RU2280448C2 - Composition with antioxidant property and method for treatment of disease in mammals - Google Patents

Abstract

FIELD: veterinary science, pharmacy.

SUBSTANCE: invention proposes a composition for antioxidant protection of cells, tissues and a whole body against hyperproduction of free radicals in acute inflammation, chemical thermal and radiation damages. The composition comprises peroxyredoxin Prx VI and, additionally, lipoic acid and pharmaceutically acceptable additives. The composition comprises peroxyredoxin Prx VI and dihydrolipoic acid taken in the effective amount in the ratio peroxyredoxin Prx VI to dihydrolipoic acid in the range (w/w) from 1:1 to 50:1 wherein peroxyredoxin Prx VI can represents human recombinant peroxyredoxin Prx VI. Also, invention relates to a method for enhancing antioxidant protection of mammals involving delivery of indicated pharmaceutical composition is carried out into intercellular space of tissue, organ or a whole body of mammal. The delivery can be carried out by passive or active diffusion in application or spraying, by parenteral or endolumbal administration by injection, by parenteral administration, infusion, inhalation, drainage, by sublingual, vaginal or rectal administration, by nasal or ophthalmic drops. Except for, the delivery can be carried out with using other therapeutic agent, in particular, interferon simultaneously. Invention provides prophylaxis of secondary alternative damages, recovery of epithelial tissue, protection of biomacromolecules against effect of irradiation.

EFFECT: valuable medicinal properties of composition.

6 cl, 9 tbl, 11 dwg, 45 ex

Description

Technical field

The present invention relates to a method and pharmaceutical composition intended for the prevention and / or treatment of pathologies that are partially or completely caused by a violation of the balance between oxidative and antioxidant processes in cells and organisms of mammals.

State of the art

A wide range of diseases is known for the treatment of which antioxidant drugs have been shown to be effective (Babish J.G. et al. PCT Appl. WO 0230419, 2002). Such diseases include: a) lung disease, b) liver disease, c) kidney disease, d) intestinal tract disease, e) immune system disease, e) nervous system disease, g) eye disease, h) inflammatory processes, and) heart disease, j) viral infections, such as AIDS and others.

A new class of natural antioxidants that are highly soluble in water and have a wide spectrum of antioxidant activity and have been studied in vitro has now been discovered. This class includes thiol-specific antioxidants or peroxyredoxins, which are able to neutralize both organic and inorganic compounds in a wide range of their concentration (Chae H.Z. et al., 1994; McGonigle S. et al., 1998). It is known from in vitro experiments that many of the peroxyredoxins are involved in cell proliferation (Prosperi M.T. et al., 1993; Pesenko I.V. et al., 1998; Novoselov S.V. et al., 1999).

Publications are known in which the role of peroxyredoxins in cells in dysfunctions caused by arteriosclerosis (Butterfield LH, 1999), cancer (Chung YM et al., 2001; Kinnula VL et al., 2002), and nervous diseases (Kirn SH et al., 2001), lung diseases (Das KC et al., 2001; Kim HS et al., 2002), kidney diseases (Fujii T., 2001), skin diseases (Lee SC et al., 2000), ionizing radiation (Park SH et al., 2000).

Based on the homology of amino acid sequences and immunological affinity, all currently known mammalian peroxyredoxins can be classified into the following groups: Prx I-Prx IV, Prx V and Prx VI. The homology of proteins belonging to the same group is more than 90%. Peroxyredoxins Prx I-Prx V belong to the group of 2-Cys thiol-specific antioxidants. The smallest group of Prx VI, represented by 1-Cys peroxyredoxins, is very different from other groups and has only 20-40% of the total amino acid sequences with peroxyredoxins of other groups.

The peroxyredoxin family is highly conservative, and its representatives are found in the genomes of almost all living organisms from bacteria to humans. According to immunohistochemical studies obtained using light microscopy and in situ hybridization, Proxy VI peroxyredoxin is found in almost all animal tissues. However, its maximum concentration was detected mainly in tissues directly in contact with the atmosphere, namely, in the olfactory epithelium, trachea, lung bronchi, skin epidermis, and hair follicles (Novoselov S.V et al., 1999). Immunohistochemical studies using electron microscopy showed that Prx VI is the only currently identified secretory peroxyredoxin. It is synthesized in specialized cells (goblet cells of the respiratory epithelium and supporting cells of the olfactory epithelium) and is secreted into the mucus covering the cells of these epithelial tissues. Direct biochemical and immunological studies in rats showed that in trachea, bronchial tubes of the lungs and olfactory epithelium, the contribution of 28 kDa Prx VI to the neutralization of reactive oxygen species is 70-80%. Similar results were obtained for the trachea and human bronchi. For example, in the mucus of the tracheal epithelium, the concentration of Prx VI is at least 15 μg / mg protein.

A known treatment method based on increasing the level of peroxyredoxin in the cell using gene therapy. For this purpose, a vector created by genetic engineering method is used, with the help of which animal cells are transformed. A gene has been introduced into the vector that encodes the sequence of peroxyredoxin isolated from helminths (Chandrashekar R. et al., US Patent 6352836, 2002). Along with the use of the vector, the authors propose the additional use of protein as a means to stimulate the immune system of animals. It should be noted that the use of gene therapy for the treatment of mammals has not received rapid development due to the high cost and the identification of a large number of side effects.

There are known studies on the isolation of natural peroxyredoxin (Prx VI) from the olfactory organs of rats and the subsequent use of the isolated preparation to influence the chemical burn of respiratory organs of rats (Novoselov V.I. et al., 2000).

The disadvantage of this method of isolating the drug from the olfactory organs is the impossibility of its use in the preparation of natural human peroxyredoxin in preparative quantities.

There is a method for the treatment of viral diseases, including AIDS (HIV-1), in which natural and recombinant peroxyredoxins having protease activity are used as the main component of the drug (Walker B.D., Lynn R.G. PCT appl. WO 02077294, 2002). The authors found the antiviral properties of peroxyredoxins to protect cell membranes from viruses entering the cell with a single dose of 100 to 1000 mg of peroxyredoxin, depending on the patient’s weight.

The objective of the present invention is to expand the range of effective pharmacological compositions and increase their effectiveness for the treatment and prevention of diseases associated with the overproduction of free radicals caused by exogenous and endogenous factors.

SUMMARY OF THE INVENTION

The subject of the present invention is a pharmaceutical composition for antioxidant protection of cells, tissues and the body as a whole from the overproduction of free radicals in acute inflammation, chemical, thermal and radiation injuries, which as the main active substance contains an effective amount of peroxyredoxin Prx VI and dihydrolipoic acid and pharmaceutically acceptable additives . Moreover, the ratio between peroxyredxine Prx VI and dihydrolipoic acid is in the range (w / w) from 1: 1 to 50: 1.

Another object of the invention is a method of prevention and treatment, in accordance with which the delivery of the composition to the intercellular space of a tissue, organ or organism as a whole is carried out either by passive or active diffusion during applications or by spraying, or by delivery by injection into the blood, lymph, dorsal cerebral fluid.

For delivery, parenteral or endolumbar injection is used, or parenteral administration is by infusion, inhalation, drainage, or sublingual, vaginal, rectal administration is used, or drops in the nose or eyes are used.

Additionally, a therapeutic agent is used, which is used simultaneously, before or after applying a composition comprising peroxyredoxin Prx VI and dihydrolipoic acid.

List of figures

Figure 1. Inclusion of ³ H-thymidine in mouse concanavalin A stimulated lymphocytes in the presence of Prx VI. Where: 1 - medium, 2 - T cells without stimulation with concanavalin A, 3 - T cells stimulated by concanavalin A, 4 - T cells in the presence of Prx VI, 5 - T cells stimulated by concanavalin A in the presence of Prx VI.

Figure 2. The effect of Prx VI on the spectral characteristics of irradiated oxyhemoglobin. Where: 1 - oxyhemoglobin; 2 - oxyhemoglobin after gamma irradiation in the presence of 0.05 mg / ml Prx VI; 3 - oxyhemoglobin after gamma irradiation in the presence of 0.01 mg / ml Prx VI; 4 - oxyhemoglobin after gamma irradiation. It can be seen that at a concentration of 0.05 mg / ml Prx VI completely prevents the conversion of oxyhemoglobin to methemoglobin.

Figure 3. Appearance of rats 12 months after gamma irradiation (600 x-rays). Where: A - control; B - a rat with Prx VI introduced before irradiation.

Figure 4. Rat tracheal epithelium after burns with hydrochloric acid vapor and subsequent application of natural rat or recombinant human Prx VI. Application was carried out 1 hour after the burn. Where: A - control; B - epithelium one day after a chemical burn; C - epithelium one day after a chemical burn with the application of Prx VI one hour after the burn; E - epithelium of the trachea; BM is the basement membrane. Almost complete preservation of all epithelial cells is observed.

Figure 5. The restored cellular epithelium of the rat trachea 14 days after burns with hydrochloric acid vapor. Where: A - control; B - epithelium 14 days after a chemical burn; C - epithelium 14 days after a chemical burn with the application of Prx VI, E - epithelium of the trachea; VM - basement membrane, F - phagocytes. Therapy was performed one day after the burn for 5 days. Recombinant human Prx VI based pharmaceutical composition solution was applied once a day.

6. Rat tracheal epithelium after application of lipopolysaccharide. Where: A is the norm; B - one hour after application; C - 3 hours after application; E - epithelium of the trachea; BM - basement membrane; F - phagocytes. There is a significant death of epithelial cells and the appearance of a large number of phagocytic cells.

7. Rat tracheal epithelium after application of lipopolysaccharide into it and subsequent application of recombinant human Prx VI. Where: E - epithelium of the trachea; BM - basement membrane; F - phagocytes. The application of Prx VI was carried out immediately after the application of the lipopolysaccharide.

Fig. 8. Comparative characteristics of the activation of peroxyredoxin Prx VI with different thiols. Antioxidant activity was determined by the degree of protection of glutamine synthetase against the metal-catalyzed oxidative system. The degree of activation of peroxyredoxin Prx VI was determined at a molar concentration at which 50% protection of glutamine synthetase occurred. Types of thiols: 1 - dithiothreitol; 2 - dihydrolipoic acid, 3 - lipoic acid.

Fig.9. The effect of the application of Prx VI on the wound, where a) control, b), c), d) - the concentration of peroxyredoxin is respectively: 1.0; 0.1; 0.2 mg / ml.

Figure 10. The effect of the application of dihydrolipoic acid on the wound, where a) control, b), c), d) - the concentration of dihydrolipoic acid is, respectively: 1.0; 0.5; 0.1 mg / ml.

11. The effect of the application of peroxyredoxin Prx VI and dihydrolipoic acid, where a) control, b), c), d) are options in which the ratio between the constant concentration of peroxyredoxin, (1.0 mg / ml) and the variable concentration of dihydrolipoic acid is: 20: one; 2: 1; 5: 1, respectively.

Description of the invention

Noting the important role of the synthesis of peroxyredoxins in cells in response to oxidative stress, the authors of well-known publications proposed to increase the content of different types of peroxyredoxins in the cell itself.

In the course of experiments on modeling exogenous and endogenous effects on the animal organism, an unobvious fact was discovered related to the fact that delivery of a water-soluble antioxidant peroxyredoxin Prx VI and an activator in the form of dihydrolipoic acid to the intercellular space of the mammal leads to high efficiency of prevention and / or treating a large group of diseases caused by overproduction of free radicals.

This fact makes it possible to implement the claimed technical result associated with the expansion of the spectrum of effective pharmacological compositions for the treatment and prevention of diseases caused by exogenous and endogenous factors.

Another claimed technical result of the invention, related to increasing the effectiveness of prophylaxis and / or treatment, is achieved by the fact that the introduction of PrxVI peroxyredoxin and dihydrolipoic acid into the composition does not lead to the additive, but to the non-obvious, synergistic effect of the interaction of antioxidants in the intercellular space. The results of experiments on the use of different variants of the compositions (with different concentration ratios between peroxyredoxin and dihydrolipoic acid in the range (w / w) from 1: 1 to 50: 1) for treating wounds showed that the synergistic effect of the simultaneous effect on the intercellular space of peroxyredoxin Prx VI and dihydrolipoic acid can increase the effectiveness of antioxidant protection in mammals. In addition, it is possible to prevent the development of secondary alterative disorders in chemical and thermal burns and from exposure to radiation.

Increased antioxidant protection of mammals from overproduction of free radicals caused by exogenous and / or endogenous factors.

The multifunctionality of the use of peroxyredoxin Prx VI and dihydrolipoic acid as components of effective compositions for the prevention and treatment of diseases associated with the overproduction of free radicals is confirmed by examples of the prevention and treatment of mammalian diseases. Diseases can be caused by both exogenous and endogenous factors included in the group: a) ionizing radiation, b) chemical burns, c) acute inflammatory process, d) wounds. Examples given include, but are not limited to other uses of peroxyredoxin Prx VI and dihydrolipoic acid for prophylaxis and / or treatment.

In the General case, the method of increasing the antioxidant protection of mammals from the overproduction of free radicals in tissues or organs or throughout the body of mammals, is to deliver the active principle of a pharmaceutical composition comprising peroxyredoxin Prx VI and dihydrolipoic acid, as well as pharmaceutically acceptable additives in the intercellular space of tissues, organs or the body of a mammal as a whole.

The proposed method relates to different options for the prevention or treatment of tissues, organs or the whole organism of mammals in general, including:

A) preventive protection of the body, individual organs and individual areas of normal tissue from the overproduction of free radicals. The occurrence of overproduction of radicals is caused by: a) ionizing radiation, for example, when treating cancer, or when flying at high altitudes and in outer space, b) by thermal and / or chemical burns during the elimination of catastrophes and fires, c) a combination of factors according to a), b).

B) the protection of tissues, organs and the organism of mammals, in which, due to endogenous or exogenous factors, an inflammatory and / or immune process occurs associated with the overproduction of free radicals. Hyperproduction of radicals accompanies or makes the main contribution to the process of diseases of the lungs, liver, kidneys, gastrointestinal tract, immune system, nervous system, eye disease, inflammatory processes caused by bacterial or viral infections, or the protection of body cells while preventing the effects of chemotherapy in the treatment of cancer , leukemia, AIDS, or protection against the effects of ozone or other exogenous factors.

C) the protection of organs intended for transplantation, or to improve the possibility of cryopreservation.

D) protection of organs from inflammatory processes caused by mechanical damage to the skin and tissue as a result of injuries, injections of drugs or surgical operations.

Depending on the type of therapy or prophylaxis, the duration of use and dose characteristics of drugs containing the composition are determined. To clarify the dose characteristics, the concentration of Proxy VI peroxyredoxin in biological samples can be measured (see Example 8). Based on the severity of the inflammatory process, the minimum effective amount of the pharmaceutical composition is determined and treatment is carried out. It is possible to use the composition for prophylactic purposes.

The treatment procedure is selected from the group of: a) single or multiple application or spraying of a solution of the pharmaceutical composition or medicine on the affected area; b) single or multiple injections of a solution of the pharmaceutical composition; c) single or multiple administration of tablet, powder or liquid forms sublingually, or in the form of powders, pastes, suppositories, ointments, gels for application to the surface of the skin and epithelium, or by a combination of these methods.

During etiotropic therapy, the administration of drugs containing peroxyredoxin Prx VI and dihydrolipoic acid is carried out through a dropper during the period of excretion of poisons or other exogenous substances from the body. Most preferred is the use of compositions and drugs containing peroxyredoxin Prx VI and dihydrolipoic acid for complex therapies. Recently, due to the widespread prevalence of cardiovascular diseases, the immune system, treatment of AIDS, alcohol and drug addiction, drugs based on peroxyredoxin Prx VI and dihydrolipoic acid can be used in maintenance therapy along with other long-term drugs.

Delivery of the composition into the intercellular space of a tissue, organ or organism can be carried out in various ways. Since peroxyredoxin is prone to breakdown and loss of biological activity when passing through the gastrointestinal tract, the parenteral route is the main route of administration. With parenteral administration, injections, infusions, inhalations, and drainage are used. The injection solution is administered intramuscularly, intravenously in the blood, lymph, cerebrospinal fluid, in shock doses or by prolonged infusions, intraarterially; intrathecal, intraventricular, endolumbar. It is possible to use a solution, powder or tablet form sublingually. In some cases, vaginal and rectal administration of drugs is required. It is possible to administer the composition by drops in the nose or eyes, or by washing or enemas.

The methods for delivering the composition to the site of inflammation of the epithelial tissues described herein do not limit the use of other known methods for delivering biologically active polypeptides to the site of inflammation or damage.

For example, in the treatment of inflammatory processes of the upper respiratory tract, a composition containing peroxyredoxin Prx VI and dihydrolipoic acid is administered to the nose and / or trachea and / or to the bronchi of the lung. For application, either a solution of the composition is used, or the solution is sprayed first, turning it into an air-drop mixture. You can use a composition made in the form of a dry fine powder, or a composition that is immobilized in fine granules or nanoparticles with a diameter of 0.1 to 7000 nm (for example, Esenaliev, 2000), or a composition immobilized in liposomes (for example, Thibeault DWet al., 1991 )

In the treatment of skin lesions, the composition is applied in a thin layer and / or an application is made at the site of the lesion. The methods for delivering the composition to the site of inflammation of the epithelial tissues described herein do not limit the use of other known methods for delivering biologically active polypeptides to the site of inflammation or damage.

Peroxyredoxin Prx VI and dihydrolipoic acid as a radioprotector can be used as a component of drugs and pharmaceutical compositions for the prevention or treatment of a wide range of diseases. Diseases can be caused by: a) ionizing radiation (primarily during radiotherapy, preventive research, when working with radiation sources); b) cosmic radiation, which primarily affects cosmonauts and pilots; c) radionucleotide infection due to ingestion of food, water or air contaminated with radionucleotides; d) exposure to non-ionizing radiation, for example, during tomographic examination; e) exposure to the body of sources of ultraviolet radiation (sunlight, welding, pulsed light sources, displays). To prevent or reduce the effect of overproduction of free radicals, it is possible to use antidotes that contain peroxyredoxins. The composition of antidotes should be focused on the type of exogenous exposure that exists in the area in which it is necessary to carry out repair work on equipment, or eliminate accidents, or eliminate the consequences of disasters.

In the prevention or treatment of diseases associated with an ionizing or non-ionizing effect on a mammalian organism, an effective amount of peroxyredoxin Prx VI and dihydrolipoic acid is selected depending on the intensity of the ionizing or non-ionizing effect, taking into account the parameters of the mammal (its weight, age, body condition). During treatment or prevention, the selected composition is administered once or repeatedly before exposure, during exposure, or after exposure to an organism with radiation or non-ionizing radiation.

For the antioxidant protection of cells, tissues and the body as a whole from the influence of several factors (for example, from the action of ionizing radiation, and / or thermal and / or chemical burns, and / or from infection of the body with radionuclides through food, water or air), use a combined the method of applying the compositions, which consists of intravenous injections and additional applications at the site of the lesion, which reduces the toxicity of the body and prevents the process of uncontrolled cell death.

Pharmaceutical composition

Known methods for producing natural and recombinant peroxyredoxins. In the framework of the present invention, natural Prx VI was isolated from mammalian organs (Pesenko I.V. et al., 1998). Recombinant protein can be obtained using various expression systems (Sang W.K. et al., 1998; Chen J.W. et al., 2000; Peshenko I.V. et al., 2001). Options for obtaining natural and recombinant Prx VI using known methods are given in the materials and methods section (examples 1-2).

The primary structure of rat Prx VI (Andreeva S.G. et al., 1998) contains 223 amino acids with a calculated molecular weight of 24,630 Da. (EMBL / GenBank, Y17295). The gene encoding human Prx VI (Nagase T. et al., 1995) was isolated from myeloblast cells (EMB / GenBank D1 4662). According to electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight is defined as 28 kDa.

The synthesized recombinant Prx VI (see example 2) was compared with the natural rat Prx VI, which was isolated from the olfactory epithelium of a Wistar rat (see example 1).

The test results (see example 3) showed that recombinant Prx VI has antioxidant characteristics similar to the characteristics of Prx VI, which is isolated from the olfactory rat epithelium.

In the pharmaceutical composition, it is possible to use other types of peroxyredoxins (Prx I-Prx IV) since the concentration distribution of different types of peroxyredoxins is different depending on the type of tissue or organ (Knoops B. et al., 1999). It is preferable to use a recombinant human peroxyredoxin type Prx VI, which has stronger antioxidant characteristics compared to I-V types of peroxyredoxins.

For the treatment of inflammatory processes, the minimum concentration of Proxy VI peroxyredoxin in the composition is determined, firstly, based on the effective concentration of Proxy VI peroxyredoxin when protecting biomacromolecules from active oxygen species in vitro and, secondly, based on the normal tissue concentration of Prx VI peroxyredoxin .

Depending on the severity of the inflammatory process and the treatment method chosen, the concentration of Proxy VI peroxyredoxin in the liquid form of the pharmaceutical composition can be selected from 0.01 to 10.0 mg / ml.

For example, in the case of damage to the respiratory organs of the rat with a lipopolysaccharide solution and in the case of a chemical burn, the empirically obtained concentration of Proxy VI peroxyredoxin in the composition was 0.5-1.0 mg / ml with 20-50 μl of its solution applied directly to the trachea, which corresponded to 10 to 50 μg peroxyredoxin Prx VI in rat. Given the surface area of the rat trachea, this corresponds to the application of 5-10 μg of peroxyredoxin Prx VI per 1 cm ^{2 of} affected tissue. This amount of Proxy VI peroxyredoxin can be used to apply to affected tissue for any mammal, including humans.

In the case of using peroxyredoxin Prx VI and dihydrolipoic acid as a radioprotector, the amount of peroxyredoxin Prx VI introduced into the body before irradiation can be selected from 1 to 10 mg / kg of animal weight and depends on the radiation power. In the case of a sublethal dose of gamma irradiation, the empirically obtained amount of peroxyredoxin Prx VI was 2-5 mg / kg of the animal’s weight when 1 ml of its solution was injected into a vein. This amount of peroxyredoxin Prx VI can be used when administered to any mammal, including humans.

Since peroxyredoxin Prx VI is a highly soluble protein, aqueous solutions, phys. Ringer's solution, Ringer's solution and other balanced salt solutions (Dawson R.M., 1986), as well as solutions based on mono- or polysaccharides, for example glucose, and / or containing vitamins. The same solvents can be used to create the composition in airborne form, for example in the form of a spray.

In the process of experiments on modeling exogenous and endogenous effects on the animal organism, various types of activators were studied. The classic activator of peroxyredoxins is dithiothreitol (Novoselov S.V. et al., 1999). However, due to its toxicity, its use in dosage forms is excluded. One of the natural activators of peroxyredoxin VI is dihydrolipoic acid, which is non-toxic and can be obtained by restoring the SS bond of alpha-lipoic acid (1,2-dithiolan-3-pentanoic acid) widely used in medicine (Sang W.K. et al., 1998 ) Dihydrolipoic acid as an activator of peroxyredoxin, was studied in the work (Peshenko I.V. Shichi H., 2001) in in vitro experiments. The synergistic effect of the use of dihydrolipoic acid as an in vivo peroxyredoxin activator is demonstrated in Example 5.

The concentration of dihydrolipoic acid in the liquid form of the composition is selected in the range from 0.01 to 10 mg / ml depending on the concentration of peroxyredoxin (see Fig. 9). The ratio of peroxyredoxin to dihydrolipoic acid (w / w) is from 1: 1 to 50: 1.

Liquid form of the composition:

a) for intravenous administration contains from 0.01 to 0.5% (from 0.1 to 5 mg / ml), preferably from 0.01 to 0.1% (from 0.1 to 1 mg / ml), the main the active substance peroxyredoxin and dihydrolipoic acid;

b) for the treatment of wound surfaces, contains from 0.1 to 1.0% (from 1 to 10 mg / ml) of the main active substance peroxyredoxin and dihydrolipoic acid;

c) for eye drops contains from 0.1 to 0.3% (from 1 to 3 mg / ml) of the main active substance;

When creating liquid media, a solvent is used that is selected from the group of: a) balanced saline solution, b) balanced saline solution and peroxyredoxin activator in a concentration of from 0.01% to 1.0% (w / w).

The ointment or suppository form (suppositories for rectal or intravaginal administration) contains from 0.1 to 1.0 wt.% Of the main active ingredients of peroxyredoxin and dihydrolipoic acid.

The concentration of peroxyredoxin in the composition of the powder is from 0.1 to 90 wt.%. In tablet form, which is used sublingually, the concentration of peroxyredoxin can be from 0.01 to 1.0 wt.%.

When creating lyophilized forms, one or more are used as stabilizers: mono- or polysaccharides or sugar alcohols, amino acids, low molecular weight proteins, which are used for stabilization and subsequent lyophilization of the composition.

Lyophilized forms can be used to prepare liquid forms of the composition and powders selected from the group: powder for injection, infusion solutions, powder for inhalation, powder for use in ointments, gels, suspensions, powder for the preparation of tablet forms. The concentration of the active principle of peroxyredoxin in lyophilized forms can be from 10.0 to 90.0 wt.%.

A composition comprising peroxyredoxin and an activator may be used in conjunction with at least one broad-spectrum therapeutic agent.

The therapeutic agent is selected from the group consisting of: a) antibacterial, antiviral, antifungal, antihistamines, hormones, vitamins, cytokines; b) high molecular weight enzymes that provide additional protection against free radicals (superoxide dismutase, catalase, glutathione peroxidase);

c) low molecular weight compounds that provide an additional reduction in the level of free radicals inside the cell (tocopherol glutathione, ubiquinone); d) preparations used for transplantation or cryopreservation of organs; e) biologically active proteins, for example, insulin.

A therapeutic agent used in conjunction with peroxyredoxin should not inhibit the biological activity of peroxyredoxin as an enzyme.

When generalizing the inflammatory process (for example, when sepsis occurs) involving several organs and / or systems, it is recommended to carry out combination therapy and use antibiotics and / or glucocorticoids in combination with the use of peroxyredoxin and activator.

The presented forms of the composition and methods of its use do not limit other options that are known from the field of medicine or veterinary medicine when using antioxidant therapy. Variations arising from this invention and obvious to every specialist having an average level of knowledge in this field should be considered within the scope of the invention.

1. Materials and methods for producing peroxyredoxin and studies of its properties.

Several methods for producing peroxyredoxins are known. In the framework of the present invention, peroxyredoxin was isolated from mammalian organs (Pesenko I.V. et al. 1998), or a recombinant protein was obtained using various expression systems (Sang Won Kang et al. 1998, Chen J.W. et al. 2000).

Example 1. Obtaining natural Prx VI rat peroxyredoxin

Obtaining natural rat Prx VI peroxyredoxin was performed according to the following procedure. Natural Prx VI rat peroxyredoxin was isolated from the Wistar rat olfactory epithelium, which contains the maximum amount of this protein. The isolated olfactory epithelium was washed twice in physiological saline and homogenized in it. The homogenate was centrifuged for 5 min at 500 g, the supernatant was repeatedly centrifuged twice at 20,000 g and dialyzed for 12 hours against solution A, which included: 12 mM Tris / HCl, pH 7.8, 1 mM MgCl ₂ , 1 mM dithiothreitol. After dialysis, the extract was applied to a chromatographic column (305 × 12.5 mm) filled with DEAE-Sepharose gel (DEAE-Sepharose, Pharmacia) and pre-equilibrated with solution A. Proteins were eluted with a linear NaCl gradient from 0 mM to 500 mM in solution A at 17 at ° C. The volume of the solution was 750 ml; the elution rate was 0.7 ml / min. Fractions were analyzed by antioxidant activity. Fractions containing Prx VI peroxyredoxin were concentrated and then chromatographed on a column filled with Sephacryl S-200 gel (820 × 16 mm). The column was eluted with buffer B: 25 mM Tris / HCl, pH 7.8, 100 mM NaCl, 1 mM MgCl ₂ , 1 mM dithiothreitol. The elution rate is 0.6 ml / min at 4 ° C. Fractions containing Prx VI peroxyredoxin were collected, concentrated, dialyzed against saline and used in further experiments.

Example 2. Obtaining human recombinant Prx VI peroxyredoxin

Human recombinant Prx VI peroxyredoxin was prepared as follows. The complementary DNA encoding human Prx VI peroxyredoxin was isolated from clone HA0683 (T. Nagase et al., 1995) (GenBank, D 14662) by known methods. DNA was then cloned into the expression plasmid pET-23a (+) from Novagen at the NdeI and EcoRI restriction sites using MBI Fermentas restriction enzymes. The obtained plasmid was used to transform E. coli cells (strain BL 21 (DE3), from the library of strains of IBPM RAS) to obtain recombinant human Prx VI peroxyredoxin. E. coli cells expressing peroxyredoxin were destroyed by ultrasound on a UZDN-2 generator at a frequency of 22 kHz for five minutes at 0 ° C. The homogenate was centrifuged for 5 min at 500 g, the supernatant was repeatedly centrifuged twice at 20,000 g and dialyzed for 12 hours against solution A (see example 2). Recombinant Prx VI peroxyredoxin was isolated by isolation of a natural rat Prx VI peroxyredoxin (see Example 1).

Example 3. Comparative characteristics of rat natural and human recombinant natural peroxyredoxins Prx VI.

Recombinant human peroxyredoxin Prx VI (GenBank, D 14662) is compared in terms of antioxidant activity to natural rat peroxyredoxin Prx VI (EMBL / GenBank, Y 17295). To determine the activity of peroxyredoxin Prx VI use its ability to protect glutamine synthetase against DTT / Fe ⁺³ / O ₂ oxidative system. The 60 μl incubation mixture, which contains 5 μg glutamine synthetase, 3 mM DTT, 3 μM Fe ³⁺ , 50 mM HEPES, pH 7.3, is incubated for 10 min at 37 ° C. As an activator of Prx VI peroxyredoxin, dihydrolipoic acid is used at a concentration of 3 mM. The residual activity of glutamine synthetase is determined by pouring 200 μl of the reaction mixture into the sample, which contains: ADP - 0.4 mm, glutamine - 150 mm, K-AsO ₄ - 10 mm, NH ₂ OH - 20 mm, MnCl ₂ - 0.4 mm, HEPES - 100 mm pH 7.4. After incubation for 10 min at 37 ° C, 100 μl of dye is added to the sample. The composition of the dye includes: 5.5 g of FeCl ₃ · 6H ₂ O, 2 g of TCA, 2.1 ml conc. HCl (38%) per 100 ml of H ₂ O. The degree of peroxyredoxin activity is determined by the value of the protein concentration at which a 50% preservation of glutamine synthetase activity was observed. Recombinant peroxyredoxin Prx VI has antioxidant characteristics similar to natural rat peroxyredoxin Prx VI. Subsequently, the human recombinant peroxyredoxin Prx VI is used to develop treatment methods or as a component of pharmaceutical compositions.

Example 4. Determination of cytotoxicity of peroxyredoxin Prx VI

The cytotoxicity of peroxyredoxins is determined by the effect of Prx VI peroxyredoxin on the proliferation level of L929 cells of human lymphoblastoma and T lymphocytes from the spleen of NRRI mice stimulated with concanavalin A.

Cells of two lines with a concentration of 10 ⁴ cells / ml of medium were cultured in RPMI 1640 medium containing 5% bovine fetal serum. T-lymphocyte stimulation was performed at a concanavalin A concentration of 0.1 μg / ml. In the case of T-lymphocytes, the proliferation level was determined by the inclusion of ³ H-thymidine. The number of living cells of the L929 line was determined in a 96-well plate using a trenanic dye followed by scanning with a Multiscan multichannel photometer (LKB, Sweden).

Natural rat and recombinant human Prx VI peroxyredoxins (at a concentration of 0.1-10 mg / ml) had no effect on the number of living cells of the L929 line.

In the case of T-lymphocytes stimulated with concanavalin A, the cell proliferation level increased approximately 2-fold in the presence of natural rat or recombinant human Prx VI peroxyredoxin (at a concentration of 0.1-1.0 mg / ml) compared to simply stimulated with concanavalin A cells (see Fig. one). Thus, we can conclude that the tested compounds are low toxic.

Example 5. Comparative characteristics of peroxyredoxin activators.

To determine the effectiveness of Prx VI activation with dithiothreitol and dihydrolipoic acid (a natural activator), the ability of peroxyredoxin to protect glutamine synthetase from inactivation caused by a metal-catalyzed oxidation system was used. The 60 μl incubation mixture contained 5 μg glutamine synthetase, 50 μg Prx VI, 3 mM ascorbic acid, 3 μM Fe ⁺³ , 50 mM HEPES, pH 7.3, and dithiothreitol or dihydrolipoic acid of various concentrations, incubated for 10 min at 37 ° C. The residual glutamine synthetase activity was determined by pouring 200 μl of the reaction mixture into the sample (ADP - 0.4 mM, glugamine - 150 mM, K-AsO ₄ - 10 mM, NH ₂ OH - 20 mM, MnCl ₂ - 0.4 mM, HEPES - 100 mM, pH 7.4 ) After incubation for 10 min at 37 ° C, 100 μl of dye was added to the sample. The dye contains 5.5 g of FeCl ₃ · 6H ₂ O, 2 g of TCA, 2.1 ml of conc. HCl (38%) per 100 ml of H ₂ O. The degree of peroxyredoxin activity was determined by the value of the protein concentration at which a 50% preservation of glutamine synthetase activity was observed.

On Fig presents the degree of protection of glutamine synthetase depending on the concentration of thiols. As can be seen from the graphs, the antioxidant effectiveness of dihydrolipoic acid is almost identical to dithiothreitol.

Example 6. The distribution of peroxyredoxin Prx VI in the animal after injection.

To determine the distribution of exogenous peroxyredoxin Prx VI in various tissues of the body, recombinant human Prx VI, labeled with fluorescentisothiocyanate in the amount of 10 mg per animal, was introduced into the rat vein. At certain time intervals, animals were killed, tissues of various organs were isolated, and luminescent analysis of the obtained samples was performed. The data obtained showed that 15 minutes after the injection of labeled peroxyredoxin Prx VI, the latter is evenly distributed over all organs of the rat, including the brain. The exception is bone marrow, where the amount of exogenous peroxyredoxin Prx VI was small compared with other tissues. Thus, the method of introducing peroxyredoxin Prx VI by injection into a vein allows to increase its content in almost all body tissues.

2. Materials and methods for determining peroxyredoxin in samples and analysis of the properties of peroxyredoxin in in vitro and in vivo studies.

Example 7. The protection of oxyhemoglobin from the action of ionizing radiation using peroxyredoxin Prx VI.

The experiments showed that after exposure to mammals with a lethal dose of radiation, the cellular capabilities of the synthesis of antioxidant enzymes are limited and their concentration is not enough even with prior stimulation of the cellular synthesis of peroxyredoxin.

To test the possibility of peroxyredoxin protection of biomacromolecules from degradation caused by ionizing radiation, we studied the characteristics of irradiated oxyhemoglobin in the control and in the presence of peroxyredoxin Prx VI. In the control, a solution of oxyhemoglobin (0.16 D at 540 nm) in 0.1 M Tris-HCl (pH 7.0) was used, which was irradiated in a GUBE unit with a total radiation dose of 10 Gy. Peroxiredoxin Prx VI was used as a radioprotector at various concentrations (0.005-0.1 mg / ml) in the presence of 50 μM dithiothreitol as a peroxyredoxin activator. The formation of oxidized forms of oxyhemoglobin (methemoglobin) was recorded at 635 nm, and aggregation of methemoglobin was recorded at 700 nm.

Irradiation of a hemoglobin solution leads to significant changes in the structure. hemoglobin (the appearance of peaks at 635 nm and an increase in light scattering, see figure 2), which indicates the conversion of oxyhemoglobin to methemoglobin with its subsequent aggregation.

Adding Prx VI to a hemoglobin solution at a concentration of about 0.05 mg / ml before irradiation significantly reduces the formation of oxidized forms of hemoglobin, the concentration of which decreased to almost zero.

Example 8. Determination of peroxyredoxin Prx VI in biological samples.

Polyclonal rabbit antibodies against recombinant peroxyredoxin were obtained in a standard manner by double immunization of an animal with recombinant human peroxyredoxin. The titer of the obtained antibodies by ELISA was 1: 10000, by immunoblotting - 1: 2000. The specificity of the antibodies was checked by immunoblotting using a water-soluble extract of the human trachea and immunohistochemical methods using paraffin sections of the human lungs, which showed a high degree of specificity of the obtained antibodies.

Purified rabbit antibodies were prepared from serum by precipitation with ammonium sulfate, followed by chromatography on DEAE cellulose. The conjugates of antibodies with sewn horseradish peroxidase were weighed by the standard method of periodic oxidation according to the method of Wilson and Nakane with a molar ratio of immunoglobulin to peroxidase equal to 1: 3.

The conjugate titer for human peroxyredoxin was: for ELISA 1: 1000 and for immunoblotting and immunohistochemistry 1: 500. The specificity of the conjugate corresponded to the specificity of the serum that was used to produce IgG.

Enzyme immunoassay protocol for peroxyredoxin Prx VI in biological samples:

1. 200 μl of a solution of immunoglobulin G against human recombinant peroxyredoxin (1: 1000 dilution) is poured into the wells of the plate and incubated for 30 minutes at room temperature.

2. The plates are washed with saline (3 × 5 min) and blocked with 1% milk powder for 30 min at room temperature.

3. 200 μl of test samples are poured into the wells and incubated for 1 hour at room temperature.

4. The plates are washed with saline containing 0.1% Tween 40 (3 × 5 min).

5. 200 μl of conjugate solution (1: 500 dilution) is added to the plate wells and incubated for 30 minutes at room temperature.

6. The plates are washed with saline containing 0.1% Tween 40 (3 × 5 min).

7. ABTS solution is poured into the wells (25 mg ABTS in 25 ml citrate buffer, pH 4.0 + 40 μl 3% peroxide).

8. The plates are scanned on a Multiscan at 405 nm.

The sensitivity of the system is tens of nanograms per milliliter of sample.

Example 9. Immunohistochemical detection of peroxyredoxin Prx VI in human tissues.

The isolated tissue was fixed in 4% formaldehyde and 0.25% glutaraldehyde in 20 mM phosphate buffer containing 0.15 M NaCl. Fixation was carried out for 12 hours at 37 ° C. The tissue was washed in saline 4 times for an hour. The tissue was dehydrated by sequential wiring through ethanol with increasing concentration (40-100%). The tissue was washed with xylene-alcohol solution (1: 1) for an hour, xylene and xylene-paraffin (1: 1) for 12 hours. The tissue was embedded in paraffin and received 5 micron sections on a microtome. Slices were dewaxed in xylene and with decreasing alcohol concentrations. Sections were washed with saline, blocked with 5% fetal serum, endogenous peroxidase was inhibited with 0.3% hydrogen peroxide and incubated with the conjugate for an hour, washed three times with saline and stained with diaminobenzidine (6 mg per 10 ml of 50 mM Tris-HCl buffer, pH 7.6 plus 200 μl of 3% hydrogen peroxide).

3. Examples of pharmaceutical compositions (options), which include peroxyredoxin or peroxyredoxin and dihydrolipoic acid.

The forms of the composition include: dry preparation, liquid form, ointment or gel form.

In general, the technology for preparing compositions consists of the following operations: preparation of a room for work, preparation of equipment for packaging, sterilization of dishes, preparation of the main components of the composition, packaging and control.

3.1. Preparation of dry formulations

3.1.1. Lyophilized peroxyredoxin preparation

The technology for producing a dry lyophilized preparation includes the following operations: isolation of natural or synthesis of recombinant peroxyredoxin; purification of the substance using known methods of purification of biologically active polypeptides and proteins, the introduction of at least one stabilizer, followed by lyophilization of a solution of peroxyredoxin and a stabilizer.

The stabilizers can be selected from the group consisting of: a) one or more mono- or polysaccharides or sugar alcohols, b) amino acids, c) polypeptides or proteins. As examples of the first group, for example, maltose, glucose, lactose, trehalose, sucrose, mannitol, inositol, galactose, ribose, xylose, mannose, sucrose, cellobiose, raffinose and maltotriosis can be mentioned. The amino acid group may include, for example, alanine, glycine. Polypeptides and proteins may be, for example, albumin.

Stabilizers are necessary to maintain the activity of peroxyredoxin for a longer period, at least one year, and in more affordable conditions, at temperatures from +4 to -20 ° C, depending on the shelf life.

Example 10. Obtaining a lyophilized preparation of peroxyredoxin for the preparation of liquid, solid and ointment pharmaceutical compositions.

Fractions containing peroxyredoxin VI (see Examples 1, 2) were concentrated to a concentration of 5-10 mg / ml using Ultrafree-15 10K concentrators using a centrifuge (15 min, 2000 g) and dialyzed against deionized water. At least one stabilizer is added to the peroxyredoxin solution and lyophilized at -20 ° C until a dry powder is formed.

The lyophilized preparation is packaged in bottles sterilized in an autoclave at 120 ° C (depending on the application, in doses from 1 to 100 mg and stored at -20 ° C).

Get the finished product of the following composition:

Lyophilized preparation for the preparation of liquid, solid and ointment pharmaceutical compositions using peroxyredoxin and stabilizers (wt.%):

Peroxyredoxin - about 90;

The stabilizer is about 10.

3.1.2. Peroxyredoxin Powder

The technology for producing a dry powder, for example, intended for inhalation or for preparing other forms of the composition, includes the following operations: isolation of natural or synthesis of recombinant peroxyredoxin; purification of the substance using known methods for purifying biologically active polypeptides and proteins, introducing at least one stabilizer into the peroxyredoxin solution, adding nanoparticle additives to the peroxyredoxin and stabilizer solution (for example, microcrystalline cellulose, hydroxypropyl methylcellulose, lactose monohydrate), granules or liposomes, followed by lyophilization or drying of the resulting preparation.

For the manufacture of aerosol preparations using known methods of dispensing biologically active polypeptides in the form of powders, aqueous solutions or using liquefied gas (Bekstrom et al. Patent RU 2175866, 2001). A person of ordinary skill in the art can easily determine the appropriate dosages of these aerosol formulations without undue experimentation.

Example 11. Obtaining peroxyredoxin in powder form for the preparation of pharmaceutical compositions using additives in the form of nanoparticles. granules or liposomes

Fractions containing peroxyredoxin VI (see examples 1, 2) were concentrated to a value of 5-10 mg / ml using Ultrafree-15 10K concentrators and using a centrifuge (15 min, 2000 g). The fraction is dialyzed against deionized water. Stabilizers and additives in the form of nanoparticles, granules or liposomes are introduced into the peroxyredoxin solution and then lyophilized at -20 ° C until a dry powder is formed. The lyophilized preparation is packaged in bottles sterilized in an autoclave at 120 ° C (depending on the application, in doses from 1 to 100 mg and stored at -20 ° C).

Powdered preparation for the preparation of pharmaceutical compositions using additives in the form of nanoparticles, granules or liposomes (wt.%):

Peroxyredoxin - 0.1-90.0;

The stabilizer is 0.01-10;

Additive - the rest.

3.1.3. Tablet peroxyredoxin in tablet form

The technology for producing a tablet form of peroxyredoxin includes the following operations: isolation of natural or synthesis of recombinant peroxyredoxin; purification of the substance using known methods for the purification of biologically active polypeptides and proteins; introducing into the solution at least one stabilizer; lyophilization or drying of the drug; the stage of mixing the active principle with the main fillers; wet and dry granulation; dusting of dry granulate and tableting.

As a filler, you can use lactose monohydrate, corn starch, talc, for dusting, you can use magnesium stearate.

Example 12. A tablet form of peroxyredoxin for the preparation of pharmaceutical compositions

The drug in tablet form for the preparation of pharmaceutical compositions (wt.%):

Peroxyredoxin - 0.1-1.0;

Stabilizer - 0.01-0.1;

Additives and fillers - the rest.

3.2. Preparation of liquid forms of the composition

The technology for producing liquid forms is based on the use of a dry lyophilized form of the composition.

Example 13. A solution for creating a liquid form of a composition for intravenous administration, inhalation and irrigation based on peroxyredoxin.

A dry form preparation of a peroxyredoxin preparation is prepared in accordance with Example 10. Immediately before use, a dry powder of lyophilized peroxyredoxin is dissolved in distilled water or in saline in the required concentration. Solutions are used immediately or stored before use at + 4 ° C. For prevention from the damaging effects of ionizing radiation, a composition of the following composition is selected (wt.%):

Peroxyredoxin 0.1;

The stabilizer is about 0.01;

Saline solution - the rest.

Examples 14-16.

The process described in Example 13 is repeated, but using the mass amounts of ingredients indicated in Table 1 below, instead of the corresponding amounts used in this example:

Table 1.
Peroxyredoxin Content in Liquid Form Example Peroxyredoxin (wt.%) The stabilizer (wt.%) Saline (wt.%) fourteen 0.01 0.001 99,99 fifteen 0.5 0.05 99.45 16 1,0 0.1 98.9

Example 17. A solution for intravenous administration, inhalation and irrigation using dihydrolipoic acid

A peroxyredoxin powder preparation is prepared in accordance with Example 10. Immediately before use, the lyophilized peroxyredoxin is dissolved in saline in the required concentration. A solution of dihydrolipoic acid is prepared. Solutions are used immediately or stored before use at + 4 ° C. Immediately before use, solutions of peroxyredoxin and dihydrolipoic acid are mixed. For the treatment of small wounds, a composition of the following composition is selected (wt.%):

peroxyredoxin 0.05;

stabilizer 0.005;

dihydrolipoic acid 0.01;

saline - the rest.

Examples 18-24.

For the preparation of compositions with other objects of treatment or prophylaxis, the process described in example 17 is repeated, but the mass amounts of ingredients indicated in the following table 2 are used instead of the corresponding quantities used in the indicated example:

Table 2.
The content of peroxyredoxin and dihydrolipoic acid in the liquid form of the composition Example Peroxyredoxin (wt.%) The stabilizer (wt.%) Dihydrolipoic acid (wt.%) Saline (wt.%) eighteen 0.01 0.001 0.01 99.98 19 0.5 0.05 0.5 98.95 twenty 1,0 0.1 1,0 97.9 21 0.5 0.05 0.02 99.43 22 1,0 0.1 0.04 98.86 23 0.5 0.05 0.01 99.44 24 1,0 0.1 0.02 98.88

Example 25. A solution for creating a pharmacological composition for intravenous administration of inhalation or irrigation using peroxyredoxin and a therapeutic agent.

A peroxyredoxin powder preparation is prepared in accordance with Example 10. Immediately before use, the lyophilized peroxyredoxin is dissolved in saline in the required concentration. An interferon solution is prepared. Solutions are used immediately or stored before use at + 4 ° C. Mix solutions of peroxyredoxin and a solution of interferon (wt.%):

peroxyredoxin 0.5;

stabilizer 0.05;

interferon ME 100000;

Saline solution - the rest.

For the preparation of compositions with other objects of treatment or prophylaxis, the process described in Example 25 is repeated, but the mass amounts of ingredients indicated in Table 3 below are used instead of the corresponding quantities used in this example:

Table 3.
The content of peroxyredoxin and interferon in liquid form Example Peroxyredoxin (wt.%) The stabilizer (wt.%) interferon (ME) Saline (wt.%) 26 0.01 0.001 100,000 99.98 27 1,0 0.1 100,000 98.9

Example 28. A solution for intravenous administration of inhalation or irrigation using peroxyredoxin, dihydrolipoic acid and a therapeutic agent

A peroxyredoxin powder preparation is prepared in accordance with Example 10. Immediately before use, the lyophilized peroxyredoxin is dissolved in saline in the required concentration. An interferon solution is prepared. A solution of dihydrolipoic acid is prepared. Solutions are used immediately or stored before use at + 4 ° C. Mix solutions of peroxyredoxin, dihydrolipoic acid and a solution of interferon (wt.%):

peroxyredoxin 0.5;

stabilizer 0.05;

dihydrolipoic acid 0.05;

interferon ME 200000;

Saline solution - the rest.

For the preparation of other compositions, the process described in Example 28 is repeated, but using the mass amounts of ingredients indicated in Table 4 below, instead of the corresponding amounts used in this example:

Table 4.
The content of peroxyredoxin, dihydrolipoic acid and interferon in the liquid form of the composition Example Peroxyredoxin (wt.%) The stabilizer (wt.%) Dihydrolipoic acid (wt.%) Interferon (ME) Saline (wt.%) 29th 0.01 0.001 0.01 100,000 99.98 thirty 0.1 0.01 0.01 100,000 99.88 31 1,0 0.1 0.5 500,000 98.4

Example 32 Cosmetic

It is known that antioxidant additives introduced into the cream prevent oxidative reactions, protect the skin from adverse external influences (for example, UV rays), carry anti-inflammatory, antibacterial activity, and increase immunity. In addition, they prevent the peroxidation of oils contained in the cream or emulsion. The following example relates to a composition consisting of an aqueous base in which the active principle of a composition containing peroxyredoxin and superoxide dismutase (SOD) is dissolved, a fat base that improves the quality of cream and excipients that contain biologically active and antioxidant substances, for example, wheat germ oil , grape seed oil and additionally contain low molecular weight antioxidants such as tocopherol in the following ratio of ingredients (wt.%):

fat base 6-10;

peroxyredoxin 0.1-0.5;

superoxide dismutase 0.01-0.05;

excipients 2-6;

water base - the rest.

The interaction of inexpensive low molecular weight antioxidants, which restore or protect against oxidation of high molecular weight antioxidants during storage, can reduce the concentration of expensive and highly effective antioxidants such as peroxyredoxin or superoxide dismutase and maximize the protective effect of the cream.

The examples given should not be considered in the context of limiting the areas of use of the present invention. Variations arising from the present invention and obvious to every specialist having an average level of knowledge in this field should be considered within the scope of the alleged invention.

Example 33. The protective effect of peroxyredoxin Prx VI with the damaging effect of ionizing radiation.

Simultaneous irradiation of all animals (Wistar rats weighing 200 g) was carried out on the installation of HUBE. The radiation dose is 6 Gy. Rats were divided into two groups. The first group of animals (n = 6) was used for control. Animals of the second group (n = 6) 30-60 minutes before irradiation were injected intravenously with 1 ml of saline with a concentration of peroxyredoxin of 1 mg / ml. After irradiation, the animals were kept in a common room with sufficient feed, lighting and ventilation. For histological examination, animals were selected from each group with signs of deteriorating general condition.

results

Data on mammalian mortality after irradiation are presented in Table 5, which shows the number of surviving rats in each group at the indicated time.

Table 5.
The survival of rats with the introduction of peroxyredoxin Prx VI before irradiation. Experiment time 3 months 6 months 12 months 15 months Survival in the control group (pcs.) four 3 2 one Survival in the group in which Prx VI (pcs.) Was used as a tread 6 5 four four

As can be seen from the table, the mortality of rats, which were introduced peroxiredoxin Prx VI before irradiation, is significantly lower than in the control group. The general condition of the surviving animals one year after irradiation was significantly different in favor of the second group compared with the control group (figb). In animals from the first group, compared with animals of the second group, there is a large loss of body weight (cachexia) (weight 180-200 g), poor body hair growth, low locomotor activity, and the presence of a tumor process.

Example 34. Therapeutic and protective properties of peroxyredoxin Prx VI in the treatment of rat upper respiratory tract after a chemical burn

The therapeutic properties of peroxyredoxin Prx VI in the treatment of rat upper respiratory tract one day after a burn was tested on a group of 4 Wistar rats weighing 150-200 grams.

Chemical burn of the upper respiratory tract.

Rats were analgesic with analgin (100 mg / kg body weight) and placed in a desiccator saturated with hydrochloric acid vapors for 10 min (desiccator volume - 10 liters) (20 animals were used in total). After a chemical burn, the rats were kept for a certain time (40 min, 6 hours, 1 day, 2 days, 14 days, 30 days), euthanized with hexenal (300 mg / kg body weight) and a sample of tracheal epithelial tissue was isolated. For histochemical studies, the isolated sample was placed in a fixing solution containing 2% formaldehyde and 0.5% glutaraldehyde. The tissue was dehydrated by sequential wiring through ethanol with increasing concentration (40-100%). The fabric was poured into LR white resin. The resin was polymerized at room temperature. Sections with a thickness of 0.5 μm were obtained on an LKB ultratome, Sweden, using glass knives. Sections were stained with a mixture of hematoxylin-eosin, analyzed using a light microscope.

To develop a more effective method of treatment, the stages of the development of pathology after exposure to a burn were studied.

At the first stage, we studied the dynamics of changes in the level of neutrophils and antioxidant enzymes in the cellular epithelium of rat trachea after burns with hydrochloric acid vapors. Two periods of a sharp increase in the number of active neutrophils were observed: 40 minutes and 6 hours after the burn. A sharp increase in the number of active neutrophils aggravates the degree of damage, since the release of hydrogen peroxide by neutrophils.

Biochemical studies (see example 9) showed that the level of peroxyredoxin activity in epithelial cells increases approximately twice immediately after a burn. At the same time, the activity of superoxide dismutase, catalase and glutathione peroxidase, which are the main antioxidant enzymes in the body, decreases by about 30% in the mucus of the trachea. After a day, a decrease in the activity level of all of the listed enzymes, including peroxyredoxin, was observed.

Histochemical studies (see example 10) showed that 40 minutes after the burn in the epithelium of the trachea and bronchi, the processes of death of the cells of the ciliary epithelium begin, which subsequently have an avalanche-like character. The maximum death of epithelial cells of the mucosa of the upper respiratory tract is observed one day after the burn. In this case, almost complete death of the cells of the ciliated epithelium occurs, which carries out mucociliary transport in the trachea and bronchi. This condition of the epithelium was taken as a control for a comparative assessment of the degree of burn and the effectiveness of the treatment.

Without treatment, a partial restoration of the epithelium of the tracheal mucosa after a chemical burn is observed after 30 days.

In Fig.4b shows a section of the epithelium of the trachea of the rat one day after the burn, Fig.7 - 14 days after the burn. The maximum destruction of the epithelium is observed a day after the burn, while the ciliated cells that provide the mucus flow in the trachea are practically absent. After two weeks, partial restoration of ciliary cells is observed.

Protective and therapeutic properties of peroxyredoxin

The application of the solution of the composition was started one hour after the burn (the protective effect of peroxyredoxin Prx VI) or one day after the chemical burn (time of maximum destruction of the epithelium of the tracheal mucosa, therapeutic effect of peroxyredoxin Prx VI). In the latter case, the application was performed once a day for 5 days. Rats were pre-euthanized by intraperitoneal injection of hexenal (70 mg / kg), the animals were fixed on the operating table and under visual control using a binocular loupe through a catheter with a diameter of 1 mm they were injected into the trachea with a solution of peroxyredoxin from 20 to 100 μl. The concentration of peroxyredoxin was selected in the range from 0.5 to 5.0 mg / ml. A 0.9% NaCl solution was used as a solvent.

Histological studies were performed one day after the burn (in the case of the protective effect of peroxyredoxin and 14 days in the case of the therapeutic effect of peroxyredoxin. Histochemical studies were carried out according to the procedure described in example 10.

results

As one of the treatment options for mammals after a chemical burn, the application of the rat upper respiratory tract with a solution of peroxyredoxin was used. Figure 5 shows a section of the epithelium of the rat trachea 14 days after the burn with daily 5-fold application of peroxyredoxin. Almost completely preserved epithelium of the tracheal mucosa is observed, which indicates almost complete regeneration of ciliated epithelial cells. The presence of elements of the mucous secretion, in which phagocytes are determined, indicates the presence of a residual inflammatory process in the trachea.

Example 35. Therapeutic properties of peroxyredoxin Prx VI in the treatment of rat upper respiratory tract immediately after exposure to an LPS solution.

The therapeutic properties of peroxyredoxin Prx VI in the treatment of rat upper respiratory tract immediately after application of LPS was tested on a group of 4 Wistar rats weighing 150-200 grams.

Application of lipopolysaccharide in rat trachea.

Rats were anesthetized with hexenal and 100 μl of a lipopolysaccharide solution was injected through a catheter (concentration of lipopolysaccharide 10–9 - 10–11 mg / ml, 20 animals were used in total). After application of the lipopolysaccharide, a certain time was kept (1 hour, 3 hours, 6 hours, 7 hours, 9 hours), euthanized with hexenal (300 mg / kg body weight) and a sample of tracheal epithelial tissue was isolated. For histochemical studies, the isolated sample was placed in a fixing solution containing 2% formaldehyde and 0.5% glutaraldehyde. The tissue was dehydrated by sequential wiring through ethanol with increasing concentration (40-100%). The fabric was poured into LR white resin. The resin was polymerized at room temperature. Sections with a thickness of 0.5 μm were obtained on an LKB ultratome, Sweden, using glass knives. Sections were stained with a mixture of hematoxylin-eosin, analyzed using a light microscope.

After the application of a lipopolysaccharide causing an acute inflammatory reaction in the trachea, the dynamics of changes in the level of neutrophils, TNF-α and peroxyredoxin Prx VI in the cellular epithelium of rat trachea was studied. To determine the characteristics of the treatment method, we studied the dynamics of the level of neutrophils and antioxidants, as well as biochemical and histochemical studies of the effects of the inflammatory process on the body of a mammal.

A sharp increase in the number of active neutrophils was observed an hour after the administration of LPS. A sharp increase in the number of active neutrophils, in turn, exacerbates the degree of damage, since there is an emission of H ₂ O ₂ neutrophils.

Biochemical studies (see example 9) showed that the level of activity of peroxyredoxin Prx VI in epithelial cells increases about two times an hour after the application of lipopolysaccharide.

Histological studies (see example 10) showed that in the case of applying an LPS concentration of 10 ^-7 mg / animal, there was a massive accumulation of neutrophils in the tracheal wall, the development of edema, followed by delamination of the mucosa into the lumen of the trachea and cell death after 3 hours (see Fig. 6).

Immunohistochemical studies showed that in many areas of the tracheal epithelium an mass death of cells secreting Prx VI peroxyredoxin is observed already in an hour, which led to the absence of Prx VI peroxyredoxin in the mucus covering these tracheal regions. This condition of the epithelium was taken as a control for a comparative assessment of the degree of inflammatory reaction and the effectiveness of the treatment.

On figb shows sections of the epithelium of the trachea of the rat after 3 hours after the application of lipopolysaccharide. Histological studies showed that in the case of the use of LPS 10 ^-7 mg / animal, massive accumulation of neutrophils in the tracheal wall, development of edema with subsequent exfoliation of the mucosa into the lumen of the trachea and cell death after 3 hours were observed.

Immunohistochemical studies showed that in many areas of the tracheal epithelium an mass death of cells secreting Prx VI peroxyredoxin is observed already in an hour, which led to the absence of Prx VI peroxyredoxin in the mucus covering these tracheal regions.

For biochemical studies, the tracheal epithelium was scraped off with a thin spatula, the scraping was washed off with saline and centrifuged at 10,000 g for 15 minutes. The concentration of hydrogen peroxide was determined by luminescence. The dynamics of the activity of peroxyredoxin Prx VI, TNF-α and the respiratory explosion of neutrophils after applying LPS to the rat trachea showed that the maximum secretion of peroxyredoxin was observed one hour after the application of LPS, while the maximum expression of TNF-α was observed after 3 hours and a respiratory explosion of neutrophils through 6 o'clock.

Therapeutic properties of peroxyredoxin Prx VI in the treatment of LPS

After LPS, a solution of the peroxyredoxin-based Prx VI composition was applied to the trachea. Pre rats were euthanized by intraperitoneal administration of hexenal (70 mg / kg). The animals were fixed on the operating table and under visual control using a binocular loupe through a catheter with a diameter of 1 mm they were injected into the trachea a solution of peroxyredoxin (20-100 μl). The concentration of peroxyredoxin is 0.5-5.0 mg / ml, 0.9% NaCl was used as a solvent. The application of the solution of the composition was carried out once immediately after the application of LPS. As a control, we used the application of a pure solvent, solutions of serum albumin (10 mg / ml) and glutathione (10 mM).

A day after the application of LPS, a histological examination of the trachea was performed. Histochemical studies were carried out by the method described in the section of example 3.

results

Direct single application of human peroxyredoxin Prx VI to the rat trachea immediately after the application of lipopolysaccharide led to almost complete restoration of epithelial tissue after 2 weeks (see Fig. 7), while in animals of the control group that did not receive treatment after application of LPS, the ciliated epithelium practically not recovered (see example 3, Fig.6).

The introduction of peroxyredoxin into the lesion of epithelial cells prevents the development of secondary alterative disorders, thereby limiting the amount of pathological changes. Neutralization of reactive oxygen species restores proliferative cellular processes, promotes rapid regeneration of damaged epithelium, reduces the risk of infectious complications in the lesion.

The use of other antioxidants, for example glutathione, did not produce a significant effect (see table 6).

Table 6.
Preservation of the tracheal epithelium during the application of various antioxidants after damage to the tracheal epithelium (according to histological data). Time after exposure to LPS Glutathione Prx VI 1 hour thirty% 90% 3 hours twenty% 80% 6 o'clock 10% 65% Treatment every other day for 5 days and the safety of the epithelium after 2 weeks twenty% 80%

7 shows a section of the epithelium of the trachea of the rat one day after the application of LPS with the application of peroxyredoxin immediately after LPS. The complete preservation of the epithelium is observed. The only difference from the intact trachea is that the number of macrophages and mast cells has increased. As can be seen from the photograph, almost complete conservation or restoration of the tracheal epithelium was observed.

Example 36. Therapeutic properties of peroxyredoxin Prx VI and dihydrolipoic acid in the treatment of wounds.

A) Previously, rats were euthanized by intraperitoneal administration of hexenal (70 mg / kg). The animals were fixed on the operating table and cuts of the skin of both paws with a depth of 2-3 mm and a length of 1 cm were applied using a blade. Control cuts were washed with saline and a gauze bandage was applied.

B) Prepared solutions of peroxyredoxin according to example 14, but using the indicated in the following table 7 mass quantities of ingredients instead of the corresponding quantities used in this example:

Table 7.
Peroxyredoxin content in liquid solution Example Peroxyredoxin (wt.%) The stabilizer (wt.%) Saline (wt.%) 37 0.01 0.001 99,99 38 0.02 0.002 99.98 39 0.1 0.01 99.89

C) Solutions of dihydrolipoic acid were prepared, using the mass amounts of ingredients indicated in Table 8 below.

Table 8.
The dihydrolipoic acid content in liquid solution Example Dihydrolipoic acid (wt.%) The stabilizer (wt.%) Saline (wt.%) 40 0.01 0.001 99,99 41 0.05 0.005 99.95 42 0.1 0.01 99.89

D) Prepared solutions of peroxyredoxin and dihydrolipoic acid according to example No. 17, using the mass amounts of ingredients indicated in Table 9 below.

Table 9.
The content of peroxyredoxin and dihydrolipoic acid in the liquid form of the composition Example Peroxyredoxin (wt.%) The stabilizer (wt.%) Dihydrolipoic acid (wt.%) The ratio of peroxyredoxin and dihydrolipoic acid Saline (wt.%) 43 0.1 0.01 0.05 2: 1 99.84 44 0.1 0.01 0.02 5: 1 98.87 45 0.1 0.01 0.005 20: 1 99,885

The wounds were washed with solutions prepared according to examples 37-45 and a gauze dressing soaked in the same solutions was applied. The dressing was changed once a day.

results

The effects of the solutions: a) peroxyredoxin Prx VI, b) dihydrolipoic acid, c) peroxyredoxin PrxVI and dihydrolipoic acid on wound healing after one day are shown in figures 9-11. On figa, 10a, 11a presents the control.

Figure 9 presents options for the impact on the wound of a solution of peroxyredoxin. On figb, 9c, 9g - presents experimental options in which the concentration of peroxyredoxin in solutions are: 1.0; 0.1; 0.2 mg / ml, respectively.

The effect on the wound of a dihydrolipoic acid solution is shown in FIG. 10. On figb, 10c, 10g - presents the options for experiments in which the concentration of dihydrolipoic acid in solutions are, respectively: 1.0; 0.5; 0.1 mg / ml.

A synergistic effect on the wound of peroxyredoxin and dihydrolipoic acid is presented in Fig.11. 11b, 11c, 11g show compositional variations in which the ratio between the constant concentration of peroxyredoxin (1.0 mg / ml) and the variable concentration of dihydrolipoic acid is: 20: 1; 2: 1; 5: 1, respectively.

In appearance, the wounds affected by dihydrolipoic acid (see Fig. 10) show that although dihydrolipoic acid is an antioxidant, in its pure form it prevents healing and, in high concentrations, irritates the wound.

Peroxyredoxin promotes wound healing (see Fig. 9), however, the best results were obtained when treating wounds with compositions comprising peroxyredoxin and dihydrolipoic acid (see Fig. 11).

According to the external signs of wounds and a shorter healing time, we can talk not about the additive, but about the synergistic effect of peroxyredoxin and dihydrolipoic acid on the intercellular space of the skin lesion damage site. Histological studies also showed significantly smaller scars in the case of the use of peroxyredoxin Prx VI and dihydrolipoic acid.

Almost complete wound healing in control occurs in 7 days. Wound healing with percosiredoxin occurs in 5 days. When using a composition based on peroxyredoxin and dihydrolipoic acid, complete healing takes place in 3 days.

Industrial applicability

A pharmacological composition containing at least one type of peroxyredoxin can find prophylactic and / or therapeutic applications for many diseases that are accompanied by overproduction of free radicals. The higher efficiency of the composition based on peroxyredoxin compared with the well-known antioxidants can increase efficiency and reduce treatment time.

The pharmacological composition is not toxic and biocompatible with the body of mammals, including humans, since it uses the recombinant human peroxyredoxin as the main element. The low toxicity of the composition allows to increase the effectiveness of treatment due to the simultaneous combined effect on local areas of the body and the whole organism as a result of applications and intravenous administration of the composition. This is especially important in the treatment of multifactorial effects on the body, such as radiation, thermal, chemical burns, wounds, bruises that occur during disasters and fires. The composition has high solubility and quickly penetrates into the intercellular space of the body, which allows you to treat diseases of almost all organs. The composition is compatible with any pharmaceutically acceptable carriers that do not reduce the biological activity of the enzyme.

Information sources

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Claims (6)

1. A pharmaceutical composition for antioxidant protection of cells, tissues and the body as a whole from the overproduction of free radicals in acute inflammation, chemical, thermal and radiation injuries, containing peroxyredoxin Prx VI, characterized in that it also contains dihydrolipoic acid and pharmaceutically acceptable additives, and contains peroxyredoxin Prx VI and dihydrolipoic acid in an effective amount, and the ratio between peroxyredoxin Prx VI and dihydrolipoic acid is in the range (w \ w) from 1: 1 to 50: one. 2. The pharmaceutical composition according to claim 1, characterized in that the specified peroxyredoxin Prx VI is a recombinant human peroxyredoxin Prx VI. 3. A method of increasing the antioxidant protection of mammals, characterized in that the delivery of the pharmaceutical composition according to any one of claims 1 or 2 is carried out in the intercellular space of the tissue, organ or entire organism of a mammal. 4. The method according to claim 3, characterized in that the delivery to the intercellular space of a tissue, organ or organism as a whole, is carried out by passive or active diffusion during application or spraying, by parenteral or endolumbal administration by injection, by parenteral administration, by infusion, inhalation, drainage, through sublingual, vaginal, or rectal administration, through drops in the nose or eyes. 5. The method according to claim 3, characterized in that the delivery is carried out simultaneously with the use of another therapeutic agent. 6. The method according to claim 6, characterized in that said therapeutic agent is interferon.

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