JPH0819736A - Desorbing agent for endotoxin - Google Patents

Abstract

PURPOSE:To desorb endotoxin which is harmful for living bodies by using a water-insoluble solid body prepared by solidifying an organosilicon quaternary ammonium salt with a specified structure as an desorbing agent for endotoxin. CONSTITUTION:A water-insoluble solid body prepared by solidifying an organosilicon quaternary ammonium salt expressed by a formula; R<1>3N<+>R<2> SiR<3>nX3-n.Y<-> (wherein X stands for a halogen atom, an alkoxy group or so which can be hydrolyzed, Y for chlorine atom or bromine atom, R<1> for the same or a different univalent aliphatic hydrocarbon group with 1-22 carbon number, R<2> for divalent aliphatic hydrocarbon group, R<3> for a lower alkyl group with 1-3 carbon atoms a phenyl group, etc., and n for 0, 1, or 2) is used as an effective ingredient of a desorbing agent for endotoxin. The desorbing agent can effectively desorb endotoxin which is harmful for living bodies.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an endotoxin adsorption remover and an adsorption removal method using the same. More specifically, the present invention relates to an endotoxin adsorption-removing agent for removing endotoxin from a system containing endotoxin, which is harmful to organisms, and an adsorption-removing method using the same.

[0002]

BACKGROUND OF THE INVENTION Endotoxin is a lipopolysaccharide and its protein complex, which are chemically constituents of the cell surface of Gram-negative bacteria, and when they enter the body of a mammal, they produce fever and It is a toxic substance. With the progress of bioreactor, cell culture, various infusion and blood transfusion therapies targeting organisms and biological components, it is desirable to remove endotoxin that may be contained in the fluids used in these techniques. It is rare. Further, diseases associated with endotoxin existing locally in the living body are also known, and in this case, removal of endotoxin is also desired.

[0003] Conventionally, as a method for removing or inactivating endotoxin, treatment with heat, acid, alkali, or a kind of surfactant has been known, but most of these methods are laborious and useful. It is also possible to deactivate the substance. Therefore, in recent years, an ultrafiltration membrane (Japanese Patent Laid-Open No. 4-2
No. 2491) and a ligand having an affinity for endotoxin, for example, an insoluble carrier having a nitrogen-containing heterocyclic compound immobilized thereon (Japanese Patent Laid-Open No. 183712/1982) has been attempted. There are many things that still have problems to be solved in terms of ability and price.

On the other hand, some of the water-insoluble solids having the organosilicon quaternary ammonium salt immobilized have bactericidal activity, and their bactericidal properties are different in the alkaline chain length in the drug and the kind of test bacteria. It depends on (J.
Antibact. Antifung. Agents Vol. 19, p53-59, 1991)
Has been reported. There is also a report on a composition for oral cavity, which is intended to be stably blended without losing the bactericidal property and to exhibit an excellent plaque control effect (Japanese Patent Laid-Open No. 5-310545). As described above, regarding the water-insoluble solid on which the organosilicon quaternary ammonium salt is immobilized, research on bactericidal activity and stable formulation thereof has been conducted, but research on adsorption removal of endotoxin has not yet been conducted.

Therefore, an object of the present invention is to provide an endotoxin adsorption / removal agent capable of effectively adsorbing and removing endotoxin. Another object of the present invention is to provide an endotoxin adsorption removal method for efficiently removing endotoxin using this adsorption removal agent.

[0006]

Means for Solving the Problems As a result of intensive studies conducted by the present inventors in view of such circumstances, the mechanism of action thereof is not clear, but a water-insoluble solid on which an organosilicon quaternary ammonium salt is immobilized is not found. The inventors have found that the endotoxin-containing system adsorbs the endotoxin extremely effectively, and completed the present invention.

That is, the gist of the present invention is: (1) Adsorption of endotoxin characterized in that it contains a water-insoluble solid having an organosilicon quaternary ammonium salt represented by the general formula (I) immobilized thereon as an active ingredient. removing _{^{agent, R 1 3 n + R 2}} SiR 3 nX 3-n · Y - (I) ( wherein, X is a halogen atom, an alkoxy group, hydrolyzable group such as an acyl group, Y represents a chlorine atom Or a bromine atom, R ¹ is the same or different monovalent aliphatic hydrocarbon group having 1 to 22 carbon atoms, R ² is divalent hydrocarbon group, and R ³ is lower alkyl having 1 to ³ carbon atoms. Group, a phenyl group, or CF ₃ CH ₂ CH ₂ and n represents 0, 1, or 2.) (2) In the organosilicon quaternary ammonium salt represented by the general formula (I), three R ¹ Two of them are methyl groups and the other one is carbon number Is a ~ 22 alkyl group, wherein (1) the adsorption removal agent according, (3) the general formula (I)
In the organosilicon quaternary ammonium salt represented by, two of three R ¹ are methyl groups, the remaining one is an alkyl group having 16 to 20 carbon atoms, and R ² is an alkylene group having 2 to 4 carbon atoms. or -CH ₂ CH ₂ CH ₂ NH
CH ₂ CH ₂ — The adsorption-removing agent according to (1) above,
(4) The organosilicon quaternary ammonium salt represented by the general formula (I) is represented by the general formula (II),
An adsorption remover according to (1) above,

[0008]

Embedded image

(In the formula, R ¹ represents an alkyl group having 16 to 20 carbon atoms.) (5) A column is filled with the adsorbent / eliminator for endotoxin according to any one of (1) to (4) above. A method for adsorbing and removing endotoxin, which comprises conducting a containing solution through the column, (6) mixing and stirring the endotoxin adsorbing and removing agent according to any one of (1) to (4) above, and then stirring the mixture. A method for adsorbing and removing endotoxin, which comprises separating the endotoxin adsorption-removing agent, and (7) bringing the endotoxin adsorption-removing agent according to any one of (1) to (4) above into contact with an object surface to which endotoxin adheres. A method for adsorbing and removing endotoxin, which is characterized.

Hereinafter, the present invention will be described specifically. In the present invention, an organosilicon quaternary ammonium salt is immobilized and used. The organosilicon quaternary ammonium salt has the general formula _{^{(I): R 1 3 N}} + R 2 SiR 3 nX 3-n · Y - in known compound represented by (I), is commercially available as a normal alcohol solution .

In the formula, X is a halogen atom, an alkoxy group,
A hydrolyzable group such as an acyl group, preferably-
OCH ₃ or a -OCH ₂ CH _3. Y is a chlorine atom or a bromine atom, which may be either but is preferably a chlorine atom. R ¹ is the same or different and has 1 to 2 carbon atoms
It is preferable that it is a monovalent aliphatic hydrocarbon group of 2, particularly two of three R ¹ are methyl groups and the other one is an alkyl group having 8 to 22 carbon atoms, and further three R ¹
Two of them are methyl groups and the other one has 16 to 2 carbon atoms
More preferably, it is an alkyl group of 0. R ² is a divalent hydrocarbon group, and particularly preferably an alkylene group having 2 to 4 carbon atoms or —CH ₂ CH ₂ CH ₂ NHCH ₂ CH ₂ —. R ³ has 1 to 3 carbon atoms such as a methyl group
Lower alkyl group, phenyl group, or CF ₃ CH ₂ C
Represents H ₂ . n represents 0, 1 or 2, and preferably 0
Is.

Among these organosilicon quaternary ammonium salts, those represented by the general formula (II) are preferred, but the present invention is not limited thereto.

[0013]

Embedded image

Here, R ¹ is preferably an alkyl group having 16 to 20 carbon atoms.

In the present invention, the water-insoluble solid on which the organosilicon quaternary ammonium salt is immobilized may be any water-insoluble solid including inorganic substances and organic substances, and the solid may be synthetic or natural. Good.
Examples thereof include metals, metal oxides and carbonates, siliceous substances, resins and plastics, cellulose substances, chitin / chitosan substances and the like. Examples of particularly preferred water-insoluble solids include metals, metal oxides and metal carbonates such as iron, aluminum, copper, nickel, titanium dioxide, alumina, zinc oxide, magnesium oxide, iron oxide, calcium carbonate, phosphoric acid. Calcium hydrogen and magnesium carbonate; as siliceous substances such as glass, silica, diatomaceous earth, ground quartz, mica, zeolites, aluminum silicates, aluminum calcium silicates, magnesium silicates and zirconium silicates; as resins and plastics, such as polyesters, Polyamide, cellulose acetate, rayon, polystyrene,
Polyethylene, polypropylene, epoxy resins, phenolic resins, silicone resins, and polycarbonate resins; cellulosic materials such as wood, cotton, and hemp; chitin-chitosan materials such as chitin-chitosan polypolymers. Other natural organic substances include, for example, silk and wool. The particle size of these water-insoluble solids is usually 10-25.
Those having a thickness of 0 μm, preferably 50 to 200 μm are used.

The organosilicon quaternary ammonium salt can be immobilized on the water-insoluble solid by a method known per se. For example, the water-insoluble solid can be coated by dipping or spraying with a solution of an organosilicon quaternary ammonium salt and then immobilized by drying the coating material or heating after the coating step. The treatment solution used here is preferably an aqueous or organic solvent solution of an organosilicon quaternary ammonium salt. Such processing solutions are readily prepared by adding the organosilicon quaternary ammonium salt to water or an organic solvent such as methanol, ethanol or hexane. The concentration of the organosilicon quaternary ammonium salt in the treatment solution is 0.25 to 10
Weight% gives satisfactory results.

After applying the treatment solution to the water-insoluble solid, the surface of the treated solid is air-dried or heated to immobilize a coating of organosilicon quaternary ammonium salt on the surface of the solid. When this treated solid is heated at 65 to 100 ° C. for several minutes, the coating film of the organosilicon quaternary ammonium salt is more firmly fixed on the surface of the solid. If desired
An ordinary silanol condensation catalyst (for example, water) can be added to the treatment solution to enhance the silanol condensation property and improve the immobilization rate on the surface. The amount immobilized on a water-insoluble solid is
0.1 to 100 μmole / g.

Since the water-insoluble solid (hereinafter also referred to as an adsorbent) on which the organosilicon quaternary ammonium salt obtained as described above is immobilized effectively adsorbs endotoxin, the adsorbent can be adsorbed on a system containing endotoxin. After adsorbing endotoxin to the adsorbent by contacting the adsorbent, by separating the adsorbent from the system or removing the adsorbent from the system,
Endotoxin can be effectively removed from the system. The adsorption treatment of endotoxin is usually performed at 0 to 70 ° C, preferably 20 to 50 ° C, and the pH of the treatment liquid is usually preferably 3 to 9.

As a specific method for adsorbing and removing endotoxin on the adsorbent, when the endotoxin-containing system is a solution containing endotoxin, a usual column method or batch method can be mentioned. 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, the endotoxin-containing solution is added to the adsorbent and mixed, and the mixture is stirred to adsorb the endotoxin to the adsorbent, and then the adsorbent is separated from the solution by filtration or centrifugation of the solution. As a result, a solution containing no endotoxin is obtained.

When the system containing endotoxin is local in the body or on the surface of an object to which endotoxin is attached, the solution or composition containing an adsorbent is brought into contact with the system to adsorb and remove endotoxin. be able to. In this case, during or after contact,
By using a mechanical method such as water washing or using a brush, cloth, non-woven fabric, paper, etc., the adsorption rate of endotoxin can be increased to increase the removal efficiency, and the treated adsorbent can be removed from the system. Further, in the present invention, the adsorbent can be used by impregnating it with brush bristles, cloth, non-woven fabric, paper, etc., kneading it with fibers or adsorbing it on the fiber surface.

Examples of the composition include ointment, pasta,
It can be prepared by a known method in the form of paste such as cream, liquid such as rinse, or gum. In this case, the adsorbent is used alone or in combination of two or more, and 0.001 to 50% by weight, preferably 0.01 to 20% by weight of the organosilicon quaternary ammonium salt-immobilized water-insoluble solid based on the entire composition It is blended so that it becomes%. If the blending amount is less than this range, a sufficient endotoxin adsorption / removal effect cannot be obtained, and if it is more than this range, the properties of the composition become unstable. Further, other compounding ingredients are not particularly limited, and those which are usually compounded in a composition of this kind can be appropriately used, including medicinal agents such as bactericides and perfumes.

The endotoxin adsorption-removing agent of the present invention is
It means the adsorbent itself, a solution containing the adsorbent, or a composition containing the adsorbent as described above. The origin of the endotoxin adsorbed in the present invention is not particularly limited, and endotoxins derived from various microorganisms such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Salmonella typhi can be adsorbed and removed. Therefore, the adsorption removing agent of the present invention, for example, water treatment, preparation of endotoxin-free water, infusion, removal of endotoxin from blood for transfusion, removal of endotoxin from foods and beverages, various containers and tubes, medical /
Used in applications such as removal of endotoxin from research equipment.

[0023]

EXAMPLES The present invention will be described more specifically below by showing experimental examples and examples, but the present invention is not limited to these. Unless otherwise specified,% is weight%
Represents

Experimental Example 1 Using three kinds of organosilicon quaternary ammonium salts having different R ¹ alkyl groups in the general formula (II) immobilized on silica, the endotoxin adsorption / removal effect was as follows. Evaluated. Table 1 shows the results.

(1) Preparation of Organosilicon Quaternary Ammonium Salt-Immobilized Silica In the general formula (II), using organosilicon quaternary ammonium salts having 1, 8 and 16 carbon atoms, 20% silicic acid suspension is used. Approximately 0.2% by weight of the organosilicon quaternary ammonium salt per dry weight was added to the suspension to form a uniform water suspension. This suspension was spray-dried at 120 ° C. to give organosilicon quaternary ammonium salt-immobilized silica (immobilization amount: 4 μmole).
/ G) was prepared and used as a sample.

(2) 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. The cells obtained were washed with sterile physiological saline, and then washed with Westpha
l The warm phenol method (Methods Carbohyd. Chem. 5, 8
3-91, 1965) to extract lipopolysaccharide. Pyrogen-free distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a predetermined concentration (1 μm)
(g / ml) to prepare a test endotoxin solution.

(3) Evaluation of Adsorption and Removal Effect of Endotoxin (Batch Method) 0.02 g of the sample prepared in (1) above was placed in a 100 ml Erlenmeyer flask, and 18 ml of pyrogen-free distilled water was added.
Was added and dispersed well. This Erlenmeyer flask is 37 ℃
The test endotoxin solution (2 ml) prepared in (2) above was added to the thermostatic bath at 30 strokes at 120 strokes / minute.
Shake for minutes. 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). As a control, the same experiment was conducted using silica on which organosilicon quaternary ammonium salt was not immobilized.

The endotoxin adsorption removal rate (%) of the sample was calculated by the following formula. Endotoxin adsorption removal rate (%) = {(initial endotoxin content)-(endotoxin content in filtrate after 30 minutes contact)} / (initial endotoxin content) × 100

[0029]

[Table 1]

As is clear from the results shown in Table 1, the three types of organosilicon quaternary ammonium salt-immobilized silica are
All of them showed excellent endotoxin adsorption / removal effect on all endotoxins extracted from 4 kinds of Gram-negative bacteria. The effect of adsorption and removal was highest when the alkyl chain of R ¹ was C16, and was highest in the order of C8 and C1. Surprisingly, the adsorptive removal effect was uniformly high regardless of the origin of the target endotoxin.

Next, as described above, the water-insoluble solid having the organosilicon quaternary ammonium salt immobilized thereon has been known to have bactericidal activity against microorganisms. In order to clarify the relationship with the endotoxin adsorption / removal effect in the invention, an experiment was conducted as follows.

(4) Preparation of test bacterial solution Each of the bacterial strains shown in Table 2 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. The obtained bacterial cells were washed with sterile physiological saline, and the bacterial concentration was about 10 ⁷ cells / m 2.
What was suspended in physiological saline so as to be 1 was used as a test bacterial solution.

(5) Evaluation of Effect of Adsorbing and Removing Microorganisms 4 g of the sample prepared in (1) above was placed in a 100 ml Erlenmeyer flask and 36 ml of sterilized water was added to sufficiently disperse the sample. This Erlenmeyer flask was placed in a constant temperature bath at 37 ° C., 4 ml of the test bacterial solution of (4) was added, and 120 strokes /
It was shaken for 30 minutes. 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 sterile physiological saline. Next, 0.1 ml of this diluted solution was spread on a brain heart infusion agar medium, and after culturing at 37 ° C. for 48 hours, the number of formed colonies was counted. Thereby, the viable cell count after contact with the sample for 30 minutes can be calculated. As a control, the same experiment was conducted using silica on which organosilicon quaternary ammonium salt was not immobilized.

The microbial adsorption removal rate (%) of the sample was calculated by the following equation. Microbial adsorption removal rate (%) = {(initial viable cell count)-(viable cell count after 30 minutes contact)} / (initial viable cell count) x 100

[0035]

[Table 2]

As is clear from the results in Table 2, when the bactericidal activity of the organosilicon quaternary ammonium salt-immobilized silica against the microorganism itself is evaluated as the effect of removing and adsorbing the microorganism, the effect is determined by the length of the alkyl group and the target. It depends on the strain. In some cases, the effect was similar to that of the control silica alone, and the effect of immobilizing the organosilicon quaternary ammonium salt was not observed.

From the above, it was revealed that the effect of the organosilicon quaternary ammonium salt-immobilized silica used in the present invention on microorganisms has nothing to do with the endotoxin adsorption / removal effect. That is, the endotoxin adsorption / removal effect in the present invention is due to the specific adsorption between the organosilicon quaternary ammonium salt-immobilized silica and the endotoxin, but the specific adsorption with the endotoxin results in the release of endotoxin. It is effective when present in the state of, and its effect is not clear if endotoxin remains present on the cell surface of Gram-negative bacteria.

Experimental Example 2 In the general formula (II), the alkyl group of R ¹ is C5 or C.
18 organosilicon quaternary ammonium salts immobilized on various carriers in the same manner as in (1) of Experimental Example (immobilized amount of organosilicon quaternary ammonium salt:
4 μmole / g) was prepared. Using these, the endotoxin adsorption-removal effect was evaluated by using the endotoxin derived from Esherichia coli IFO14571 in the same manner as in the experimental example. The results are shown in Table 3.

[0039]

[Table 3]

As is clear from the results shown in Table 3, Samples 1 to 1 to which the organosilicon quaternary ammonium salt was immobilized were
5, 7, 9 to 11 showed a clearly higher endotoxin adsorption / removal effect as compared with the control samples 12 to 15 containing only the non-immobilized carrier. The sample concentration of the sample on which the organosilicon quaternary ammonium salt was immobilized was 0.
It can be seen that a high adsorption removal effect is exhibited at a concentration of 001 to 50%. Further, the chain length of the organosilicon quaternary ammonium salt is higher in C18 than in C5, which shows the effect of adsorbing and removing endotoxin.

Experimental Example 3 In the organosilicon quaternary ammonium salt represented by the general formula (I), two of three R ¹ are methyl groups, the remaining one is an alkyl group having 20 carbon atoms, and R ² is −
_{CH 2 CH 2 CH 2 NHCH 2} CH 2 - and is, R ³ is -CH ₂ CH _3, X is -OCH ₂ CH _3, Y is Br, n
An adsorbent (an amount of immobilization: 10 μmole / g) in which cellulose is 1 is immobilized on a cellulose powder, and an inner diameter of 10 m
It was packed in a column of m and 100 mm in length. The immobilization was performed by the same method as in Experimental Example 1. Then Salmonel
100 ng of endotoxin derived from la typhymurium LT2
/ Ml of endotoxin-free 20 mM buffer (pH is 3, 4, 5, 6, 7, 8, 9 respectively) was prepared, and 50 ml of each was passed through the column and collected. The endotoxin content of the liquid was measured, and the endotoxin adsorption removal rate was calculated. As a result, pH 3 ~
A high endotoxin adsorption / removal effect of 99.9% or higher was observed at any pH of 8, and a 90% adsorption / removal effect was observed at pH 9.

Example 1 In the organosilicon quaternary ammonium salt represented by the general formula (II), R ¹ is an alkyl group having 18 carbon atoms and OCH ₃ is replaced with OC ₂ H ₅ to obtain a silicone resin. It was immobilized (immobilization amount: 6 μmole / g). Approximately 3 of this on a nylon filament surface with a diameter of 0.5 mm.
%, And a brush for adsorbing and removing endotoxin was prepared by coating this at a ratio of%. The immobilization was performed by the same method as in Experimental Example 1.

Example 2 Surfactants and fragrances in concentrations normally used as cleaning liquids,
A cleaning liquid for adsorbing and removing endotoxin was prepared by mixing the adsorbent of Experimental Example 3 with a liquid containing other components at a ratio of 10%.

[0044]

EFFECTS OF THE INVENTION By using the adsorption / removal agent of the present invention, endotoxin harmful to organisms can be effectively adsorbed and removed.

Claims (7)

[Claims] 1. An endotoxin adsorption-removing agent comprising a water-insoluble solid having an organosilicon quaternary ammonium salt represented by the general formula (I) immobilized thereon as an active ingredient. _{^{R 1 3 N + R 2 SiR}} 3 nX 3-n · Y - (I) ( wherein, X is a halogen atom, an alkoxy group, hydrolyzable group such as an acyl group, Y represents a chlorine atom or a bromine atom R ¹ is the same or different monovalent aliphatic hydrocarbon group having 1 to 22 carbon atoms, R ² is divalent hydrocarbon group, R ³ is lower alkyl group having 1 to ³ carbon atoms, phenyl Represents a group or CF ₃ CH ₂ CH _2. n represents 0, 1 or 2.) 2. The organosilicon quaternary ammonium salt represented by the general formula (I), wherein two out of three R ¹ are methyl groups and the remaining one is an alkyl group having 8 to 22 carbon atoms. Item 1. An adsorption remover according to item 1. 3. In the organosilicon quaternary ammonium salt represented by the general formula (I), two of three R ¹ are methyl groups and the other one is an alkyl group having 16 to 20 carbon atoms, and R ² is an alkylene group or -CH ₂ CH ₂ CH ₂ NHCH 2 to 4 carbon atoms ₂ CH ₂ - is the adsorption removal agent according to claim 1. 4. The adsorption remover according to claim 1, wherein the organosilicon quaternary ammonium salt represented by the general formula (I) is represented by the general formula (II). Embedded image (In the formula, R ¹ represents an alkyl group having 16 to 20 carbon atoms.) 5. A method for adsorptive removal of endotoxin, which comprises filling a column with the endotoxin adsorption removal agent according to claim 1, and conducting an endotoxin-containing solution through the column. 6. A method for adsorbing and removing endotoxin, which comprises mixing and stirring the endotoxin adsorption-removing agent according to claim 1 and an endotoxin-containing solution, and then separating the endotoxin adsorption-removing agent. 7. An endotoxin adsorption-removal method, which comprises contacting the endotoxin adsorption-removal agent according to any one of claims 1 to 4 with the surface of an object to which endotoxin adheres.