JP2773412B2 - Pyrogen adsorbent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a pyrogen adsorbent.

[Conventional technology and its problems]

Pyrogens are often contained in drugs derived from living organisms and biological components such as interferon, TNF, and SOD, or in filling fluids for medical devices such as blood purifiers, dialysis machines, and plasma separators. Cause an accident. It is also known that the pyrogen Endotoxin is a causative substance of DIC (general intravascular coagulation syndrome) and sepsis associated with MOF (multi-organ failure).

The most pyrogenic substances in pyrogens are bacterial and are generally exotoxin and endotoxin (Entoxin).
dotoxin). Among these, endotoxin mainly composed of cell wall phospholipid polysaccharide (LPS) of Gram-negative bacteria has the strongest pyrogenicity.

Therefore, aseptic handling is strongly required in the manufacturing process in order to prevent pyrogen from being mixed into a filling solution or the like of a medical device such as a blood purifier or a drug such as an interferon.
However, in reality, contamination with gram-negative bacteria and consequent contamination with pyrogens often occur. Once contaminated with drugs, medical equipment, and pyrogens, normal sterilization cannot prevent the patient from generating heat. This is because pyrogen is a chemical substance, not an organism, and cannot be detoxified by ordinary sterilization operations. In order to thermally destroy the chemical structure of pyrogen, it is usually necessary to heat it to 250 ° C. or higher, but it is actually very impossible to perform such an operation for a drug or a medical device.

Therefore, conventionally, pyrogens have been removed by filtration using a reverse osmosis membrane or an ultrafiltration membrane, or by adsorption removal using a pyrogen adsorbent in which a ligand having a high affinity for pyrogen such as histidine or polymyxin B is immobilized.

However, injection water and low molecular weight drugs can be filtered through reverse osmosis membranes or ultrafiltration membranes to remove pyrogens.
Difficult for blood and plasma. In addition, even when a small amount of sample is used, such as when a test such as TNF or SOD is produced on a laboratory scale, purification by these membranes is difficult.

On the other hand, by using a pyrogen adsorbent, pyrogen can be easily removed from blood, plasma, or a small amount of a sample. However, conventionally, there was a problem that the pyrogen adsorbent itself did not have sufficient adsorption performance and adsorption selectivity. In addition, when using a pyrogen adsorbent of the type in which the ligand is immobilized on a carrier, the patient may cause a serious immune reaction due to the ligand dropping out of the adsorbent and flowing out into the patient's body. There was also a problem in terms of safety.

[Problems to be solved by the present invention]

An object of the present invention is to provide a pyrogen adsorbent which is excellent in adsorption selectivity and adsorption speed and does not use a ligand.

[Technical means for solving the problem]

The pyrogen adsorbent of the present invention is obtained by bonding a polymer compound having a hydroxyl group such as agarose to an isocyanate compound having two or more isocyanate groups.

The polymer compound having a hydroxyl group may be any compound as long as it is insoluble in water, and particularly preferred are agarose, cellulose, dextran, polyethylene glycol and the like. These may be used alone or as a mixture of two or more. Further, a crosslinked product of these compounds is also preferable.

The shape of these compounds is not particularly limited, such as a particle shape, a fibrous shape, a film shape, a hollow fiber shape, and a tubular shape. However, a particulate or fibrous material is preferred because it is convenient to react with the isocyanate compound and the adsorbent obtained by reacting with the isocyanate compound is easy to handle.

In the case of particles, the resulting adsorbent has a diameter of from 20 to 5000 μm, and more preferably from 20 to 5000 μm, from the viewpoint that the obtained adsorbent is hardly clogged when packed into a column and has a high pyrogen adsorption rate.
A 00 μm sphere is preferred.

The isocyanate compound may be any compound having two or more isocyanate groups in the molecule. Examples of such an isocyanate compound include, for example, a bifunctional compound represented by the formula: OCN-R-NCO (R represents an aliphatic chain having 1 to 12 carbon atoms or an aromatic chain having 6 to 15 carbon atoms) In addition to the functional isocyanate, a trifunctional or higher polyfunctional isocyanate may be used.

As the bifunctional isocyanate, specifically, hexamethylene diisocyanate, tetramethylene diisocyanate, o-tolidine isocyanate, tolylene diisocyanate, naphthylene-1,5-diisocyanate, 4,
4'-diphenylmethane diisocyanate and the like.

Examples of the trifunctional isocyanate include, for example, triphenylmethane 4,4 ', 4 ",-triisocyanate, 1,3,5
-Triisocyanate-2-methylbenzene, 1,3,6-
Hexamethylene triisocyanate, 1,3,5-tris (6-hydroxyhexyl) triisocyanate.

Among these compounds, hexanetylene diisocyanate is most preferred because the resulting adsorbent has particularly high adsorption activity.

The pyrogen adsorbent of the present invention can be produced, for example, as follows.

It is preferable to use a high molecular compound having a hydroxyl group in a dry state. If these compounds contain water, they may be dried by ordinary means or the replacement treatment with an anhydrous organic solvent may be repeated. The organic solvent used for the substitution may be the same as the solvent used for the reaction with the isocyanate compound, and examples thereof include dimethyl sulfoxide, dimethylacetamide, dimethylformamide, tetrahydrofuran, acetone, methyl ethyl ketone, methyl isobutyl ketone, and the like. it can.

The reaction with the isocyanate compound is performed as follows. That is, a polymer compound such as agarose having a hydroxyl group is suspended in the above organic solvent, an isocyanate compound is added, and the mixture is stirred and reacted at 10 to 200 ° C, preferably 30 to 110 ° C. The ratio between the polymer compound having a hydroxyl group and the isocyanate compound is not particularly limited, but the hydroxyl group (-OH) and the isocyanate compound (-NCO) in the polymer compound are not limited.
Is preferably such that -NCO / -OH> 1. All reactions are carried out under the condition that water does not enter the reaction system.
After stirring for a designated time and the hydroxyl group and the isocyanate group react, the obtained adsorbent is repeatedly washed with an organic solvent to remove unreacted isocyanate compounds and by-products. Next, the adsorbent is suction-filtered and repeatedly washed with water to remove the organic solvent. This adsorbent is usually stored aseptically in distilled water for injection.

There are various methods for removing pyrogen from blood and plasma of a patient using the pyrogen adsorbent of the present invention.

As the simplest method, for example, a patient's blood is led out of the body, stored in a blood bag, mixed with the pyrogen adsorbent of the present invention to adsorb and remove the pyrogen, and then the adsorbent is removed with a filter to remove the blood. There is a way to return to the patient's body. This method is preferable because it does not require a complicated device.

As another method, there is a method in which a column filled with a pyrogen adsorbent is incorporated into an extracorporeal circulation circuit, and pyrogen is adsorbed and removed online. Whole blood may be circulated in the extracorporeal circuit, or only plasma may be circulated.

As a method for removing pyrogen from drugs such as interferon, SOD, and TNF, for example, a method of mixing a crude product of these drugs with the pyrogen adsorbent of the present invention to adsorb and remove pyrogen, and removing the adsorbent with a filter is known. is there. This method is preferable because it does not require a complicated device.

As another method, there is a method in which a pyrogen adsorbent is packed in a column, and pyrogen is adsorbed and removed by passing a crude product of these drugs.

To remove pyrogen from the filling fluid of medical devices,
There is a method of incorporating a pyrogen adsorbent packed in a column into a circulation circuit of a medical device, and circulating a filler through the column.

〔Example〕

 Hereinafter, the present invention will be described in more detail with reference to examples.

Production of pyrogen adsorbent Cross-linked agarose [trade name: Sepharose CL-4B (manufactured by Pharmacia)] was repeatedly washed with distilled water, filtered by suction, and sufficiently squeezed out.

10.0 g of this wet cross-linked agarose
0.77 g) of dehydrated dimethyl sulfoxide (DMSO) 50 ml
And stirred at room temperature for 2 hours. In a system that does not enter moisture
After removing DMSO, 30 ml of dehydrated DMSO was newly added, followed by stirring at room temperature for 1 hour. Similarly, 30ml (12.5 hours), 30ml
The operation of (3 hours) was repeated, and finally 20 ml of DMSO was added. At this time, when the amount of water in DMSO in the system was analyzed by the Karl Fischer method, it was about 10 ppm.

Hexamethylene diisocyanate (HMDI) 1.
A solution in which 0 g was added to 10 ml of dehydrated DMSO was charged and stirred at 100 ° C. for 2 hours to carry out a reaction. After removing the reaction solution, add a new DM
25 ml of SO was charged, and the mixture was stirred at room temperature for 1.5 hours for washing. Thereafter, similarly, 25 ml (1.5 hours), 20 ml (1.5 hours),
20 ml (1.5 hours), 20 ml (11.5 hours), 20 ml (2.5 hours 9
Were sequentially washed. Titration analysis of isocyanate groups in the last 20 ml of the washing solution revealed no isocyanate groups. Thereafter, 30 ml of water was added to stop the reaction. The reaction product was filtered by suction and stirred overnight at room temperature in 20 ml of DMSO, then collected by filtration again and repeatedly washed with a large amount of water to obtain an adsorbent. The shape of the adsorbent is bead-shaped and the average particle size is 55
μm. This adsorbent was immersed in distilled water for injection after suction filtration.

The adsorption test was performed by squeezing water from the pyrogen adsorbent by suction filtration.

Example 1 0.5 ml (0.1 g dry weight) of a pyrogen adsorbent prepared by the above method and a pyrogen solution (E. Coli 0128: B1)
2) After mixing 1.5 ml with a vibrator,
Incubated for minutes. Then, centrifuge (7000rp
m, 15 minutes), and the supernatant was collected and pyrogen was quantified by the endospace method. Table 1 shows the results.

From these results, it was found that the pyrogen adsorbent of the present invention adsorbed pyrogen well even in a pyrogen solution having a high concentration of 100 ng.

Example 2 5.0 ml of the pyrogen adsorbent used in Example 1 was
Packed in a 0 mm column. A pyrogen solution (E. Coli 0128: B12, concentration 10.0 ng / ml) was passed through this column at a flow rate of 2.0 ml / min, and the pyrogen concentration in the effluent was measured.

 Table 2 shows the results.

From this result, it can be seen that the pyrogen adsorbent of the present invention adsorbs pyrogen well even when packed into a column and a pyrogen solution is passed through it.

Example 3 5.0 ml of the pyrogen adsorbent used in Example 1 was
Packed in a 0 mm column. 200 ml of a mixed solution of a pyrogen solution (E. coli 0128: B12, concentration 100 ng / ml) and a human albumin solution (concentration 5.0 mg / dl) was passed through this column, and the recovery rate of human albumin and the removal rate of pyrogen were measured. . As a result, the recovery of human albumin was 95.2% and the removal of pyrogen was almost 100%.

This indicates that the pyrogen adsorbent of the present invention adsorbs almost 100% of pyrogen, but hardly adsorbs albumin as a useful component.

Example 4 5.0 ml of the pyrogen adsorbent used in Example 1 was
Packed in a 0 mm column. 200 ml of a mixed solution of a pyrogen solution (E. coli 0128: B12, concentration 100 ng / ml) and an insulin solution (concentration 2.0 mg / ml) was passed through this column, and the insulin recovery rate and the pyrogen removal rate were measured. As a result, the recovery of insulin was 99% and the removal of pyrogen was almost 100%.

This indicates that the pyrogen adsorbent of the present invention adsorbs almost 100% of pyrogen, but hardly adsorbs insulin, a useful substance.

Example 5 5.0 ml of the adsorbent used in Example 1 was packed in a column having an inner diameter of 10 mm. The pyrogen solution (E.Coli
0128: B12, concentration 100 ng / ml) and TNF solution (concentration 1.0 mg / ml)
200 ml of the mixed solution was passed, and the TNF recovery rate and pyrogen removal rate were measured. As a result, the recovery rate of TNF was 95
%, And the removal rate of pyrogen was almost 100%.

From this, it was found that the pyrogen adsorbent of the present invention adsorbs almost 100% of pyrogen, but hardly adsorbs TNF as a useful substance.

Example 6 0.5 ml of the pyrogen adsorbent used in Example 1 and 1.5 ml of the plasma of a patient having a high pyrogen level were mixed with a vibrator and incubated at 37 ° C for 120 minutes. afterwards,
The mixture was centrifuged, and the pyrogen concentration of the supernatant was measured by the endospace method. Table 3 shows the results.

From these results, it was found that pyrogen was well adsorbed even when actually used for human plasma.

Comparative Example 1 Sepharose CL-4B 0.5 mg (dry weight 0.1 g) and pyrogen solution (E. Coli 0128: B12) 1.5 ml were mixed with a vibrator, and then incubated at 37 ° C. for 120 minutes. Thereafter, the mixture was centrifuged (7000 rpm, 15 minutes), the supernatant was collected, and pyrogen was quantified by the endospace method. Table 4 shows the results.

Comparative Example 2 5.0 ml of the adsorbent used in Comparative Example 1 was packed in a column having an inner diameter of 10 mm. The pyrogen solution (E.Coli
[0128: B12, concentration 100 ng / ml) was flowed at a flow rate of 2.0 ml / min, and the concentration of pyrogen in the effluent was measured. Table 5 shows the results.
Shown in

Comparative Example 3 5.0 ml of the adsorbent used in Comparative Example 1 was packed in a column having an inner diameter of 10 mm. The pyrogen solution (E.Coli
0128: B12, concentration 100ng / ml) and human albumin solution (concentration 5.
(0 mg / dl) of a mixed solution (200 ml) was circulated, and the recovery rate of human albumin and the removal rate of pyrogen were measured. As a result, it was found that the recovery rate of human albumin was 95.2%, but pyrogen was hardly removed at a removal rate of 1.0%.

Comparative Example 4 5.0 ml of the adsorbent used in Comparative Example 1 was packed in a column having an inner diameter of 10 mm. The pyrogen solution (E.Coli
0128: B12, concentration 100 ng / ml) and insulin solution (concentration 2.
(0 mg / ml) was circulated, and the recovery rate of insulin and the removal rate of pyrogen were measured. As a result, it was found that the insulin recovery rate was 99%, but the pyrogen removal rate was 1.0%, which was hardly removed.

Comparative Example 5 5.0 ml of the adsorbent used in Comparative Example 1 was packed in a column having an inner diameter of 10 mm. The pyrogen solution (E.Coli
0128: B12, concentration 100 ng / ml) and TNF solution (concentration 1.0 mg / ml)
200 ml of the mixed solution was passed, and the TNF recovery rate and pyrogen removal rate were measured. As a result, the recovery rate of TNF was 95
%, But the removal rate of pyrogen was found to be 1.0%, which was hardly removed.

(Effect of the present invention)

The pyrogen adsorbent of the present invention is excellent in pyrogen adsorption selectivity and adsorption speed. In addition, since it is not a type to which a ligand is bound, a substance that causes an immune disease such as a ligand is not eliminated from the pyrogen adsorbent during use.

Therefore, the pyrogen removal method of the present invention in which blood, plasma, or the like of a patient is adsorbed to the above-described pyrogen adsorbent is particularly effective for treating patients with endotoxin shock seen in sepsis.

In addition, the pyrogen removal method of the present invention is also effective for purifying drugs such as injection water and interferon, or filling fluid for medical devices such as blood purifiers, and greatly reduces accidents such as fever in patients. Can be.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-4-114660 (JP, A) JP-A-61-199864 (JP, A) JP-A-63-11167 (JP, A) (58) Investigation Field (Int.Cl. ⁶ , DB name) A61M 1 / 36,1 / 38 B01J 20/22-20/34 CA (STN)

Claims (2)

(57) [Claims] A pyrogen adsorbent comprising an isocyanate compound having two or more isocyanate groups bonded to a polymer compound having a hydroxyl group. 2. The pyrogen adsorbent according to claim 1, wherein the polymer compound having a hydroxyl group is selected from agarose, cellulose, and dextran.