RU2401112C2 - Pharmaceutical composition for treating autoimmune diseases associated with increased nucleic acid antibody formation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there is offered a pharmaceutical composition inducing enteral native DNA tolerance. As an active substance, the composition contains oligonucleotides immobilised on a water-soluble polymer by ionising irradiation of an aqueous polymer. The declared composition is used for treating autoimmune diseases.

EFFECT: invention provides preserving stability of the antigen responsible for the development of DNA antibodies, and protecting it from the degradation in an organism.

6 cl, 3 ex, 3 tbl

Description

Field of Invention

The invention relates to medicine, in particular to the field of creation and use of agents for the treatment of autoimmune diseases.

State of the art

Autoimmune diseases are a big problem. In some cases, antibodies are formed against nucleic acids, in particular, against DNA, as, for example, in case of systemic lupus erythematosus (SLE). However, even with chronic infectious diseases, such as hepatitis C, it is the autoimmune response to DNA that is leading in organ damage. An increased level of antibodies to DNA leads to damage to the joints, cardiovascular system, neurological disorders, obstructive bronchitis.

Known methods of treating SLE, which use hormone therapy, and in severe cases, cytostatics. However, the method has inherent disadvantages. Patients are forced to constantly take these drugs, while deep suppression of immunity leads to an increased risk of infections, damage to the intestinal mucosa, and hormonal disorders (Sentyakova TN, 2003).

The prototype of the invention is a method of inducing tolerance to a known antigen, in which the antigen is administered enterally. Induction of tolerance in this case occurs in Peyer's plaques (Kagnoff MF., 1996). A known method of preventing autoimmune diabetes in mice, in which for the formation of immunological tolerance to antigens of Langerhans cells; pancreatic hydrolyzate was introduced into mice (A.Hanninen and L.C. Harrison, 2004). This phenomenon is called enteric tolerance (oral tolerance). In this case, the emerging tolerance is not associated with total immunosuppression, therefore, there is no risk of infectious complications. However, there are currently no methods for treating SLE by inducing immunological tolerance to DNA. One of the reasons for the lack of clinically proven methods for the induction of enteric tolerance is the difficulty in delivering antigen to the lower intestine without degradation of this antigen.

An object of the present invention is to provide a method for treating autoimmune diseases associated with increased formation of antibodies to DNA based on a new pharmaceutical composition for inducing enteric tolerance to nucleic acids. The main problem associated with the well-known developments in this field is the use of “pure” DNA purified from bound protein-nucleoproteins, and this leads to the rapid degradation of nucleic acids in the gastrointestinal tract.

SUMMARY OF THE INVENTION

The present invention is directed to the creation of such a composition, which provides the induction of enteric tolerance, by maintaining the stability of the antigen responsible for the production of antibodies to DNA, protecting it from degradation in the body.

The specified technical result is achieved by the proposed pharmaceutical composition for the induction of enteric tolerance to DNA and the treatment of autoimmune diseases, due to the fact that it contains DNA of animal, bacterial, fungal or plant origin, or its fragments having antigenic properties that are immobilized on soluble polymer.

By immobilizing the DNA or oligonucleotides that make up the composition, they are protected from degradation in the body, and at the same time, they retain their antigenic properties, allowing the body to develop an immune response.

The generated immune response, including the formation of antibodies to nucleic acids, provides the formation of tolerance to native DNA.

The presence of an immune response to native DNA provides the treatment of diseases associated with increased formation of antibodies to native DNA, i.e. for the treatment of autoimmune diseases, such as systemic lupus erythematosus, bronchial asthma, vasculitis, rheumatoid arthritis, etc., for which the autoimmune response plays an important role in etiopathogenesis.

Disclosure of invention

The present invention provides a pharmaceutical composition for inducing enteric tolerance to native DNA and / or treatment of autoimmune diseases, comprising, as active substance, oligonucleotides of animal, plant, bacterial or fungal origin, where the oligonucleotides are immobilized on a water-soluble polymer.

As the active substance responsible for the development of immunity, full-sized DNA or fragments that are encompassed by the concept of oligonucleotides and which exhibit antigenic properties necessary for the formation of the body's immune response can be used. DNA or fragments thereof may be of animal, bacterial, fungal or plant origin. Typically, the oligonucleotides that are used in the composition of the invention have a molecular weight of 1000-100000 Da. It is possible to use oligonucleotides with a higher molecular weight, however, the speed and intensity of the immune response is likely to be reduced. The use of fragments of nucleic acids with a mass of less than 1000 Yes, causes a quick immune response, but not characterized by the duration of the protective effect. By varying the mass of oligonucleotides, it is possible to achieve the desired profile of the immune response, described in the coordinates "response intensity" - "duration of the protective effect."

The immobilization of oligonucleotides in the composition is carried out, in principle, on any water-soluble polymer used in pharmaceutical practice. Techniques for immobilizing oligonucleotides are known in the art. The resulting immobilized products must retain the ability to dissolve in physiological body fluids.

In the framework of the invention, various physiologically and pharmaceutically acceptable water-soluble polymers can be used as polymers. Their non-limiting list includes polymers such as polyethylene oxides (polyethylene glycols, carbovax), starches, dextrans, polyvinylpyrrolidones, sialic acids, isoprenols, acrylamides, polyvinyl acetates, acrylic acid derivatives, and the like. The determining parameter when choosing a polymer for the composition according to the invention is the solubility condition of the polymer and the resulting complex with immobilized oligonucleotides. As a rule, the molecular weight of polymers capable of dissolving in water or physiological body fluids does not exceed 10,000 Da.

As one of the options in the framework of the invention can be used polyethylene glycols with a molecular weight of 400-6000 Da. To ensure the immobilization of oligonucleotides, it is necessary that the polymer has the necessary active groups that provide binding to oligonucleotide molecules. Such groups may be, in particular, carbonyl groups.

To create such highly active groups in the polymer, radiation activation can be used, during which the initial inert polymer becomes able to interact with oligonucleotides, ensuring their immobilization. However, it should be borne in mind that the activation of a water-soluble polymer, i.e. giving it the ability to bind to oligonucleotides can be performed by other methods of the prior art, for example, traditional chemical methods.

In a particular embodiment of the invention, polyethylene glycol with a molecular weight of 400-6000 Da may be used, which is exposed to radiation for activation. In particular, a 5-50% aqueous solution of polyethylene glycol with a molecular weight of 0.4 to 6 kDa was exposed to the bremsstrahlung generated by the ILU-6 or ILU-10 accelerator with an electron energy of 2.5 MeV, the absorbed dose from 2 to 10 kGy, dose rate of 1.65 kGy / hour. However, other types of ionizing radiation can also be used (gamma radiation, laser, ultraviolet radiation) (Gonchar A.M. and Auslender V.L. 1996; Gonchar A.M. and Auslender V.L., 1998, Vereschagin E.I., et al., 2001). These types of radiation have a similar effect on a solution of polyethylene glycol, leading to the activation of this polymer.

Next, nucleic acid fragments are introduced into a solution of radiation-activated polyethylene glycol to a final concentration of 1-10 mg / ml. The mixture is stirred for 10-30 minutes until a uniform, clear or slightly opalescent solution is obtained. As the active component, purified nucleotides of animal or plant origin are used with a molecular weight of 1000-100000 Da.

The defining difference of the claimed pharmaceutical composition compared with the prototype is that the active component is not just a tissue hydrolyzate or isolated DNA, but nucleotides immobilized on a radiation-activated water-soluble polymer. When irradiated during the radiation-chemical oxidation of polyethylene glycol, highly active carbonyl groups are formed in the polymer, which ensures the immobilization of oligonucleotides.

The immobilization of oligonucleotides on polymer carriers selected from among water-soluble polymers leads to an increase in the stability of nucleic acids and an increase in the bioavailability of large biomolecules.

Thus, the composition according to the invention, due to its unique design, ensures the achievement of the claimed technical result.

The composition according to the invention, in one of the private embodiments, has the following composition:

- DNA of animal, bacterial, fungal or plant origin, or its fragments, with a molecular weight of 1000-100000 Yes;

- polyethylene glycol with a molecular weight of 400-6000 Yes, subjected to radiation-chemical oxidation.

Moreover, the ratio of components (oligonucleotides and polymer) is from 1:20 to 1: 3 by weight.

To increase the effectiveness of the composition, bacterial or animal proteinases can be added to it. The proportion of proteinases in the composition can be from 5 to 20% of the total weight of the composition (on a dry matter basis). The introduction of proteinases into the liquid composition reduces the number of foreign compounds that can cause unwanted reactions of the body. Serine proteinases, in particular subtilisins, are preferred.

The composition obtained in the form of a solution can be dried by known methods used in the field of pharmacy.

The dry product obtained by drying can be used to obtain various oral pharmaceutical forms, such as tablets, capsules, solutions, emulsions, and the like.

One of the preferred embodiments of the invention involves the preparation of tablets or capsules with a gastroenteric coating. This form of presentation of the composition contributes to additional protection of the composition and its more efficient delivery to the lower intestine.

Methods and techniques for producing dosage forms are known from the prior art and do not require detailed explanations.

The effectiveness of the proposed pharmaceutical composition in the induction of enteric tolerance to nucleic acids is demonstrated in example 1.

Example 1

The proposed pharmaceutical composition was used in animals with an induced autoimmune response to nucleic acids. The experiments were conducted on male Chinchilla rabbits (body weight 1.5-2 kg). Induction was carried out daily by subcutaneous injection of purified nucleotides at a dose of 10 mg subcutaneously in conjunction with Freund's adjuvant (0.2 ml) for 5 days. The level of antibodies to DNA was evaluated by ELISA on 10, 20 and 30 days after the first injection.

As the first control, animals were used that did not enterally enter oligonucleotides. As the second control, animals were used, which were administered enterally purified oligonucleotides isolated from salmon milk and not immobilized on PEG (25 mg / day). In the experimental groups, rabbits were injected intragastrically daily with salmon fish DNA (fragments 1000-100000 Da) immobilized on PEG 1500 using ionizing radiation at a dose of 25 mg DNA / day, however, in the second experimental group, bacterial proteinases immobilized on PEG were introduced along with DNA (subtilisins ) at a dose of 400 PE / day.

The results are presented in table 1.

Table 1 The effect of enteric-administered oligonucleotides immobilized on a water-soluble polymer on the development of an autoimmune response to nucleic acids The initial concentration of antibodies to DNA in the blood The concentration of antibodies to DNA in the blood, 10 days after 1 injection The concentration of antibodies to DNA in the blood, 20 days after 1 injection The concentration of antibodies to DNA in the blood, 30 days after 1 injection Intact 1.5 +/- 0.3 1.4 +/- 0.2 1.3 + 7-0.15 1.4 +/- 0.12 The control 1.4 +/- 0.2 11 +/- 1.3 12 +/- 0.3 12.5 +/- 0.2 Experience unimmobilized DNA 1.4 +/- 0.3 10 +/- 2.2 8 +/- 1.5 8.5 +/- 2.3 Experience immobilized DNA without proteinases 1.3 +/- 0.2 9.5 +/- 1.3 8.5 +/- 2.3 6.0 +/- 0.7 Experience immobilized DNA with proteinases 1.5 +/- 0.25 8.5 +/- 2.3 7 +/- 1.1 3.2 +/- 0.5 * ## * P <0.05 compared with the corresponding control, ## P <0.05 compared with the experiment (non-immobilized DNA)

Thus, the enteral use of oligonucleotides immobilized on water-soluble polymers is accompanied by the development of immunological tolerance to nucleic acids, the rapid extinction of the formation of antibodies to nucleic acids and can be used in the treatment of autoimmune diseases associated with the formation of antibodies to nucleic acids (systemic lupus erythematosus, vasculitis, bronchial asthma ) It is also obvious that the introduction into the composition of immobilized polyethylene oxide proteinases significantly increases the effectiveness of the composition in the induction of immunological tolerance.

Example 2

The proposed pharmaceutical composition was used in animals with an induced autoimmune response to nucleic acids. The experiments were conducted on male Chinchilla rabbits (body weight 1.5-2 kg). Induction was carried out daily by subcutaneous injection of purified nucleotides at a dose of 10 mg subcutaneously in conjunction with Freund's adjuvant (0.2 ml) for 5 days. The level of antibodies to DNA was evaluated by ELISA on 10, 20 and 30 days after the first injection.

As the first control, animals were used that did not enterally enter oligonucleotides. As the second control, animals were used, which were introduced enterically purified isolated from bifidobacteria, fragments with a molecular weight of 1000-100000 Da oligonucleotides, but not immobilized on PEG (25 mg / day). In the experimental groups, similar bacterial oligonucleotides immobilized on PEG 400 with ionizing gamma radiation at a dose of 25 mg nucleic acids / day were daily administered intragastrically to rabbits.

The results are presented in table 2.

table 2 The effect of bacterial oligonucleotides introduced enterally immobilized on a water-soluble polymer on the development of an autoimmune response to nucleic acids The initial concentration of antibodies to DNA in the blood The concentration of antibodies to DNA in the blood, 10 days after 1 injection The concentration of antibodies to DNA in the blood, 20 days after 1 injection The concentration of antibodies to DNA in the blood, 30 days after 1 injection Intact 1.5 +/- 0.1 1.4 +/- 0.3 1.3 +/- 0.15 1.4 +/- 0.5 The control 1.4 +/- 0.1 11.2 +/- 1.1 12 +/- 2.4 12.7 +/- 1.9 Experience unimmobilized DNA 1.4 +/- 0.25 9 +/- 2.1 8.5 + 7-1.7 8.1 +/- 1.9 Experience immobilized DNA 1.5 +/- 0.3 9.3 +/- 2/2 8.1 +/- 1.7 5.0 +/- 0.5 * ## * P <0.05 compared with the corresponding control, ## P <0.05 compared with the experiment (non-immobilized DNA)

Thus, bacterial oligonucleotides of molecular weight 1000-100000 Da immobilized on PEG 400 are also capable of inducing enteric tolerance and lowering the titer of antibodies to native DNA.

Example 3

Patient K., age 56 years old, sick with systemic lupus erythematosus, 24 years old. The disease is accompanied by skin lesions, arthrosis of the hip and knee joints, periodically occurring vasculitis. The level of antibodies to native DNA before taking the proposed pharmaceutical composition of 6.52 OE (normal to 1.2). The proposed pharmaceutical composition was used as a biologically active food supplement in a total daily dose of 25 mg of PEG DNA immobilized on PEG and 400 PEG subtillizin immobilized on PEG. 14 days after the start of taking the proposed pharmaceutical composition, the antibody level decreased by 40% compared to the original. The skin manifestations of the disease, joint pain, vasculitis phenomena disappeared. Remission is persistent. After another 14 days, against the background of continued administration, a further decrease in the level of antibodies was noted by another 30%, a significant improvement in well-being. With continued administration of the pharmaceutical composition, a further decrease in the titer of antibodies to native DNA was noted. The data are presented in table 3.

Table 3 The level of antibodies to native DNA in the blood serum of a patient with systemic lupus erythematosus when taking the proposed composition Initial Values (OE) 14 days 28 days 42 days The level of antibodies to native DNA in blood serum 6.52 3.91 2.75 1.92

Thus, the use of the proposed pharmaceutical composition was effective in patients with systemic lupus erythematosus, clinical improvement was combined with a decrease in the titer of antibodies to native DNA.

The claimed pharmaceutical composition can be used as an independent method for the treatment of patients with autoimmune pathologies such as systemic lupus erythematosus, as well as bronchial asthma, vasculitis, rheumatoid arthritis, i.e. autoimmune diseases associated with increased formation of antibodies to native DNA.

The above examples serve illustrative purposes and are not intended to limit the scope of claims. The scope of the claims of the present invention is determined only by the formula, and the narrative is intended solely to explain the essence of the invention. Embodiments of the invention, in light of the foregoing disclosure of the invention and illustrative examples, are obvious to specialists and can be implemented without further experimentation, using exclusively the methods and techniques described in the prior art.

Information sources

1. Sentyakova T.N. Systemic lupus erythematosus. Novosibirsk, 2003, p. 192.

2. Kagnoff MF. 1996 Oral tolerance: mechanisms and possible role in inflammatory joint diseases. Baillieres Clin Rheumatol. 1996 Feb; 10 (1): 41-54.

3. A. Hanninen, L. C. Harrison Rev Diabet Stud. 2004 Fall; 1 (3): 113-121. Mucosal Tolerance to Prevent Type 1 Diabetes: Can the Outcome Be Improved in Humans?

4. Gonchar A.M. and Auslender V.L. Immobilization of bacterial proteases on water-solved polymer by means of electron beam. Rad. Phys. Chem. 48 (1996), 795-797.

5. Gonchar A.M. and Auslender V.L. Electron beam immobilization of hydrolytic ferments having polyfunctional application. Rad. Phys. Chem. 52 (1998), 213-216.

6. E.I. Vereschagin, Do-Hung Khan, A.V. Troitskiy, O.V. Grishin, S.E. Petrov, E.P. Gulayeva, L.A. Bogdanova, M.V. Korobeinikov, V.L. Auslender Radiation Technology in the Preparation of Polyethylene Oxide Hydrophilic Gels and Immobilization of Proteases for Use in Medical Practice. Arch. Pharm. Res. 2001.V. 24, No3. P. 229-233.

Claims (6)

1. A pharmaceutical composition for the treatment of autoimmune diseases associated with increased formation of antibodies to DNA, by inducing enteric tolerance to native DNA, containing as active ingredient DNA oligonucleotides of animal or bacterial origin, immobilized on a water-soluble polymer selected from the group: polyethylene oxide, starch, dextran, polyvinylpyrrolidone, sialic acids, isoprenol, acrylamide, polyvinyl acetate, acrylic acid derivatives, by mixing these oligonucleos ide irradiated with an aqueous solution of said polymer with ionizing radiation. 2. The composition according to claim 1, additionally containing immobilized bacterial serine proteinases (subtillins). 3. The composition according to claim 1, where these oligonucleotides have a molecular weight of 1000-100000 Yes, and the ionizing radiation is gamma radiation, or electron beam radiation, or laser radiation, or ultraviolet radiation. 4. The composition according to claim 1, containing the indicated oligonucleotides with a molecular weight of 1000-100000 Yes and polyethylene glycol with a molecular weight of 400-6000 Yes in a ratio of from 1:20 to 1: 3. 5. The composition according to claim 3, containing 5-20 wt.% These oligonucleotides with a molecular weight of 1000-100000 Yes, 60-90 wt.% Polyethylene glycol with a molecular weight of 400-6000 Yes and 5-20 wt.% Proteinases of bacterial origin. 6. The composition according to any one of claims 1 to 5, presented in the form of capsules or tablets with a gastroenteric coating.