JPH0134067B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention enables the use of anti-inflammatory agents from blood, plasma, and other body fluids.
It relates to an adsorbent that removes self-flowing antibodies and immune complexes such as DNA antibodies, anti-ENA antibodies, and anti-nuclear antibodies. For more information, see systemic lupus erythematosus (systemic lupus erythematosus).
By specifically adsorbing and removing these autoantibodies and immune complexes present in the body fluids of patients with collagen diseases such as SLE (abbreviated as SLE), we purify and regenerate the body fluids and prevent the progression of the disease. The purpose is to provide a simple adsorbent that alleviates symptoms and accelerates healing. SLE is an intractable disease that affects the vascular system and connective tissue, especially in the organs of the body, and is a typical autoimmune disease. Although the cause was unknown, in recent years
The presence of anti-nuclear antibodies in the blood of SLE patients is thought to have an important relationship with the development of various symptoms, and antigen-antibody immune complexes are believed to be the cause of vasculitis and renal lesions. The mechanism of deposition is becoming more important. Recently, antigens in patient plasma,
In addition to removing malignant substances such as antibodies and immune complexes, plasmapheresis therapy, in which the patient's plasma is replaced with other fresh frozen plasma or albumin preparations, has been performed, and has been shown to be effective in preventing disease progression, alleviating symptoms, and curing the disease. It's starting to feel like this. However, this plasmapheresis therapy has two drawbacks: (1) difficulty in obtaining large quantities of fresh frozen plasma or plasma components in a sustained manner to replenish the removed plasma;
(2) Because it uses someone else's blood plasma, it has the disadvantage of being at high risk of infection with hepatitis viruses, etc., and cannot be widely used. Additionally, to remove malignant substances from patient plasma, there is a method using an ultrafiltration membrane, which has the advantage of not requiring supplementation of plasma components; however, (1) it cannot be separated cleanly due to molecular weight; (2) There is no selectivity for removal other than molecular weight, so useful substances in plasma are also removed. (3) Problems such as decreased overspeed due to membrane clogging and fluctuations in cut-off molecular weight. have. Furthermore, a method has also been proposed in which autoantibodies are removed by immobilizing biological (similar) polymers such as DNA and synthetic nucleic acid polymers on a carrier. However, this method
In practical terms, it is difficult to use it for healing because (1) the immobilized substance is expensive, and (2) the immobilized substance, which is an antigen, may be released and enter the patient's body. In addition, adsorbents for immune complexes and anti-nucleic acid antibodies have been proposed in JP-A-53-136380 and JP-A-55-38141, but none of them are practically satisfactory. I can't say that. In order to overcome these problems, the present inventors
We conducted extensive research into a simple and safe adsorbent that specifically removes malignant substances from patient plasma.
As a result, while some biological (similar) polymers have been known to have adsorption ability, surprisingly, even low-molecular substances that make up nucleic acid bases have an adsorption ability that is equal to or higher than that of polymers. We discovered that this technology has the following properties and applied for a patent. As a result of more detailed research, the present inventors surprisingly found that sugar mono- or di-orthophosphates, mono- or dipyrophosphates, triphosphates, and their homo- or di-orthophosphates with a degree of polymerization of 10 or less It has been discovered that heteropolymers, and even those in which orthophosphoric acid, pyrophosphoric acid, or triphosphate groups are bonded to homo- or hetero-oligosaccharides with a degree of polymerization of 10 or less, have an adsorption capacity equivalent to or higher than that of high molecular weight, and the present invention is based on the present invention. I was able to complete it. As a result, concerns about the detachment and outflow of the immobilized substance were resolved, and the difficulties in actual clinical application could be overcome. That is, the present invention provides mono- or di-orthophosphoric acid esters, mono- or dipyrophosphoric acid esters, triphosphates, homo- or heteropolymers thereof with a degree of polymerization of 10 or less, and homo- or hetero-oligomers thereof with a degree of polymerization of 10 or less. Anti-native DNA antibodies (anti-n-
DNA antibody), anti-double-strand DNA antibody (anti-
It is an adsorbent for autoantibodies such as ds-DNA antibodies), antinuclear antibodies, and immune complexes. The diseases targeted for removal by the adsorbent of the present invention are anti-n
- It can be applied to any antibody in which DNA antibodies, anti-ds-DNA antibodies, antinuclear antibodies, and immune complexes thereof appear. Specifically, SLE, mixed connective tissue disease (MCTD), progressive systemic sclerosis, Siegren's syndrome, dermatomyositis (polymyositis), silicosis,
Chronic rheumatoid arthritis, Hashimoto's disease, chronic hepatitis, diabetes,
Bronchiectasis, etc. Attempts have been made to remove antibodies by immobilizing antigens on carriers using antigen-antibody reactions, and it is also possible to remove antibodies with anti-DNA antibodies and the like by immobilizing DNA on carriers. However, as already mentioned, it was not suitable for practical use. According to the present invention,
Use safe substances in your body at all times,
An adsorbent with adsorption performance equal to or higher than that of DNA has been developed, which will enable a wide range of clinical applications and is expected to bring about major advances in the treatment of autoimmune diseases in the future. In the present invention, the substances to be bound to the insoluble carrier include tetroses such as erythrose and threose, aldopentoses such as arabinose, xylose, lyxose, and ribose, xylulose,
Ketopentoses such as pentulose and ligulose, aldohexoses such as galactose, glucose, talose, and mannose, ketohexoses such as sorbose, tagatose, psicose, and fructose, hexosamines such as N-acetyl-glucosamine, aldoheptose, ketoheptose, ketooctose, and ketononose polymonosaccharides such as 2-deoxypentose, 6-deoxyhexose, deoxysaccharides such as 2-deoxyhexose, 2,6-dideoxyhexose, 3,6-dideoxyhexose, sugar alcohol anhydrides, uronic acid, ketoaltonic acid , acidic sugars such as ascorbic acid, sugar methyl ethers, branched sugars, amino sugars, mono- or di-orthophosphoric acid esters, mono- or dipyrophosphoric acid esters, triphosphates, and homo- or hetero-phosphoric acid esters of these with a degree of polymerization of 10 or less. It consists of a polymer, or a homo- or hetero-oligosaccharide with a degree of polymerization of 10 or less, to which orthophosphoric acid, pyrophosphoric acid, or triphosphate groups are bonded. The above sugars can be used regardless of whether they are D, L, threo, or erythro. Further, among the substances to be bound to the above-mentioned insoluble carrier, phosphoric acid esters of pentose such as ribose and deoxyribose are particularly preferable, and 3,5 diorthophosphoric acid of deoxyribose and oligomers thereof are more preferable. Rather than simply immobilizing just one of these, a plurality of types may be immobilized on the carrier. Although any carrier can be used in the present invention as long as it is insoluble in water, it is preferable to use a hydrophilic carrier in order to avoid non-specific adsorption. The shape of the insoluble carrier can be particulate, fibrous, hollow fiber, membrane, gauze, nonwoven, etc., but depending on practical handling, amount of ligand retained, effective area of contact with plasma, etc. From this point of view, particles and fibers are preferred. As a particulate carrier, the average particle size is 50μ to 2500μ.
It is preferable that it is in the range of . Average particle size is JIS-
After classifying in running water using a sieve specified in Z-8801, the intermediate value between the upper limit particle size and the lower limit particle size of each class is taken as the particle size of each class, and the average particle size is calculated as the weight average. In addition, the particle shape is preferably spherical, but
It is not particularly limited. Average particle size is 2500μ
If it exceeds 50μ, the amount and speed of adsorption of malignant substances will decrease, and if it is less than 50μ, the pressure drop will be large and clogging will easily occur when used for extracorporeal circulation, and the coagulation system will be activated and blood cells will adhere. As particulate carriers that can be used, carriers such as agarose-based, cross-linked dextran-based, cellulose-based, cross-linked polyacrylamide-based, porous silica beads, and activated carbon can be used. Polysaccharide gel gives good results. Furthermore, immobilized enzymes and known carriers commonly used in affinity chromatography can be used without any particular limitations. Moreover, porous synthetic polymer particles can be used as the particulate carrier used in the present invention. The particles can fix the substance used in the present invention described above on their surfaces, and have an average pore diameter of 300.
Å to 9000Å, more preferably 1000Å to
It is in the range of 6000 Å. As the polymer composition of the particles, polyamide-based, polyester-based, polyurethane-based, or vinyl compound polymers can be used, but particularly hydrophilic vinyl compound-based porous composite particles give preferable results. The porous structure preferably has an average pore diameter in the range of 300 Å to 9000 Å; however, if the average pore diameter is too small, the amount of autoantibody adsorbed is small, and if it is too large, the strength of the polymer particles is reduced. This is not practical because the surface area decreases. The average pore diameter can be measured using a mercury intrusion type polysimeter. In this method, mercury is injected into a porous material, and the amount of pores is determined from the amount of mercury infiltrated, and the pore diameter is determined from the pressure required for injecting, and pores of 40 Å or more can be measured.
The average pore diameter is the value of r when the value of dv/dlogr is maximum, where r is the pore diameter and v is the cumulative pore volume measured by a porosimeter. When using a fibrous carrier, the fiber diameter is 0.02
Denier to 10 denier, more preferably 0.1
It is preferable to have a denier to 5 denier. If the fiber diameter is too large, the amount and rate of adsorption of autoantibodies will be reduced, and if it is too small, activation of the coagulation system, blood cell adhesion, and clogging are likely to occur. As the fibrous carrier that can be used, there are known hydrophilic fibers such as regenerated cellulose fibers, nylon, acrylic, and polyester. Any known method such as covalent bonding, ionic bonding, physical adsorption, crosslinking, embedding, or precipitation insolubilization on the surface of the carrier can be used to immobilize the bound ligand on the hydrophilic carrier. Covalent bonding is preferred in order to prevent elution of Covalent bonding methods usually involve immobilized enzymes,
Methods used in the manufacture of affinity columns are employed. The carrier can be activated using cyanogen halides, periodic acid, crosslinking reagents, epoxides, and the like. Between the carrier and the ligand,
A molecule (spacer) of any length can be inserted and used. In the present invention, the amount of the ligand bound to the carrier is 0.1 μmol or more per 1 g (dry weight) of the carrier.
in the range of 1000μmol, more preferably 0.5μmol
or 100 μmol. Retention amount
If it is less than 0.1 μmol, the adsorption capacity for malignant substances will decrease, and if it exceeds 200 μmol, non-specific adsorption of plasma useful components will occur, which is not preferable. The portion of the ligand to be bound to the carrier includes a hydroxyl group, a phosphate group,
Amino groups can be used. The mechanism of action of this adsorbent is thought to be similar to that of an antigen-antibody reaction, but it can be used internally without any problems without using biological polymers or similar synthetic polymers, which are the original antigens. It is surprising that small molecules that are metabolized in the body exhibit sufficient adsorption and removal ability. Furthermore, it is thought that the interaction between the remaining Fab portion of the antibody in the complex and the ligand contributes to the adsorption of the immune complex. The autobody adsorption device using the adsorbent of the present invention is such that the autoantibody adsorbent described above is filled in a container having a fluid inlet/outlet so that the body fluid passes through while coming into contact with the autoantibody adsorbent. be. A concrete example of this is shown in FIGS. 1 and 2. In Figure 1,
A cap 5 having a body fluid inlet/outlet 4 is fitted into the openings at both ends of the cylinder 1 via a packing 3 with a filter 2 stretched inside with a screw 6, and an adsorbent 7 is filled and held in the gap between the filters at both ends. It is what it is. As the adsorbent, the adsorbent of the present invention may be filled alone, or may be mixed or laminated with other adsorbents. The appropriate volume of the adsorbent layer is about 50 to 400 ml when used for extracorporeal circulation. When using the above device for extracorporeal circulation, there are roughly two methods as follows. One method is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or membrane plasma separator, and then pass the plasma components through the above device to purify them. One method is to return the blood to the body along with blood cell components, and the other method is to directly pass the blood taken out from the body through the above-mentioned device for purification. In addition, regarding the passage speed of blood or plasma, since the adsorption efficiency of the adsorbent is extremely high, the particle size of the adsorbent can be made coarser, and the degree of packing can be lowered, so the shape of the adsorbent layer can be changed. Regardless, a high passing speed can be provided. Therefore, a large amount of body fluid can be treated. The method for passing body fluids may be continuous or intermittent depending on clinical needs or the installation status of the equipment. As described above, the adsorbent of the present invention can highly and specifically adsorb and remove autoantibodies and immune complexes such as anti-n-DNA antibodies, anti-ds-DNA antibodies, and anti-nuclear antibodies in body fluids. , it is very compact, simple and safe. It also has the effect that sterilization operations can be carried out easily and reliably. In addition, the adsorbent of the present invention can be used not only as a treatment device by filling it into a device, but also as an anti-N-
It can also be used extremely effectively as an adsorbent for separating and purifying autoantibodies such as DNA antibodies, anti-ds-DNA antibodies, and antinuclear antibodies and immune complexes, and as a substrate for measuring these. Hereinafter, embodiments of the present invention will be explained in more detail with reference to Examples. Example 1 Deoxyribose 3,5-diphosphoric acid was bound to CNBr-activated Sepharose 4B (manufactured by Pharmacia, Sweden, average particle size 260μ) by a conventional method (“Experiments and Applications of Affinity Chromatography” p. 48). , 1976 Kodansha Scientific Publishing), and the usual active groups were blocked with trishydroxymethylaminomethane. The amount retained is
It was 60 μmol/g. After thoroughly washing the adsorbent with physiological saline, it was dehydrated and used for the experiment. The adsorption experiment was conducted by mixing 3 volumes of patient plasma and 1 volume of adsorbent and incubating at 37°C for 3 hours. In addition, as a control, unactivated Cepharose
4B was used. The anti-ds-DNA antibody titer was measured using an indirect fluorescent antibody method using Crithidia luciliae. Antinuclear antibody titer is determined by dropping a serially diluted sample (primary antibody) onto a slide glass smeared with cells, performing an antigen-antibody reaction, dropping a peroxidase-labeled anti-human immunoglobulin antibody (secondary antibody), and measuring the enzyme concentration. The color reaction was observed using an optical microscope. For measurement,
A kit of "Enzyme ANA Test" [manufactured by Medical and Biological Research Institute, Inc.] was used. The antibody titer was expressed using the highest dilution factor indicating positivity. To measure the amount of immune complexes, a method was used in which immune complexes were precipitated with polyethylene glycol (PEG) and combined with complement hemolysis reaction. The operating method and conditions for this method were as follows. (1) Pour 0.3ml of sample into a separation tube. next,
Add 50μ of 0.2NEDTA and stir, then add 50μ of boric acid buffer and stir. 12.5% PEG
Add 0.1ml of (molecular weight 6000) and stir at 4℃, 90℃.
Let stand for a minute. (2) Centrifuge at 1700g at 4℃ for 10 minutes to reduce the precipitate to 2.5%.
Wash with 1.0ml of PEG. Centrifuge at 1700g for 15 minutes and discard the supernatant. (3) Add 30μ of GVB ⁺⁺ (gelatin veronal buffer containing divalent cations) at 37°C to dissolve the precipitate. Add 10 µl of pooled healthy human serum as a complement source. The immune complex and complement are allowed to react at 37°C for 30 minutes. (4) Add 1.0 ml of 1.5×10 ⁸ /ml EA (antibody-sensitized red blood cells) and shake at 37°C for 60 minutes to promote hemolytic reaction due to residual complement. (5) After the reaction, add 6.5 ml of physiological saline at 4°C, centrifuge, and measure the absorbance (OD ₄₁₄ ) of the supernatant. (6) Using healthy human serum as a control, calculate the inhibition rate of hemolysis. The unit is PEG-CC%. Rejection rate = (control) - (analyte absorbance) / (control absorbance)
×100 Removal rate = (untreated) - (treated plasma inhibition rate) / (untreated plasma inhibition rate) × 100 Sensitized red blood cells (KW) for complement value measurement manufactured by Japan Freeze Drying Institute were used for EA. The amount of albumin was measured using an albumin measurement method using bromcresol green (BCG), and the reagent used was [A/GB B-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.]. The ratio of the reduced amount of alumin to the patient's plasma after the adsorption experiment was defined as the removal rate. The results are shown in Table 1. Example 2 All experiments were carried out under the same conditions as in Example 1, except that ribose 3,5-diphosphate was used as the binding ligand. The results are shown in Table 1. Example 3 All experiments were carried out under the same conditions as in Example 1 except that deoxyribose 5-diphosphoric acid and 3-diphosphoric acid were used as binding ligands. The results are shown in Table 1. Example 4 All experiments were carried out under the same conditions as in Example 1, except that a deoxyribose 5-phosphate dimer (3,5-linked dimer) was used as the binding ligand. The results are shown in Table 1. Example 5 0.5 denier Bemberg long fiber (Asahi Kasei Corporation)
Co., Ltd.) and activated with CNBr in a conventional manner.
It was fixed using deoxyribose 3,5-diphosphate. 1 g of the adsorbent and 10 ml of patient plasma were incubated at 37° C. for 3 hours. Untreated Bemberg was used as a control. All other conditions were the same as in Example 1. The results are shown in Table 1.

[Table] Example 6 An adsorption experiment was conducted using 10 ml of the adsorbent used in Example 1 using the experimental system shown in FIG. That is, 30 ml of the same patient's plasma 9 is placed in a container 8, pumped out at a flow rate of 1 ml per minute by a pump 10, sent to a malignant substance adsorption device 11, passed through a drip chamber 12 and a sampling port 13, and returned to the container 8. The tube 14 was arranged so that the After circulating blood for 3 hours using the above device,
Blood was sampled, and the amount of autoantibodies, immune complexes, etc. in plasma was measured in the same manner as in Example 1. Both anti-ds-DNA antibody titer and anti-nuclear antibody titer were 640 and 40.
It declined to . In addition, 75% of immune complexes were removed.

[Brief explanation of drawings]

FIG. 1 is a sectional view showing an example of a malignant substance adsorption device using the adsorbent of the present invention, and FIG. 2 is an explanatory diagram of a model experiment of Example 6. 1... Cylinder, 2... Filter, 3... Packing, 4... Body fluid inlet/outlet, 5... Cap, 6
...Screw, 7...Adsorbent.

Claims (1)

[Claims] 1 Mono- or di-orthophosphoric acid esters, mono- or dipyrophosphoric acid esters, triphosphates of sugars, homo- or heteropolymers thereof with a degree of polymerization of 10 or less, and homo- or hetero-oligosaccharides with a degree of polymerization of 10 or less with orthophosphoric acid 1. An adsorbent for autoantibodies and immune complexes, characterized in that at least one selected from the group consisting of , pyrophosphoric acid, and triphosphate groups is bound to an insoluble carrier.