RU2441674C1 - Method for blood purification from dna-containing immune complexes using combined sorbent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is referred to the area of medicine, namely rheumatology, immunology and biotechnology and is related to lowering of content of DNA-containing circulating immune complexes in blood. In order to do that the blood is filtered through the original combined sorbent which is a composition consisting of granulated drug with immobilized DNAse I and granulated drug with immobilized Clq-component of complement. Both drugs are being synthesized separately using the emulsion polymerization method in the gaseous nitrogen flow on the basis of polyacrylamide carrier with magnetic properties which is granulated at the 2:1 carrier: iron ratio.

EFFECT: method provides for significant decrease of release of duplex DNA antibodies that are released into the blood flow in process of destruction of DNA-containing circulating immune complexes, while maintaining the effectiveness of destruction of said complexes.

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Description

The invention relates to medicine, namely to rheumatology, immunology and biotechnology, and can be used to improve the treatment of systemic lupus erythematosus and other autoimmune diseases.

It is known that immune complexes (IR) have a pronounced effect on the severity and prognosis of various diseases. In particular, the important role of DNA-containing IR in the progression of kidney damage in systemic lupus erythematosus, and thereby in the formation of a prognosis of life in this disease, has been proved. It has been shown that IR with high molecular weight DNA is the most pathogenic, in contrast to IR containing small DNA. However, the currently existing methods for removing circulating IR (CEC) from the bloodstream (plasmapheresis, hemosorption, immunosorption using immobilized agents tropic to monomeric or aggregated immunoglobulins) are not selective enough for the eliminated substance. This leads to a significant loss of plasma components. In addition to the need for replacement therapy, the consequence of this drawback is a significant increase in the incidence of severe bacterial and viral infections, which is also facilitated by the drug immunosuppression standard for the treatment of autoimmune diseases.

A convenient tool for the destruction of CECs containing high molecular weight DNA is deoxyribonuclease type I (DNase I), an enzyme whose physiological function is the destruction of DNA and nucleoproteins. Previously, we patented a method for the destruction of DNA-containing CECs using DNase I immobilized on magnetically controlled polyacrylamide granules (PGM) [Gontar I.P., Trofimenko A.S., Zborovsky AB, Shilova L.N., Simakova E .FROM. A method of purifying blood from DNA-containing immune complexes using an immobilized granular magnetically controlled preparation: Patent RU 2356585 C2 // Application No. 2007105420/15; declared 02/13/2007; publ. 05/27/2009, bull. No. 15. - 5 p.]. In the future, this method is designated as a prototype. The prototype allows you to vary within wide limits the amount of immobilized enzyme, as well as to use the drug repeatedly after regeneration. When using the prototype in the process of blood processing does not occur significant changes in the content of uniform elements. The prototype allows with high efficiency to destroy the high molecular weight deoxyribonucleic component of the CEC. It is known that DNA-containing large CECs (greater than 20S) under the action of DNase I are cleaved through the intermediate stage of medium molecular CECs (10S-20S) to small CECs (less than 10S). The latter are much less pathogenic than high molecular weight CECs and are more rapidly eliminated by mononuclear phagocytes of the liver and spleen. However, one of the products of enzymatic cleavage of DNA-containing IR, in addition to small-sized complexes, can be free antibodies to DNA. It is also known that in systemic lupus erythematosus, the CEC contains pathogenic antibodies to double-stranded DNA, a significant release of which into the bloodstream during the processing of blood by immobilized DNase I is undesirable. This side effect of the prototype, which we found during in vivo tests on an experimental model, is a significant drawback and is the basis for the refinement of the previously patented method.

A decrease in the release of antibodies to DNA into the bloodstream during the cleavage of DNA-containing CECs with DNase I can be achieved by the combined use of an immobilized enzyme and a sorbent that fixes antibodies in the intermediate phase of the enzymatic reaction in the form of medium or low molecular weight CECs. This method is technically more advantageous than the sorption of free antibodies to DNA, due to the significantly smaller number of molecules to be sorbed and, as a result, with a lower need for sorbent. A prerequisite for the implementation of such a sorption procedure is the combined use of immobilized DNase I and a sorbent in one perfusion chamber, the most convenient option of which may be to use a composition of granules with immobilized DNase I and granules with a sorbent. Observance of this condition makes it possible to fix the short-lived intermediate product of the enzymatic reaction — medium molecular weight CEC — at the site of its formation.

A promising ligand for fixing the intermediate decay products of DNA-containing CECs is the Clq component of complement (hereinafter - Clq), which mainly reacts with medium molecular IR [Kilgallon W., Amlot P.L., Williams B.D. Anti-Clq column: ligand specific purification of immune complexes from human serum or plasma. Analysis of the interaction between Clq and immune complexes // Clin. Exp.Immunol. - 1982. - Vol. 48. - P.705-714]. The large molecular weight of Clq — about 460,000 Da — simplifies its immobilization, and the non-covalent nature of the interaction between Clq and CEC makes it possible to easily regenerate immobilized Clq using a solution with increased ionic strength. Another important advantage is the increased activity of plasma DNase I in the presence of Clq [Gaipl U.S., Beyer T. D., Heyder P. et1 al. Cooperation between Clq and DNase I in the clearance of necrotic cell-derived chromatin // Arthritis Rheum. - 2004 .-- Vol.50, N.2. - P.640-649]. When studying the prior art, data on the combined use of immobilized Clq and immobilized DNase I to reduce the level of DNA-containing CECs were not found. No data were also found on other immobilized ligands used in conjunction with immobilized DNase I to reduce the release of antibodies to DNA.

A known method of selective removal from the blood of any substance contained in it, including determining its concentration, adding specific antibodies to the solution in an amount sufficient to bind the substance to be removed as part of the IR, and extracting from the solution the CECs formed de novo using these antibodies using solid phase adsorbent based on any of the three ligands: Clq, Fc receptor, or rheumatoid factor [Liberti PA, Pollara P. Selective removal of immunospecifically recognizable substances from solution: Patent 4551435 USA // Appl. No. 06/526039; filed 08/24/1983; published November 5, 1985]. It is proposed to use a polystyrene sheet with a thickness of 0.1 mm folded into a spiral and placed in a chamber through which the processed solution is perfused in the direction corresponding to the longitudinal axis of the spiral as a carrier for these ligands. The disadvantages of this method are, firstly, a small sorption capacity due to the small working area of the carrier; secondly, the lack of magnetically controlled immobilized Clq, which complicates the manipulation of the drug. Moreover, the simultaneous use of Clq and PGM based on DNase I immobilized in this way in a single perfusion chamber is extremely difficult due to the limited distance between the turns of the spiral sheet, which disrupts the mobility of the PGM and contributes to damage to the granules.

The problem to be solved is to develop a method for reducing the content of DNA-containing circulating immune complexes by passing blood through an original combined sorbent, which is a mixture of magnetically controlled polyacrylamide granules with immobilized DNase I and magnetically controlled polyacrylamide granules with immobilized Clq (previously obtained by emulsion polymerization in a nitrogen gas stream) (hereinafter this composition is referred to as a “combined sorbent”), which reduces ejection of DNA antibodies in the bloodstream as compared to the prototype.

The technical result of the claimed invention is a decrease in the number of antibodies to DNA secreted into the bloodstream during the destruction of DNA-containing circulating immune complexes by passing blood through magnetically controlled polyacrylamide granules with immobilized DNase I. The technical result is achieved by perfusion of blood through a combined sorbent.

A method of purifying blood from DNA-containing immune complexes using a combined sorbent is as follows.

Obtaining PGM is as follows. A chemically inert gas is introduced into the vessel containing hexane and an emulsifier under pressure through a tube so as to create intense mixing. A solution of iron oxide with hydrophilic properties and ammonium persulfate is added to the flask for polymerization, then a solution containing monomers of acrylamide, methylene bisacrylamide, NNN'N'-tetra-methylethylenediamine and DNase I is added. After polymerization is complete, PGMs are washed from unreacted monomers, hexane emulsifier. PGM with immobilized Clq is obtained in the same way, the only difference from the above is the addition of Clq instead of DNase I. Next, the PGM with DNase I and PGM with Clq are mixed, the resulting combined sorbent is introduced into the column and native heparinized blood is perfused through it. The drug is regenerated by passing through a column of 1 M glycine-HCl pH 2.2 buffer twice for 10 minutes.

Example 1. Obtaining PGM with immobilized DNase I and PGM with immobilized Clq

PGM with immobilized DNase I and PGM with immobilized Clq were obtained separately. 300 mg and 100 mg of acrylamide and methylene bisacrylamide (Reanal, Hungary) were dissolved in 2 ml of a 0.15 M sodium chloride solution. To obtain PGM with immobilized DNase I, 0.6 mg of bovine pancreatic DNase I (Sigma-Aldrich, USA) was added to such a solution, and to obtain PGM with immobilized Clq, 0.6 mg of Clq (Sigma-Aldrich, USA).

At a pressure of 0.2-1 atm, nitrogen gas was introduced into a vessel containing 100 ml of hexane and 0.5 ml of SPEN-85 emulsifier through a tube with a cross section of 0.8 mm so as to create vigorous stirring for 5 minutes. In a flask for polymerization, 1 ml of physiological solution of pH 7.4 was added with 200 mg of iron oxide with hydrophilic properties and 10 mg of ammonium persulfate. After 1-2 minutes, 2 ml of a solution containing acrylamide monomers, methylene bisacrylamide, an immobilized substance (DNase I or Clq), and 20 μl of NNN'N'-tetramethylethylenediamine were added.

After completion of the polymerization, the granules were washed with acetone and physiological saline with Tween-20 detergent (0.05%) from unreacted monomers, emulsifier and hexane for 10-15 minutes. The granules had a standard spherical shape with a particle size of the gel 10-100 microns. The ratio of the polymer carrier: iron = 2: 1 (in terms of dry weight).

Example 2. Blood perfusion through a combined sorbent and prototype.

PGM was added to a 40 ml column: in the study of a combined sorbent, a mixture of PGM with immobilized DNase I and PGM with immobilized Clq in a ratio of 3: 1 (volume: volume), in the study of the prototype, PGM with immobilized DNase I. Using a column with a combined sorbent , treatment of native heparinized blood of 23 patients with systemic lupus erythematosus (SLE); for control, perfusion of blood samples of the same patients through the prototype was used. Through a column, 20 ml of native heparinized blood were perfused at a rate of 10 ml / h. Next, the combined sorbent and prototype were washed from unbound proteins with a buffered saline solution of pH 7.4 and regenerated with 1 M glycine-HCl buffer pH 2.2 twice for 10 minutes.

The serum CEC content was determined by the Haskova method of precipitation in polyethylene glycol-6000 according to Lempert modification (1988), the concentration of serum antibodies to double-stranded DNA by ELISA using commercial kits (Orgentec Diagnostika, Germany), and the content of blood cells was also evaluated ( red blood cells, white blood cells, platelets). The values of all the studied parameters for each sample were measured twice - before and after perfusion.

The dynamics of serum concentrations of CEC and antibodies to double-stranded DNA as a result of the implementation of the proposed method are shown in table 1, the dynamics of the concentration of blood cells in table 2. From these values it follows that when perfused through a combined sorbent, a decrease in the content of CEC by 69.7% is achieved the initial, and when perfusion through the prototype - by 61.8% of the original, the differences in both the absolute values of the concentration of the CEC and the rate of decrease of the latter are not statistically significant. However, when blood perfusion through the prototype, the concentration of antibodies to double-stranded DNA increases by 15.7%, and when using a combined sorbent - by 8.4%; this difference is statistically significant. When using both methods, the content of blood cells is not significantly changed. Therefore, obtained by direct comparison with the prototype, the data indicate a significant reduction in the release of antibodies to double-stranded DNA and maintaining the efficiency of the destruction of DNA-containing CECs using the proposed method.

After use, the combined sorbent and prototype were regenerated with 1 M glycine-HCl buffer, pH 2.2. After the first regeneration, the loss of specific DNase activity was 11% for both drugs; the effect on antibodies to double-stranded DNA did not change significantly. In subsequent repeated regenerations (up to 10 times), there was no loss of activity of the immobilized ligands.

MKI A61M 1/38 A61P 37/00

METHOD FOR BLOOD CLEANING FROM DNA-CONTAINING IMMUNE COMPLEXES USING A COMBINED SORBENT

Table 1 Changes in the concentrations of circulating immune complexes (CECs) and antibodies to double-stranded DNA as a result of perfusion through a combined sorbent and through a prototype Indicator Originally After perfusion p through a combined sorbent through prototype CEC 7.6 (6.7-8.5) 2.3 (1.1-3.5) 2.9 (2.0-3.8) 0.845 ∗ - value, U (M (95% CI)) - growth rate,% - -69.7 -61.8 0.671 ^ Antibodies to double-stranded DNA 46.4 (36.9-55.9) 50.3 (39.7-60.9) 53.7 (44.2-63.2) 0.029 ∗ - value, IU / ml (M (95% CI)) - growth rate,% - 8.4 15.7 0,034 ^

∗ - for the difference between the combined sorbent and prototype, unpaired student criterion

^ - for the difference between the combined sorbent and prototype, Fisher’s exact test

table 2 The change in the concentration of blood cells as a result of perfusion through a combined sorbent and through the prototype Indicator Originally After perfusion p ∗ through a combined sorbent through prototype Red blood cells, × 10 ¹² L ^-1 (M (95% CI)) 3.6 (2.4-1.8) 3.8 (2.4-5.2) 4.0 (2.6-5.4) 0.710 White blood cells × 10 ⁹ L ^-1 (M (95% CI)) 5.2 (1.8-8.6) 4.0 (1.1-6.9) 4.3 (0.6-8.0) 0.294 Platelets × 10 ⁹ L ^-1 (M (95% CI)) 272.6 (189.1-356.1) 252.7 (200.6-304.8) 245.3 (174.8-315.8) 0.924

∗ - for the difference between the combined sorbent and prototype, unpaired student criterion

Claims (1)

A method of reducing the content of DNA-containing circulating immune complexes, including passing blood through a granular preparation with immobilized DNase I based on a polyacrylamide carrier with magnetic properties, which is granulated by emulsion polymerization with a carrier: iron ratio of 2: 1, characterized in that the blood is passed through the composition consisting of, firstly, a granular preparation with immobilized DNase I based on a polyacrylamide carrier with magnetic properties and, secondly, g cured preparation with an immobilized Clq complement component based on a polyacrylamide carrier with magnetic properties, which is granulated by emulsion polymerization with a carrier: iron ratio of 2: 1.