JP2002369881A - Amination carrier and method of adsorbing cellular fibronectin-heparin composite using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material which is capable of selectively adsorbing a composite of a heparin which is injected into blood as a blood anticoagulant and an EDA(+)FN-heparin which is formed by EDA(+)FN in the blood, in extracorporeal circulation treatment. SOLUTION: The amino group content ratio of the amination carrier is controlled to be in the range of 5 to 10 μmol/ml.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an aminated carrier and a method for adsorbing a cellular fibronectin-heparin complex using the aminated carrier.

[0002]

BACKGROUND OF THE INVENTION For the treatment of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus-death, and ankylosing myelitis, Nose (Artif.
g) The cryofiltration therapy devised by 4,205-207 1980) has been performed, and its therapeutic effect has been reported. Subsequently, research on the properties of cryogels proceeded, with the main component being cellular fibronectin (EDA (+) F
N), fibrinogen (FbN), and heparin (Umemoto et al. Blood, Coagulation, Fibrinolysis (Blood, Coagul. Fibrinolysis).
4,127-131 1993). Cryogel does not occur in healthy human plasma. That is, EDA (+) FN is an essential component.

[0003] EDA (+) FN is derived from plasma fibronectin (p
It has almost the same structure as FN), but is characterized by the addition of regions called extra domain A (EDA), extra domain B (EDB), and IIICS. Especially ED
A (+) FN is said to be the causative agent of cryoprecipitation removed by cryofiltration treatment (Taka Komai, Satoshi Shimizu, Keiichi Miyashita, Keiichi Miyamoto, Masayuki Tokita, Kobayashi) Naoyuki, Eiji Sakashita Population organ 26 (1) pp1
95-199 (1997)). EDA (+) FN has high affinity for heparin, and cryogel contains heparin.

[0004] Although EDA (+) FN is present in the plasma of healthy individuals, the concentration of EDA (+) FN is usually very low due to the simultaneous presence of plasmatic fibronectin (pFN). However, in the plasma of patients with autoimmune diseases, the high concentration of EDA (+) FN is considered to be a causative agent of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and ankylosing myelitis. Have been.

[0005]

However, cryofiltration therapy requires multiple steps such as blood cell-plasma separation, plasma cooling, cryogel separation, and plasma heating. In addition, cryogels formed by cooling plasma contain not only pathogenic substances but also other normal components. Therefore, when the cryogels are separated and removed, a large amount of normal components such as albumin is removed from the body.
Therefore, it was necessary to supply the albumin preparation to the human body after the treatment.

On the other hand, Japanese Patent No. 2796645 discloses an EDA (+) FN removing agent using heparin-immobilized particles. This utilizes the high affinity of EDA (+) FN for heparin. EDA (+) FN was removed from plasma using the remover.
ATIII (a type of anticoagulant protein) that is considered not to be involved in autoimmune disease when elimination
Was also removed at the same time, and the blood coagulation system in blood was sometimes out of balance. In addition, in the removal method, since expensive heparin is immobilized on a gel and used, the cost of producing an adsorbent is high. Furthermore, when the remover is used for extracorporeal circulation treatment, in order to prevent clogging of blood cell components, a blood cell-
A plasma separation step was required.

[0007]

Means for Solving the Problems The present inventor has conducted intensive studies in view of the above-mentioned problems of the prior art, and has found that heparin injected into blood as a blood anticoagulant in extracorporeal circulation treatment,
We have been diligently studying how to remove the complex of EDA (+) FN-heparin formed by EDA (+) FN in blood.
As a result, the content ratio of the amino group was 5 to 10 μmol / m
It has been found that an aminated carrier having a range of 1 selectively adsorbs a cellular fibronectin-heparin complex, and based on this finding, has completed the present invention.

The present invention has the following constitutions (1) to (12). (1) An aminated carrier having an amino group content in the range of 5 to 10 μmol / ml.

(2) The aminated carrier according to the above (1), wherein the content of the amino group is controlled by the reaction between the protecting group and the amino group.

(3) The aminated carrier according to the above (1) or (2), wherein the aminated carrier is an aminated polysaccharide.

(4) The aminated carrier according to the above (3), wherein the aminated polysaccharide is an aminated cellulose.

(5) The aminated carrier according to any one of the above items 1 to 4, which is water-insoluble.

(6) The aminated carrier according to any one of the above (1) to (5), wherein the carrier is in the form of particles.

(7) The aminated carrier according to the above (6), which has a sphericity of 0.9 or more.

(8) The aminated carrier according to the above (6) or (7), wherein the particle size is in the range of 240 to 500 μm.

(9) The aminated carrier according to any one of the above items 6 to 8, having an exclusion limit molecular weight in the range of 500,000 to 5,000,000.

(10) The aminated carrier according to any one of the above items 6 to 8, which has an exclusion limit molecular weight in the range of 800,000 to 3,000,000.

(11) Any one of the above items 1 to 10
A method for adsorbing a cellular fibronectin-heparin complex, which comprises using the aminated carrier according to the above item.

(12) Any one of the above items 1 to 10
A method for adsorbing a cellular fibronectin-heparin complex, which comprises adsorbing a cellular fibronectin-heparin complex from a whole blood component using the aminated carrier according to the above item.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, the carrier having an amino group is not particularly limited, but specifically, an aminated polysaccharide, an inorganic carrier such as an aminated silica gel, an aminated styrene-divinylbenzene crosslinked product, polyallylamine particles, polylysine particles , Polyvinylamine particles, aminated polymethylglutamic acid, and aminated polyvinyl alcohol. Among them, aminated polysaccharides can be preferably used in the present invention because they have polyols and have high biocompatibility.

Examples of the aminated polysaccharide include aminated cellulose, deacetylated chitin, chitosan, aminated agarose, aminated dextran, aminated mannan, and aminated starch. Among them, aminated cellulose is preferably used in the present invention because it has high mechanical strength and is usually insoluble in water.

The method of synthesizing the aminated carrier is not particularly limited. For example, in the case of a compound having no amino group in its chemical structure, such as cellulose,
An amino group may be chemically introduced into the structure. In the case of an amino sugar polymer such as chitin, an aminated carrier that can be used in the present invention can be obtained by deacetylation.

The method for producing an aminated carrier will be described in more detail by taking aminated cellulose as an example. An epoxy-activated cellulose gel can be obtained by reacting epichlorohydrin with cellulose at 30 ° C. under alkaline conditions, and an aminated cellulose can be obtained by reacting the epoxy-activated cellulose gel at 40 ° C. in aqueous ammonia.

As another method for aminating a polysaccharide having no amino group such as cellulose, a sugar hydroxyl group is activated by using paratoluenesulfonic acid chloride, 2-fluoro-1-methylpyridinium or the like. Thereafter, an aminated polysaccharide can be obtained by reacting with ammonia. Also, an aminated polysaccharide can be obtained by using a divalent or higher valent amine such as ethylenediamine instead of ammonia.

The amino group content of the aminated carrier of the present invention is in the range of 5 to 10 μmol / ml, particularly preferably in the range of 7 to 9 μmol / ml.

The method of controlling the amino group content is not particularly limited. As the control method, specifically, when the aminated carrier is produced, the amino group content is 5 to 1%.
A method of adjusting reaction conditions and the like so as to be in the range of 0 μmol / ml, and a method of reacting an amino group of an aminated carrier containing an excess amino group with a protective group to block the amino group, A method of controlling the content ratio can be mentioned. Furthermore, a method of removing excess amino groups by treatment with nitrous acid can also be mentioned.

Among them, the method of controlling the amino group content using a protecting group is preferable in the present invention because the amount of free amino groups can be finely adjusted.

Specific examples of the protecting group that can be used in the present invention include organic acids such as acetic acid and propionic acid, aldehydes such as acetaldehyde, carbobenzoxy chloride and di-t-butyl dicarbonate. Among them, those in which the protection product does not show ionicity are preferable, and those having a carboxyl group are more preferable. Preferred protecting groups include acetic anhydride. Acetic anhydride is easily available and can be preferably used in the present invention.

The aminated carrier of the present invention is preferably water-insoluble. The term "water-insoluble" means that the amount dissolved in water at 100 ° C is 1 mg or less.

The shape of the aminated carrier of the present invention is not particularly limited, and the effects of the present invention can be obtained in any shape such as a particle shape, a film shape, a hollow fiber shape, a rod shape, and a lump shape. Among them, in the present invention, the shape of the aminated carrier is preferably particulate.

When the aminated carrier of the present invention is in the form of particles, it is preferable in the present invention because the preparation and handling are easy and the amount of ligand bound per aminated carrier is large. Among the aminated carriers in the form of particles, spherical cellulose particles are particularly preferably used in the present invention because they are inexpensive, have high biocompatibility, high strength, good column pressure resistance, and can be autoclaved. can do.

Further, when the aminated carrier of the present invention is in the form of particles, the sphericity is preferably high. If the sphericity is high, it is easy to pack uniformly in a column or the like. The sphericity as referred to in the present invention is a ratio (minor axis / major axis) between the shortest diameter (minor axis) and the longest axis (major axis) of a particle, and the sphericity increases as this value approaches 1.0. Indicates that

In the present invention, the sphericity of the particulate aminated carrier is preferably 0.9 or more, more preferably 0.95 or more.

The particle size of the particulate aminated carrier is not particularly limited, but in the present invention, the particle size is preferably in the range of 240 to 500 μm, more preferably in the range of 240 to 270 μm. is there. With this particle size, solid components such as red blood cells, white blood cells, and platelets can also pass through the particle gap.

The aminated support in the form of a membrane is a support having a flat plate-like shape, a porous shape, and a certain range of exclusion limit molecular weight, such as a commercially available membrane filter.

The exclusion limit molecular weight (measured using polyethylene oxide as a standard substance) of the above-mentioned particulate aminated carrier and film-like aminated carrier is not particularly limited, but is preferably 500,000 to 5,000,000. , More preferably 800,000 to 3,000,000. When the exclusion limit molecular weight of polyethylene oxide is within this range, problems such as physical adhesion of blood components in the column can be suppressed.

The method for producing a porous carrier that is water-insoluble and has the above-mentioned exclusion limit molecular weight is not particularly limited, but the case of spherical cellulose particles will be described as an example. Spherical cellulose particles are described, for example, in
As described in -8649, cellulose acetate is dissolved in an organic solvent, and the cellulose acetate is spheroidized by suspending the solution in an aqueous medium.
Then, the organic solvent is evaporated to obtain cellulose ester particles, which are converted into cellulose particles after saponification, whereby porous spherical cellulose particles preferable for the present invention can be obtained.

The hollow fiber-like aminated carrier refers to a fibrous carrier having a continuous or discontinuous cavity therein.
A cavity is formed inside the spinning solution by adding a foaming agent or using a special die.

The adsorbent used in the method for adsorbing the cellular fibronectin-heparin complex of the present invention may be any compound and in any form as long as it is the aminated carrier of the present invention. Yes, it has a remarkable effect. The specific form of the adsorption method is not particularly limited, but when the aminated carrier of the present invention is in the form of particles, it may be a form in which the aminated carrier is packed in a column. preferable.

Further, in the aminated carrier of the present invention,
It is possible to selectively adsorb cellular fibronectin-heparin complex directly from whole blood. According to the adsorption method of the present invention, a device for separating a blood cell component and a plasma component and an operation are not required, so that the device can be simplified.

The specific mode of the adsorption method is not particularly limited, but is a mode in which the aminated carrier of the present invention is packed in a column and incorporated into an on-line circuit of a device for extracorporeal circulation treatment. Is preferred. More specifically, blood extracted from the patient's body and added with heparin as an anticoagulant, or a blood plasma component separated from blood cells, is filled with the aminated carrier of the present invention in an extracorporeal circulation device. It is preferable that the blood from which the cellular fibronectin-heparin complex has been removed is returned to the patient through a cellular fibronectin-heparin complex adsorption column.

After adsorption, EDA (+) FN eluted and recovered from the aminated carrier can be used as a reagent for research.
In addition, the reagent can be used effectively for cell culture, as a component of a therapeutic agent for wounds, for adhering cells.

Among the aminated carriers of the present invention, aminated cellulose particles can be preferably used in the adsorption method of the present invention. Among them, aminated cellulose particles having a particle size in the range of 240 to 500 μm are particularly preferably used. be able to.

[0044]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples. The “%” described in the examples is
Unless otherwise specified, it means "% by weight". 1. Preparation of Adsorbent Example 1 Cellulose particles (CPB-06, manufactured by Chisso Corporation) were classified into 200 to 500 μm with a wet vibrating sieve. 100 g (wet weight) of the classified cellulose particles were dispersed in 150 ml of a 3% aqueous sodium hydroxide solution and stirred at 30 ° C.
After adding 50 g of epichlorohydrin and stirring at 30 ° C. for 2 hours, the filtrate was washed with pure water until the filtrate became neutral, and 95 g (wet weight) of a gel in which an excessive amount of water was removed by suction filtration was obtained. After 100 ml of 25% aqueous ammonia was added to the gel and stirred at 50 ° C. for 2 hours, the gel was washed with pure water until the filtrate became neutral to obtain aminated cellulose particles. The amino group content was 20 μmol / ml of gel.

Example 2 Example 1 except that the concentration of sodium hydroxide was 2%.
The same operation was performed. Amino group content is 9 per ml of gel
μmol

Example 3 The aminated cellulose particles obtained by the above method were washed with a 40% aqueous ethanol solution. The aminated cellulose particles were dispersed in a 40% aqueous ethanol solution, acetic anhydride in an amount shown in Table 1 was added, and the mixture was reacted at 30 ° C. for 1 hour. After the reaction, the gel was washed with 0.1 N sodium hydroxide, then with pure water until the filtrate became neutral, and finally with 0.1 M saline to obtain an adsorbent. Table 1 shows the amino group content of the adsorbent.

[0047]

[Table 1]

Example 4 The adsorption behavior of various proteins on the adsorbent prepared in Example 2 was examined. Method for adjusting model plasma [0107] Purified cellular fibronectin was added to citrated plasma obtained from a healthy person to adjust the plasma. In addition, heparin was added to prepare two concentrations of model plasma of 3 unit / ml and 30 unit / ml. The components of the model plasma other than heparin are shown in Table 2.

[0049]

[Table 2]

Adsorption experiment operation 0.5 ml of the adsorbent was added to 6.25 ml of model plasma to which Fusan (Torii Pharmaceutical) (2 mg / ml) and calcium chloride (10 mM) were added, and the mixture was shaken at 30 ° C for 4 hours. The reaction solution is centrifuged (2000 g, 5 minutes, 25 ° C.), and the supernatant is sampled. The concentration of each component in the sampled liquid was measured, and the concentration before the treatment was defined as 100%, and the amount adsorbed was represented based on the remaining amount after the adsorption. The results are shown in Figs.
The results are shown in FIG.

As a result of the adsorption experiment, it was clear that albumin, fibrinogen and immunoglobulin (M, A, G) hardly adsorbed. Cellular fibronectin is remarkably adsorbed when the amino group content of the adsorbent is in the range of 7 to 9 μmol / ml. On the other hand, plasma fibronectin hardly adsorbs on all adsorbents. ATIII has an amino group content of 7 only when the concentration of heparin is 30 unit / ml.
Adsorbed with ９9 μmol / ml adsorbent. But,
At a heparin concentration of 3 unit / ml, ATIII is hardly adsorbed.

Example 5 The adsorbent of Example 2 was applied to a 2 cm column having an inner diameter of 1.1 cm.
Filled to height. The mesh at the top and bottom of the column is 2
The mesh was replaced with a nylon mesh having a mesh size of 00 μm. Column containing 3 μmol / ml heparin in PBS
To equilibrate. 3 μmol / ml heparin in human blood,
After adding calcium chloride (10 mM) and Fusan (Torii Pharmaceutical Co., Ltd.) at a concentration of 10 μg / ml, the mixture was added to the column at a speed of about 0.3 ml / mim, and the eluted solution was collected to count the number of blood cells. Was measured. The results are shown in Table 3.

[0053]

[Table 3]

[0054]

The aminated carrier of the present invention selectively adsorbs cellular fibronectin-heparin complex. Further, if the method for adsorbing a cellular fibronectin-heparin complex using the aminated carrier of the present invention, the complex can be selectively adsorbed and removed.

[Brief description of the drawings]

FIG. 1. Adsorption behavior of albumin, fibrinogen, and immunoglobulin (M, A, G). The subscript in the legend indicates the unit / ml of heparin.

FIG. 2: Adsorption behavior of cellular fibronectin.

FIG. 3 shows the adsorption behavior of plasma fibronectin.

FIG. 4 shows the adsorption behavior of antithrombin III.

Continuing on the front page (72) Inventor Takashi Komai 145 Idoyama-cho, Hisai-shi, Mie Prefecture Joint lodgings Hisai 5-201 (72) Inventor Keiichi Miyamoto 145, Idoyama-cho, Hisai-shi, Hisai, Mie prefecture 5-401 Hisashi-ho, joint housing (72) Inventor Masami Todori 4-318, Tsukiji, Minamata-shi, Kumamoto F-term (reference) 4C077 AA12 BB03 CC03 EE01 HH03 MM03 NN05 NN06 PP30 4D017 AA11 BA07 CA14 CB01 DA01 DB02 EA05 4G066 AC01B AC02B AD10B AE19BA20 CA20 DA12 EA04 FA03 GA11

Claims (12)

[Claims] 1. An amino group content of 5 to 10 μmol.
Aminated carriers in the range of / ml. 2. The aminated carrier according to claim 1, wherein the content of the amino group is controlled by the reaction between the protecting group and the amino group. 3. The aminated carrier according to claim 1, wherein the aminated carrier is an aminated polysaccharide. 4. The aminated carrier according to claim 3, wherein the aminated polysaccharide is an aminated cellulose. 5. The method according to claim 1, which is water-insoluble.
Item 3. The aminated carrier according to item 1. 6. The aminated carrier according to claim 1, which is in the form of particles. 7. The aminated carrier according to claim 6, which has a sphericity of 0.9 or more. 8. The aminated carrier according to claim 6, wherein the particle size is in a range of 240 to 500 μm. 9. The aminated carrier according to claim 6, which has an exclusion limit molecular weight in the range of 500,000 to 5,000,000. 10. The aminated carrier according to claim 6, which has an exclusion limit molecular weight in the range of 800,000 to 3,000,000. 11. A method for adsorbing a cellular fibronectin-heparin complex, comprising using the aminated carrier according to any one of claims 1 to 10. 12. A cellular fibronectin-heparin complex characterized by adsorbing a cellular fibronectin-heparin complex from whole blood components using the aminated carrier according to any one of claims 1 to 10. Adsorption method.