JP3453623B2 - Endotoxin adsorption remover and removal method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an endotoxin adsorption / removal agent and an endotoxin adsorption / removal method for removing endotoxin from a system containing endotoxin harmful to organisms.

[0002]

2. Description of the Related Art Endotoxin is a lipopolysaccharide and its protein complex that are chemically constituents of the cell surface of Gram-negative bacteria and are substances that show fever and toxicity when they enter the body of mammals. Is. With the progress of technical fields targeting organisms and biological components, such as bioreactors, cell culture, various infusion and transfusion therapies, removal of endotoxin that may be contained in the fluids used in these techniques Is desired. Further, diseases associated with endotoxin existing locally in the living body are also known, and in this case, removal of endotoxin is also desired. Conventionally, as a method for removing or deactivating endotoxin, heat,
Treatment with an acid, an alkali, or a surfactant of some kind is known, but most of these methods are troublesome and may deactivate useful substances. Therefore, in recent years, an ultrafiltration membrane (JP-A-4-22491) and an insoluble carrier having a ligand having an affinity for endotoxin, for example, a nitrogen-containing heterocyclic compound (JP-A-57-18371).
Although the use of 2) and the like has been attempted, there are many problems that remain to be solved in terms of safety, removal capacity and price. On the other hand, a method of adsorbing and treating dyeing wastewater by using a mixture of an insoluble calcium salt and an aluminum compound and baking it (Japanese Patent Publication No. 58-894) has been disclosed, but no study on endotoxin adsorption and removal has been conducted yet. .

[0003]

[Means for Solving the Problems] The inventors of the present invention have made extensive studies in view of the above-mentioned circumstances, and as a result, the mechanism of action thereof is not clear. However, in water-insoluble calcium salts such as calcium carbonate, The present inventors have found that the endotoxin-containing system is extremely effectively removed from the system, and have completed the present invention.

The present invention will be specifically described below. Examples of the water-insoluble calcium salt of the present invention include calcium carbonate, calcium phosphate, and hydroxyapatite, which are easily available. Of these water-insoluble calcium salts, calcium carbonate is particularly preferable.

Since these water-insoluble calcium salts effectively adsorb endotoxin, the adsorbent is brought into contact with a system containing endotoxin to adsorb the endotoxin to the adsorbent, and then the adsorbent and the system are separated or the system is separated. The endotoxin can be removed from the system by removing the adsorbent from.

As a method of actually utilizing the adsorbent, when the system containing endotoxin is a solution containing endotoxin, a usual column method or batch method can be considered. For example, in the case of the column method, the adsorbent is packed in a column, washed with a salt solution, water, a buffer solution, etc., and then the endotoxin-containing solution is conducted. Obtained as a liquid. On the other hand, in the case of the batch method, an endotoxin-containing solution is added to the adsorbent, the mixture is stirred to adsorb the endotoxin to the adsorbent, and then the solution is separated by filtration, centrifugation, etc. Is obtained. Further, if the system containing endotoxin is local in vivo or is a solid surface to which endotoxin is attached, when a solution or composition containing an adsorbent is brought into contact with the system or after the system is mechanically treated, For example, by using water washing, brush, cloth, non-woven fabric, paper, etc.,
The efficiency of endotoxin removal can be increased, and the adsorbent after treatment can be removed from the system. Further, the adsorbent can be used by impregnating the bristles of brush, cloth, non-woven fabric, paper or the like, or by kneading into the fiber or adsorbing onto the fiber surface. As the composition, for example, ointment, pasta,
It can be made into a paste form such as cream, a liquid form such as rinse, and a gum form such as chewing gum by a known method, and the adsorbent may be used alone or in a combination of two or more kinds, and may be 0.001% based on the entire composition. ˜50% by weight, preferably 0.01% to 20% by weight. If the blending amount is less than 0.001% by weight, sufficient endotoxin removing activity cannot be obtained, and if it is more than 50% by weight, the properties of the composition become unstable. Other compounding ingredients are not particularly limited, and those which are usually compounded in this type of composition can be used including medicinal agents such as bactericides and perfumes.

Hereinafter, the present invention will be described more specifically by showing experimental examples and examples, but the present invention is not limited to these. Unless otherwise specified,% means% by weight. (Experimental Example 1) Three kinds of water-insoluble calcium salts of calcium carbonate, calcium phosphate, and hydroxyapatite, and PyroSe which is commercially available as an endotoxin adsorbent as a control.
using p C ^R and silica, adsorption sterilizing effect against endotoxin extracted from four gram negative bacteria shown in Table 1 was evaluated by the following experiments. (1) Preparation of test endotoxin Each strain shown in Table 1 was cultured in a brain heart infusion liquid medium at 37 ° C. for 24 hours and then centrifuged (7
The cells were collected at 000 rpm for 10 minutes, and the obtained cells were washed with sterile physiological saline. Westp from the fungus body
Hal et al.'s Warm Phenol Method (Methods Carb
ohyd Chem 5,83-91, 1965) lipopolysaccharide extracted with pyrogen-free distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.) to an initial endotoxin concentration of 1 g / ml was used as a test endotoxin solution. (2) Evaluation of Adsorption and Removal Effect of Endotoxin (Batch Method) A predetermined sample was placed in a 100 ml Erlenmeyer flask, and 18 ml of pyrogen-free distilled water was added and sufficiently dispersed. This Erlenmeyer flask was placed in a constant temperature bath at 37 ° C., 2 ml of the test endotoxin solution of (1) above was added, and
Shake for 30 minutes at stroke / min. Then, the Erlenmeyer flask was taken out from the constant temperature bath, the sample and the filtrate were separated by filtration, and 1 ml of the filtrate was diluted with pyrogen-free distilled water. The endotoxin content in this diluted solution was quantified using an endotoxin colorimetric quantification kit (Toxicolor System, Seikagaku Corporation). The endotoxin adsorption removal rate (%) of the sample was calculated by the following formula.

[0008]

[Formula 1]

The results are shown in Table 1.

[0010]

[Table 1]

As is clear from the results shown in Table 1, all of the three water-insoluble calcium salts exhibited excellent endotoxin adsorption / removal effects on all endotoxins extracted from four Gram-negative bacteria. The effect of adsorption and removal was found to be highest in calcium carbonate. Surprisingly, the adsorptive removal effect was uniformly high regardless of the origin of the target endotoxin.

(Experimental Example 2) Next, the effect of adsorbing and removing endotoxin by adding sodium chloride to a water-insoluble calcium salt was used in the same manner as in Experimental Example 1 using endotoxin derived from Esherichia coli IFO14571. And evaluated. Here, the amount of endotoxin in the initial stage was set to 100, and the ratio (%) of the amount of endotoxin in the filtrate after contact was determined. The results are shown in Fig. 1.

[0013]

[Figure 1]

As is clear from the results shown in FIG. 1, when the sodium chloride was added during the treatment, the endotoxin adsorption-removal activity was higher in a concentration-dependent manner as compared with the case where sodium chloride was not added. The concentration of sodium chloride added is 0.00
It can be seen that a high adsorption removal effect is exhibited at a concentration of 1% to 20%.

Example 1 Calcium carbonate, calcium phosphate, and hydroxyapatite were packed in a column having an inner diameter of 10 mm and a length of 100 mm, respectively. An endotoxin derived from Salmonella typhymurium LT2 was suspended in an endotoxin-free 20 mM buffer solution (pH: 3, 4, 5, 6, 7, 8, 9) at 100 ng / ml, and 50 ml of each suspension was prepared. The solution was passed through the column, the endotoxin content of the recovered solution was measured, and the endotoxin adsorption removal rate was calculated. As a result, a high endotoxin adsorption / removal effect of 99.9% or more was observed with any of the water-insoluble calcium salts.

Example 2 Calcium carbonate, calcium phosphate, and hydroxyapatite were coated on a surface of a nylon filament of 0.5 mm at a ratio of about 3% to prepare a brush for adsorbing and removing endotoxin.

Example 3 Surfactants and fragrances in concentrations normally used as cleaning liquids,
A cleaning liquid for removing endotoxin was prepared by adding the water-insoluble calcium salt of Example 1 in a ratio of 10% to a liquid containing other components.

[0018]

INDUSTRIAL APPLICABILITY According to the present invention, the endotoxin can be effectively removed from the system containing the endotoxin, which is harmful to the organism.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the concentration (%) of residual endotoxin and the concentration of sodium chloride.

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Claims (2)

(57) [Claims] 1. A calcium carbonate of a water-insoluble calcium salt .
An adsorbent / removal agent for endotoxin, comprising one or two selected from the group consisting of calcium and calcium phosphate as an active ingredient. 2. Adsorption removal of endotoxin, characterized in that during the treatment of the endotoxin adsorption removal agent according to claim 1, sodium chloride is added in an amount of 0.001 to 20.0% by weight. Method.