JP2850086B2 - Exothermic substance adsorbent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pyrogen-containing adsorbent containing bile acid as an active ingredient.

[0002]

2. Description of the Related Art Pyrogenic substances (pyrogen, Pyrog)
en) is generally bacterial and is referred to as a bacterial toxin, and is an exotoxin (Exotoxin)
And endotoxin (Endotoxin, Endotoxin
). Among these toxins, cell wall phospholipid polysaccharides of gram-negative bacteria (lipopolysaccharide, L
Polysaccharides (PS) and phospholipids (lipid A, Lipi) constituting ipopolysaccharide (LPS)
dA), and it has been revealed that the exothermic active center is lipid A. Conventionally, as a method for removing the exothermic substance, a method of physically adsorbing or a method of chemically decomposing are known. Among them, in the physical adsorption method, there is little possibility that the main drug is decomposed, and even as an operation method, It is simple, and an ion exchanger adsorption method or the like is used. However, these methods have problems in adsorption performance, and it is difficult to completely remove exothermic substances. In addition, this pyrogen is removed under physiological conditions when mixed into the formulation due to its heat resistance and chemical stability,
Inactivation was extremely difficult.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a technique useful for adsorbing and removing exothermic substances in view of such circumstances.

[0004]

Means for Solving the Problems In order to solve such problems, the inventors of the present invention have studied the removal and inactivation of pyrogenic substances under physiological conditions, and as a result, have completed the present invention described below.

[0005] That is, the present invention relates to an exothermic substance adsorbent comprising an immobilized bile acid.

The bile acids used in the present invention are classified according to their structures as follows: 1) monohydroxycholanoic acid; for example, lithocholic acid 2) monohydroxycholenic acid; for example, 3β-hydroxy-Δ ⁵ -cholenic acid 3 ) Monooxocholanic acid; for example, 3-oxo-5β-cholanic acid 4) dihydroxycholanic acid; for example, ursodeoxycholic acid, deoxycholic acid 5) monohydroxymonooxocholanic acid; for example, 3α
-Hydroxy-6-oxo-5α-cholanic acid 6) dioxocholanic acid; for example, 3,12-dioxo-5β-cholanic acid 7) trihydroxycholanic acid; for example, cholic acid 8) dihydroxymonooxocholanic acid; for example, 3α,
7α-dihydroxy-12-oxo-5β-cholanic acid 9) Tetrahydroxycholanic acid; for example, 3α, 7α,
12α, 23-Tetraoxy-5β-cholanoic acid These classified bile acids are a kind of steroid compounds, and have a basic skeleton of 24 carbon atoms of colanic acid, and are oxy derivatives of the colanic acid. It has 1 to 4 hydroxyl groups or keto groups on the side chain and is mostly a natural product, but is also partially synthesized and is metabolized and decomposed in vivo. Some bile acids are used as pharmaceutical raw materials and can be said to be extremely safe substances.

[0007] The exothermic substance adsorbent of the present invention utilizes the affinity between bile acids and exothermic substances. Bile acids are supported on a carrier and immobilized. By supporting and immobilizing the bile acid on the carrier in this way, it can be useful for adsorption removal of pyrogenic substances. As the carrier to be used, any carrier may be used as long as the above-mentioned bile acid is bound directly or via a spacer. A typical example of such a carrier is a water-insoluble carrier having a hydroxyl group, an amino group, a carboxyl group, or a halogen atom. The method of selecting the water-insoluble carrier and the method of binding to the bile acid as a ligand are not particularly limited, and can be performed by an existing method. After the adsorbent of the present invention is brought into contact with the exothermic substance-containing solution, when the adsorbent and the solution are separated, the exothermic substance is favorably adsorbed on the adsorbent side to obtain a solution containing no exothermic substance. be able to. Examples of the method of removing and removing a pyrogenic substance from a pyrogenic substance-containing solution using the adsorbent of the present invention include a batch method and a column method.
The former is to mix the exothermic substance adsorbent of the present invention and the exothermic substance-containing solution, and sufficiently stirred by a round mixer,
This is a method of obtaining a solution containing no exothermic substance by separating the exothermic substance adsorbent and the solution.The latter is a method of filling the exothermic substance adsorbent of the present invention into a column and passing the exothermic substance-containing solution. This is a method for obtaining a solution containing no exothermic substance.

[0008]

EXAMPLES The present invention will be specifically described based on examples, but the present invention is not limited to these examples. The measuring method used in the examples is as follows. 1. Bile acid measurement method When the coenzyme NAD and 3α-hydroxysteroid dehydrogenase (3α-HSD) act on bile acid, the hydroxyl group at the 3α-position of bile acid is specifically oxidized to 3-ketosteroid and simultaneously to NADH. The generated NADH
Reduces nitrotetrazolium blue (NTB) via diaphorase to produce magenta diformazan. The content of total bile acids in the sample is determined by measuring the absorbance of the purple-red color. (Total bile acid-Test Wako: From the manual) [References: Fumiko Maeshi, Manabu Yamanaka: Clinical Chemistry, 8, 191 (1)
985)] 2. Method for measuring endotoxin by endotoxin colorimetric method Toxicolor (manufactured by Seikagaku Corporation) was used as a measuring reagent. This reagent was prepared from a chromogenic synthetic substrate and a horseshoe crab blood cell extract based on the method of Iwanaga et al. Endotoxin can be quantified by the same method. (Literature; Tamunori Obayashi et al .: Clinical Pathology, 33, 639-644, 198)
5)

Production Example 1 Amino-Cellulofine (porous spherical cellulose which was activated by epoxy and then used as an aminated carrier) was sufficiently washed with pyrogen-free water on a glass filter and filtered. 4 per gram of wet weight)
After mixing and shaking with 5 ml of ursodeoxycholic acid solution (previously dissolved in a weak alkaline solution to adjust to pH 9) and reacting at room temperature for 2 hours, dimethylaminoborane (3.
0.5 mg of a solution containing 5 mg) with shaking,
The reaction was performed at 4 ° C. for 18 hours. The solution was filtered on a glass filter to separate the carrier and the solution, and sufficiently washed using a 50 mM phosphate buffer (pH 7.2). An exothermic substance adsorbent having ursodeoxycholic acid as a ligand was obtained on the glass filter. The ursodeoxycholic acid content of this product was 7.3 μmole / g (wet weight) adsorbent. However, the content of the ligand compound in the product was determined from the difference in the bile acid enzyme colorimetric method between the ligand compound solution before the reaction and the washing solution (including the filtrate) of the product. The same shall apply hereinafter.

Example 1 20 ml of the exothermic substance adsorbent prepared in Production Example 1 was dispersed in 100 ml of a pyrogen-free 1 M (molar) saline solution, filled in a column, and allowed to pass through 100 ml of a 0.1 N / sodium hydroxide solution. And then equilibrate with 10 mM sodium acetate buffer (pH 7.2). Next, E. co
li UKT-B, LPS of control 881 was replaced with pyrogen-free human-derived albumin (the body temperature rise per bird was 0.2 ° C or less in a fever test based on biologics) and the concentration of albumin in pyrogen-free water was 2%. And 370 ng of the LPS was added to make 100 ml. This solution was passed through a prepared column (parallelizing the exothermic substance adsorbent with a buffer solution), and the albumin solution was recovered. LPS was not detected. Further, the adsorbent was washed by passing through a 10 mM sodium acetate buffer solution (pH 7.2). However, LPS was not detected in the recovered washing solution. Next, 1 M saline solution was passed through the column to collect the effluent. LPS was detected at 369 ng.

Production Example 2 Production Example 1 is the same as Production Example 1, except that EAH-Sepharose 4B (a carrier prepared by epoxy-activating sepharose gel and covalently bonding 1,6-diaminohexane) is used instead of amino-cellulofine. The processing is performed in the same manner as 1. Ursodeoxycholic acid content of this product is 5.6 μm
ole / g (wet weight) adsorbent.

Example 2 20 ml of the exothermic substance adsorbent prepared in Production Example 2 was dispersed in 100 ml of a pyrogen-free 1 M (molar) saline solution, and the mixture was packed in a column.
And then equilibrate with 5 mM sodium acetate buffer (pH 7.2). Next, E. coli
UKT-B, LPS of control 881 was replaced with a pyrogen-free human-derived albumin (the body temperature rise per bird was 0.2 ° C or less in a fever test based on the product formulation) and 2% albumin concentration in pyrogen-free water. And 430 ng of the LPS was added to make 100 ml. This solution was passed through a prepared column (equilibrated with a pyrogen adsorbent with a buffer solution) to recover an albumin solution.
LPS was not detected. Further, the adsorbent was washed by passing through a 5 mM sodium acetate buffer (pH 7.2). However, LPS was not detected in the recovered washing solution.
When the saline solution was passed through the column and the effluent was collected, LP
429.6 ng of S was detected.

Production Example 3 The procedure of Production Example 1 was repeated except that cholic acid was used instead of ursodeoxycholic acid. The product had a cholic acid content of 6.0 μmole / g (wet weight) adsorbent.

Example 3 25 ml of the exothermic substance adsorbent prepared in Production Example 3 was dispersed in 125 ml of a pyrogen-free 1 M (molar) saline solution, and the mixture was packed in a column.
5 ml, and 10 mM phosphate buffer (pH
Equilibrate in 6.5). Next, E. E. coli 055 LPS was diluted with pyrogen-free human-derived albumin (the body temperature rise per bird was 0.2 ° C. or less in a fever test based on biological preparations) by diluting albumin concentration to 2% with pyrogen-free water. Add 85ng LPS and 50ml
And This solution was passed through a prepared column (equilibrated with a pyrogen adsorbent with a buffer solution) to recover an albumin solution. As a result, 90% of LPS was adsorbed and removed.

Example 4 1 ml of the exothermic substance adsorbent prepared in Production Example 2 was dispersed in 5 ml of a pyrogen-free 1 M (molar) saline solution and packed in a column, followed by 15 ml of a 0.1 N / sodium hydroxide solution.
And further pass through a 10 mM phosphate buffer (pH 7.
Equilibrate in 0). Next, a 10 mM phosphate buffer aqueous solution (pH 7.0) containing a pyrogen (LPS) derived from various bacteria was passed through a column (a pyrogen-adsorbent was equilibrated with a buffer), and the pyrogen was added. Flow down until is detected. Further, a 10 mM buffer solution (pH 7.0) is allowed to flow down, and washing is performed until no pyrogenic substance is detected. In order to elute the adsorbed exothermic substance from the exothermic substance adsorbent, a 1 M saline solution was allowed to flow down, the solution was recovered, and the amount of the exothermic substance was measured. Table 1 shows the results.

[0016]

[Table 1]

Embodiment 5 FIG. 1 ml of the exothermic substance adsorbent prepared in Production Example 2 was dispersed in 5 ml of a pyrogen-free 1 M (molar) saline solution, packed in a column, passed through 15 ml of a 0.1 N / sodium hydroxide solution, and further subjected to 5 mM phosphate buffer. Equilibrate with liquid (pH 7.0). Next, a 5 mM phosphate buffer aqueous solution (pH 7.0) containing a pyrogen (LPS) derived from various bacteria was passed through a column (a pyrogen-adsorbent was equilibrated with a buffer), and the pyrogen was added. Flow down until is detected. Further, a 5 mM phosphate buffer solution (pH 7.0) is allowed to flow down and the washing is caused to flow down until no pyrogenic substance is detected. In order to elute the adsorbed exothermic substance from the exothermic substance adsorbent, a 1 M saline solution was allowed to flow down, the solution was recovered, and the amount of the exothermic substance was measured. Table 2 shows the results.

[0018]

[Table 2]

[0019]

As described above, according to the present invention, it is possible to provide a technique useful for removing and adsorbing exothermic substances, and it is difficult to use a conventional physical adsorption method or a chemical decomposition method as a method for removing exothermic substances. It is difficult to completely remove pyrogens due to problems in adsorption performance, and the pyrogens may be mixed under physiological conditions when mixed in the preparation due to their heat resistance and chemical stability. Removal and inactivation was extremely difficult,
The present invention has solved these problems.

Claims (2)

(57) [Claims] 1. An exothermic substance adsorbent comprising an immobilized bile acid. 2. The exothermic substance adsorbent according to claim 1, wherein the bile acid is supported on a water-insoluble carrier having a hydroxyl group, an amino group, a carboxyl group or a halogen atom.