hand, if the amount is too large, the stability of the carrier gel decreases. The amino group content is preferably in the range of 0.05 to 3.00 meq / g, and more preferably 0.20 to 1.50 meq / g.

The average particle size of the adsorbent is 20
In particular, if the particle size is too small during blood purification, it is easily affected by blood cell components and pressure loss. On the contrary, if it is too large, the surface area becomes insufficient, and therefore 50 to 1000 μm. Is more preferable. Further, in the case of purifying water, an aqueous solution, etc., the range of 20 to 200 μm is more preferable.

Regarding the fine pores, those having an average fine pore diameter of 200 to 30,000 Å are preferable, but in the case of blood purification, the range of 300 to 3,000 Å is more preferable,
1000 to 2000 when purifying water, aqueous solution, etc.
The range of 0Å is more preferable.

The method of introducing chitosan into the water-insoluble carrier in the present invention is not particularly limited, such as covalent bonding and grafting by electron beam irradiation, but [cellulose]-[chitosan] as described in claim 4 is obtained. In this case, the following method is preferable. (1) Modification of Cellulose A solution of sodium periodate in 0.1 to 5.0 N, preferably 0.5 N in 1.5 N sulfuric acid has an average particle size of 20 to 2000 Å and an average pore size. Is 20
0 to 30000Å granular porous cellulose is added, and the reaction is carried out at 10 ° C to 50 ° C, preferably 20 ° C to 20 ° C for 5 hours to 30 hours, preferably 10 hours to 24 hours. The sodium periodate concentration of the sodium periodate-sulfuric acid solution is 2% by weight to 15% by weight, preferably 4% by weight to 10% by weight. The bath ratio of the granular porous cellulose to the sodium periodate solution is 10% by volume to 30% by volume, preferably 15% by volume to 25% by volume.

The reaction mixture is filtered to collect the purified product, which is washed thoroughly with water to give an aldehyde content of 0.1 in the swollen state.
0 to 4.00 meq / g, preferably 0.20 to 3.00 meq / g of aldehyde cellulose are obtained.
This aldehyde cellulose has a structure in which some glucose units are ring-opened, as shown in the partial structure in Chemical formula 1.

[0018]

[Chemical 1]

(2) Introduction of chitosan 1 in terms of polyethylene glycol (hereinafter referred to as PEG) by gel permeation chromatography (hereinafter referred to as GPC)
000 to 20000, preferably 2000 to 1
Chitosan of 0000 is dissolved in a buffer solution of pH 9.5, and the aldehyde cellulose obtained in the above (1) is added thereto and stirred at 10 ° C to 50 ° C, preferably 20 ° C.
The reaction is carried out at 30 ° C to 30 ° C for 5 to 30 hours, preferably 10 to 24 hours. The concentration of the chitosan-buffer is 0.2 wt% to 5.0 wt%, preferably 0.4.
From 3% to 3.0% by weight, the bath ratio of aldehyde cellulose to this solution is 3% to 20% by volume, preferably 5% to 15% by volume.

The reaction mixture is filtered to recover the product,
It is washed with water, dispersed in a buffer solution having a pH of 9.0, and sodium borohydride is added thereto at 10 ° C to 50 ° C, preferably 20 ° C to 30 ° C for 5 hours to 30 hours, preferably 10 hours to 24 hours. The Schiff base is hydrogenated by reacting for a time. The bath ratio of the Schiff-containing base [cellulose]-[chitosan] to the buffer is 3% to 20% by volume, preferably 5% to 15% by volume, and the Schiff-containing base [cellulose]-[chitosan] and hydrogenation are used. The charging ratio of sodium borohydride is 20/1 to 3/2, preferably 15/1 to 3
/ 1 (both volume / weight ratio). Thus, [cellulose] -having an amino group content of 0.05 to 3.00 meq / g, preferably 0.20 to 1.50 meq / g.
Obtain [Chitosan].

[0021]

EXAMPLES The present invention will be described below with reference to examples. Parts in the examples mean parts by weight unless otherwise noted. <Example 1> A solution prepared by dissolving 325 parts of sodium periodate in 5000 parts of 1N sulfuric acid, 1000 parts (volume) of several kinds of granular porous cellulose having a particle diameter of 20 to 2000 μm and an average pore diameter of 200 to 3000 Å. To 18 ° C., reacted at 25 ° C. for 18 hours, filtered and washed with water to obtain aldehyde cellulose having an aldehyde content of 0.5 to 1.4 meq / g (hereinafter referred to as CA-1).

Determination of aldehyde was carried out by the oxime method. That is, about 1.0 ml of CA-1 was accurately weighed (this amount is V1 ml), and 0.5 N hydroxylamine hydrochloride solution (hydroxylamine 35
35 g was dissolved in 160 ml of water and diluted with 95% ethanol to give 11). This suspension (hereinafter referred to as "Sample 1") was combined with 10 ml of 0.5N hydroxylamine hydrochloride solution and 35 ml of pyridine-bromophenol blue solution (hereinafter referred to as "blank") without adding CA-1 and ultrasonicated. It was left in the washer for 15 minutes. Sample 1 and the blank were taken out, and a 0.1N sodium hydroxide-methanol solution was added dropwise until the color exhibited by sample 1 became the same as the blank. When the amount of sodium hydroxide dropped at this time is W1 and the titer is F1, the aldehyde content x1 meq / g can be obtained by the following formula. x1 = (0.1 x F1 x W1) / V1

P according to GPC (Shimadzu LC-6A)
PH of 30 parts of chitosan with a molecular weight of about 5,000 converted to EG
The carbonate buffer solution of 9.5 was dissolved in 5000 parts (volume), 250 parts (volume) of CA-1 obtained above was added thereto, and the mixture was reacted at 25 ° C. for 18 hours while stirring. The reaction mixture was filtered to collect the product, washed with water,
Disperse in 5000 parts (volume) of carbonate buffer solution of H9.0,
Add 70 parts of sodium borohydride and add 18 at 25 ° C.
The Schiff base was hydrogenated by reacting for a period of time. Thus, the content of amino groups derived from chitosan is 0.10 to 1.2.
0 meq / g of [cellulose]-[chitosan] (hereinafter C
C-1).

The quantification of amino groups was carried out by the following method. About 0.1 ml of CC-1 was accurately weighed (this amount was V2 g), 10 ml of 0.1 N hydrochloric acid aqueous solution (titer was F2) was added, and this suspension was diluted with dioxane. Make up to 40 ml in total (this suspension is referred to below as Sample 2). After stirring Sample 2 for about 30 minutes, it is titrated with a 0.1 N aqueous sodium hydroxide solution (titer is F2 ') using an automatic titrator (COMITITE 101 manufactured by Hiranuma Sangyo). Assuming that the amount of 0.1N aqueous sodium hydroxide solution required to neutralize sample 2 is W2 ml, the amino group content x2 meq / g of CC-1 can be obtained by the following formula. V2 x x1 +0.1 x F2 'x W2 = 0.1 x F2 x 1
0

The adsorbent obtained by the above method was washed with physiological saline and then packed in a column having an inner diameter of 4 cm and a length of 8 cm, and the adsorbing performance was evaluated by supplying bovine blood to which endotoxin was added. did. Endotoxin is lipopolysaccharide B (Difco, E. coli
0111: B4 derived) was used. In addition, endotoxin is quantified using a Toxinometer ET201 (manufactured by Junko Wako)
Was performed by the Limulus method.

In the evaluation with bovine blood supplemented with endotoxin, the inside of the column and the blood circuit were washed with 150 ml of physiological saline containing 100 U / ml heparin, and subsequently with 150 ml of physiological saline containing 1 U / ml heparin. On the other hand, the citrate-added bovine blood 11 to which the above endotoxin was added at a concentration of 100 mg / ml was pooled to 5
The amount of endotoxin in blood after circulating for 2 hours at a flow rate of 0 ml / min was quantified.

The total amount of protein in blood before and after circulation,
The amount of albumin and the number of white blood cells were quantified. The results are as shown in Tables 1 and 2 below. The total protein amount was measured by the Lowry method, the albumin amount was measured by Albumin B Test Wako (manufactured by Wako Pure Chemical Industries), and the white blood cell count was measured by Coulter Counter ZM type (manufactured by Coulter Electronics). The endotoxin amount was shown as an actual measured value, and the total protein amount, albumin amount, and white blood cell count were shown as residual ratios.

Example 2 Bovine serum albumin (Wako Pure Chemical Industries, Ltd., hereinafter referred to as BSA) obtained by packing the adsorbent in a column having an inner diameter of 10 mm and a length of 200 mm and adding endotoxin.
The adsorption performance was evaluated by using the physiological saline solution of When the endotoxin-added BSA aqueous solution was evaluated, 500 mg of the above endotoxin was added.
BSA physiological saline solution 11 having a concentration of 1 mg / ml added at a concentration of 1 / ml was passed through at a rate of 1 ml / min, and the endotoxin concentration and albumin concentration of the eluate were quantified in the same manner as in Example 1. The results are shown in Table 3 below. The endotoxin amount was shown as the actual measured value, and the albumin amount was shown as the residual rate.

[0029]

[Table 1]

[0030]

[Table 2]

[0031]

[Table 3] In addition, N. D. Indicates that measurement is not possible, and the numerical values in Table 2 indicate the residual rate (%).

<Comparative Example 1> Granular porous cellulose having a particle diameter of 150 μm and an average pore diameter of 100 Å was processed by the method shown in Example 1 to have an aldehyde content of 0.05 meq / g and 1.2 meq / g (hereinafter referred to as CA. −
I got 2). Furthermore, chitosan was introduced into CA-2 in the same manner as in Example 1 to obtain [cellulose]-[chitosan] having chitosan-derived amino group contents of 0.02 meq / g and 0.50 meq / g. After the adsorbent thus obtained was washed with physiological saline, the adsorption performance of the endotoxin-added bovine blood was evaluated in the same manner as in Example 1. The results are as shown in Tables 1 and 2.

<Comparative Example 2> The adsorbent shown in Comparative Example 1 was
According to the method shown in Example 2, the above endotoxin-added B
The adsorption performance of the SA solution was evaluated. The results are shown in Table 3.
As shown in.

<Comparative Example 3> Granular porous cellulose having a particle diameter of 150 μm and an average pore diameter of 10000 Å was converted into aldehyde cellulose having an aldehyde content of 0.05 meq / g (hereinafter referred to as CA-3) by the method shown in Example 1. Obtained.
Furthermore, chitosan was introduced into CA-2 in the same manner as in Example 1, and the content of amino groups derived from chitosan was 0.02 meq.
/ G of [cellulose]-[chitosan] was obtained. After the adsorbent thus obtained was washed with physiological saline, the adsorption performance of the endotoxin-added bovine blood was evaluated in the same manner as in Example 1. The results are as shown in Tables 1 and 2.

<Comparative Example 4> The adsorbent shown in Comparative Example 3 was
According to the method shown in Example 2, the above endotoxin-added B
The adsorption performance of the SA solution was evaluated. The results are shown in Table 3.
As shown in.

[0036]

The endotoxin adsorbent according to the present invention has excellent endotoxin adsorption specificity. It has good blood compatibility and can be used for treatment or prevention of endotoxemia. It can also be used for purification of water and aqueous solutions, and can be expected to be applied not only in the medical field but also in a wide range of applications.

Front page continuation (72) Inventor Masakazu Tanaka, 1-1 1-1 Katata, Otsu City, Shiga Prefecture, Toyobo Co., Ltd.

Claims (4)

[Claims] 1. A water-insoluble carrier having a molecular weight of 1,000 to 20.
Chitosan with an amino group content of 0.05 to 3.
An endotoxin adsorbent that is immobilized in the range of 00 meq / g. 2. The endotoxin adsorbent according to claim 1, wherein the water-insoluble carrier is cellulose, crosslinked polyacrylic acid, polyvinyl alcohol, polystyrene or a derivative thereof. 3. The water-insoluble carrier has a particle size of 20 to 200.
The endotoxin adsorbent according to claim 1, wherein the adsorbent has an average pore size of 0 μm and an average pore size of 200 to 30,000 liters. 4. Aldehyde content of 0.1 to 4.0 me.
q / g cellulose has a molecular weight of 1,000 to 20,000
The endotoxin adsorbent according to claim 1, which is obtained by condensing an amino group of chitosan to form a Schiff base, and reducing this to immobilize chitosan.