Composition with antioxidant property and method for treatment of disease in mammals

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

 

The 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 fully caused by an imbalance between oxidative and antioxidative processes in cells and organisms mammals.

Prior art

Known for a wide range of diseases for which treatment antioxidant drugs have proven effective (J.G. Babish et al. PCT Appl. WO 0230419, 2002). Such diseases include: (a) lung, (b) liver disease, kidney disease, diseases of the gastrointestinal tract, e) disease of the immune system, (e) diseases of the nervous system, W) diseases of the eye, C) inflammation, and heart disease, viral infections, such as AIDS and others.

Now open a new class of natural antioxidants, soluble in water with a wide range of antioxidant activity and investigated in vitro experiments. To this class belong the thiol-specific antioxidants or peroxiredoxin that can neutralize both organic and inorganic compounds in a wide range of concentrations (H.Z. Chae et al., 1994; S. McGonigle et al., 1998). From in vitro experiments it is known that many of peroxiredoxins participate in the process of cell proliferation (Prosperi M.T. et al.,1993; Pesenko I.V. et al., 1998; S.V. Novoselov et al., 1999).

Known publications that have studied the role of peroxiredoxins in cells with dysfunction due to arteriosclerosis (L.H. Butterfield, 1999), cancer (Y.M. Chung et al., 2001; Kinnula V.L. et al., 2002), nervous diseases (Kirn S.H. et al., 2001), lung disease (Das K.C. et al., 2001; Kim H.S. et al., 2002), kidney disease (T. Fujii, 2001) diseases of the skin (S.C. Lee et al., 2000), ionizing radiation (S.H. Park et al., 2000).

On the basis of amino acid sequence homology and immunological affinity of all known to date peroxiredoxin mammals can be classified into the following groups: Prx I-Prx IV, Prx Prx V and VI. Homology of proteins belonging to the same group, constitutes more than 90%. Peroxiredoxin Prx I-Prx V belong to the group of 2-Cys thiol-specific antioxidants. Group Prx VI, the smallest, presents 1-Cys-peroxiredoxin, very different from the other groups and has only 20-40% of the total amino acid sequence with peroxiredoxin other groups.

Family peroxiredoxins has a high conservatism, and its representatives are found in the genomes of virtually all living organisms from bacteria to man. According to immunohistochemical studies using light microscopy and in situ hybridization, peroxiredoxin Prx VI is found practically is in all tissues of the animal. However, its maximum concentration detected predominantly in tissues in direct contact with the atmosphere, namely: in the olfactory epithelium, trachea, bronchi of the lungs, skin epidermis and hair follicles (Novoselov S.V et al., 1999). Immunohistochemical studies using electron microscopy showed that Prx VI is the only identified to date secretory peroxiredoxins. It is synthesized in specialized cells (goblet cells of the respiratory epithelium and supporting cells of the olfactory epithelium and secreted into the mucus covering the cells of these epithelial tissues. Direct biochemical and immunological studies in the rat have shown that in the trachea, the bronchi of the lungs and olfactory epithelium of the contribution of the 28-kDa Prx VI in the neutralization of reactive oxygen species is 70-80%. Similar results were obtained for the trachea and bronchi of a human. For example, in the composition of the mucus epithelium of the trachea concentration Prx VI is not less than 15 µg/mg protein.

There is a method of treatment based on raising the level peroxiredoxin in the cell via gene therapy. For this purpose we use the vector created by a genetic engineering method, which transforms the cells of animals. The composition of the vector introduced the gene encoding the sequence peroxiredoxin is a, selected from helminths (R. Chandrashekar et al., US Patent 6352836, 2002). Simultaneously with the application 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 been rapid development due to the high cost and identify a large number of side effects.

Known studies on the allocation of natural peroxiredoxin (Prx VI) of the olfactory organs of rats and subsequent use of the selected drug to influence chemical burns of the respiratory system of rats (Novoselov V.I. et al., 2000).

The disadvantage of this method of preparation of the olfactory organs is the impossibility of its application in obtaining natural peroxiredoxin person in preparative amounts.

There is a method for the treatment of viral diseases, including AIDS (HIV-1), which as a main component drugs use natural and recombinant peroxiredoxin with protease activity (B.D. Walker, Lynn R.G. PCT appl. WO 02077294, 2002). The authors found antiviral properties peroxiredoxins for the protection of cell membranes against viral penetration into the cell by introduction of a single dose of from 100 to 1000 mg peroxiredoxin depending on the patient's weight.

The purpose of this image is the shadow is expanding the range of effective pharmacological compositions and increase their effectiveness for the treatment and prevention of diseases associated with hyperproduction of free radicals caused by exogenous and endogenous factors.

The invention

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

Another object of the invention is a method of prevention and treatment, in accordance with which the delivery composition in the extracellular space of the tissue, organ or whole organism are carried out either through passive or active diffusion in application or spraying, or by delivery by injection into the blood, lymph, cerebral spinal fluid.

For delivery use parentrule or endolyumbalno introduction by injection or use parentrule introduction using infusions, inhalations, introduction to drainage, or use sublingual, vaginal, rectal gland, or use nose drops or eye.

the optional use of a therapeutic agent, which is used at the same time, either before or after application of the composition comprising peroxiredoxin Prx VI and dihydrolipoic acid.

List of figures

Figure 1. The inclusion of3H-thymidine in stimulated concanavalin And mouse lymphocytes in the presence of Prx VI. Where: 1 - medium, 2 - T cells without stimulation concanavalin And 3 - T-cells stimulated by concanavalin And 4 - T-cells in the presence of Prx VI, 5 - T cells stimulated by concanavalin And in the presence of Prx VI.

Figure 2. The effect of Prx VI on the spectral characteristics of the 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 is seen that at a concentration of 0.05 mg/ml Prx VI completely prevents the conversion of oxyhemoglobin to methemoglobin.

Figure 3. The appearance of rats after 12 months after gamma-irradiation (600 x-ray). Where: a - control; B - rat entered before irradiation Prx VI.

Figure 4. The epithelium of the trachea of rats after burn pairs of hydrochloric acid and subsequent application of natural or recombinant rat human Prx VI. The application was carried out 1 h after burn. Where: a - control; b - epithelium through the day after a chemical burn; With the epithelium through the day after the chemical is zhoga with applique Prx VI in an hour after burn; E - the epithelium of the trachea; BM, basal membrane. There is an almost complete preservation of all cells of the epithelium.

Figure 5. The recovered cell epithelium of the trachea in rats 14 days after burn pairs of hydrochloric acid. Where: a - control; b - epithelium through 14 days after a chemical burn; With the epithelium through 14 days after a chemical burn with applique Prx VI, E - epithelium of the trachea; BM, basal membrane; F - phagocytes. Therapy was performed one day after burn within 5 days. The application of a solution of the pharmaceutical composition on the basis of recombinant human Prx VI was performed once a day.

6. The epithelium of the trachea in rats after application of lipopolysaccharide. Where: a is the norm; In one hour after application; With - in 3 hours after application; E - epithelium of the trachea; BM, basal membrane; F - phagocytes. There is a significant loss of epithelial cells and the emergence of a large number faguoqitirute cells.

7. The epithelium of the trachea in rats after application of lipopolysaccharide and subsequent application of recombinant human Prx VI. Where: E - the epithelium of the trachea; BM, basal membrane; F - phagocytes. Applique Prx VI was carried out immediately after application of lipopolysaccharide.

Fig. Comparative characteristics of activation peroxiredoxin Prx VI different thiols. Antioxidant activity prideless according to the degree of protection is s glutamine synthase against metal-catalyzed oxidation system. The degree of activation peroxiredoxin Prx VI was determined in molar concentration, which was 50% protection of glutamine synthase. Types of thiols: 1 - dithiothreitol; 2 - dihydrolipoic acid, 3 - lipoic acid.

Fig.9. The effect of application of Prx VI to the wound, where (a) control, b), C), d) - concentration peroxiredoxin respectively: 1,0; 0,1; 0,2 mg/ml

Figure 10. The effect of application of dihydrolipoic acid on the wound, where (a) control, b), C), d) the concentration of dihydrolipoic acid respectively: 1,0; 0,5; 0.1 mg/ml

11. The effect of application peroxiredoxin Prx VI and dihydrolipoic acid, where (a) control, b), C), d) - variants where the ratio between the constant concentration peroxiredoxin, (1.0 mg/ml) and a variable concentration of dihydrolipoic acid is: 20:1; 2:1; 5:1 respectively.

Description of the invention

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

During experiments on simulation of exogenous and endogenous influences on the organism of animals found non-obvious fact, associated with the fact that shipping in the extracellular space of the tissue, organ or organism of a mammal water-soluble antioxidant peroxiredoxin the ina Prx VI and activator in the form dihydrolipoic acid, leads to high efficiency in the prevention and/or treatment of a large group of diseases caused by the overproduction of free radicals.

This fact gives the possibility to carry out the claimed technical result that is associated with expansion of the range 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 improved efficiency in the prevention and/or treatment, is achieved by the introduction into the composition peroxiredoxin PrxVI and dihydrolipoic acid does not lead to additive, and non-obvious, the synergistic effect of the interaction of antioxidants in the intercellular space. The results of experiments on the use of different versions of songs (with different concentration ratio between peroxiredoxins and dihydrolipoic acid in the interval (w/w) of 1:1 to 50:1) to treat wounds, showed that the synergistic effect of simultaneous exposure to the extracellular space peroxiredoxin Prx VI and dihydrolipoic acid can improve the efficiency of antioxidant defense mammals. In addition is the ability to prevent the development of secondary alterative disorders in chemical and thermal burns jot exposure.

The increase in antioxidant protection of mammals from hyperproductive free radicals caused by exogenous and/or endogenous factors.

Versatility of application peroxiredoxin Prx VI and dihydrolipoic acids as components of effective compositions for the prevention and treatment of diseases associated with the overproduction of free radicals, confirmed by the examples of prevention and treatment of diseases of mammals. Diseases can be caused by both exogenous and endogenous factors belonging to the group: a) ionizing radiation, b) a chemical burn in) acute inflammatory process, g) wounds. Examples include, but are not limited to other applications peroxiredoxin Prx VI and dihydrolipoic acid for the prevention and/or treatment.

In General, the method of improving the antioxidant protection of mammals from hyperproduction of free radicals in tissues or organs or in the whole organism mammals, consists of the delivery of the active agent pharmaceutical composition comprising peroxiredoxin Prx VI and dihydrolipoic acid, and pharmaceutically acceptable additives in the extracellular space of tissues, organs or the body of a mammal as a whole.

The proposed method applies to different choices of prevention or treatment of tissues, organs or whole PR is anima mammals in General, including:

A) preventive protection of the body, individual organs and certain areas of normal tissue from hyperproductive free radicals. The emergence of hyperproductive radicals is called: a) ionizing radiation, for example, during the treatment of cancer, or when flying at high altitudes and in space, b) effect of thermal and/or chemical burns with the elimination of disasters and fires, a combination of factors in paragraph a), b).

B) protection of tissues, organs and mammals, which is due to endogenous or exogenous factors is inflammatory and/or immune process associated with hyperproduction of free radicals. The overproduction of radicals accompanies or is a major contributor in the process diseases of the lungs, liver, kidneys, gastrointestinal tract, immune system, nervous system, eye disease, inflammation, 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 from the action of ozone or other exogenous factors.

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

D) protection of organs from inflammatory processes caused by mechanical damage to the skin and tissue in the grass is, injection of drugs or surgical operations.

Depending on the form of therapy or prophylaxis determine the duration of use and dose characteristics of pharmaceutical preparations containing the composition. To clarify the values of dose characteristics it is possible to measure the concentration of peroxiredoxin Prx VI in biological samples (see example 8). Based on the severity of the inflammatory process determine the minimum effective amount of the pharmaceutical composition and treatment. Perhaps the use of a composition for prophylactic purpose.

The treatment chosen from the group: a) single or multiple application or spraying of a solution of the pharmaceutical composition or drug to an affected area; b) single or multiple injections of a solution of the pharmaceutical composition; C) a single or repeated administration of a tablet, powder or liquid forms, sublingual, or in the form of powders, pastes, suppositories, ointments, gels for application to the surface of the skin and epithelium, or by combinations of these methods.

In the process of etiotropic therapy input of drugs containing peroxiredoxin Prx VI and dihydrolipoic acid, carried out via IV during the period of detoxification of poisons or other exogenous substances. Most preferably the use is giving compositions and drugs containing peroxiredoxin Prx VI and dihydrolipoic acid, for complex therapies. Recently, in connection with the prevalence of cardiovascular diseases, immune system, and AIDS treatment, alcohol and drug preparations based peroxiredoxin Prx VI and dihydrolipoic acid can be used in supportive therapy along with other drugs long-term use.

Delivery of the composition in the extracellular space of the tissue, organ or organism can be accomplished in various ways. Because peroxiredoxin prone to splitting and loss of biological activity during the passage through the gastro-intestinal tract, the main way of introduction is parenterally method. When parentelem the introduction of the use of injection, infusion, inhalation, introduction to drainage. The solution for injection is administered intramuscularly, intravenously in the blood, lymph, cerebral spinal fluid, shock doses or prolonged infusions, intraarterially; intrathecal, intraventricular endolumbalno. It is possible to use a solution, powder or pellet form sublingual. In some cases, vaginal and rectal enter drugs. Perhaps the introduction of composition using drops in the nose or eyes or care of the patient or enemas.

Delivery methods is oppozitsii to the site of inflammation epithelial tissues, described in this description do not restrict the application of other known methods of delivery of biologically active polypeptides to the site of inflammation or lesions.

For example, in the treatment of inflammatory processes of the upper respiratory tract composition comprising peroxiredoxin Prx VI and dihydrolipoic acid, is introduced into the nose, and/or in the trachea and/or bronchi of the lung. For applications using either a solution of a composition or a pre-solution is sprayed, turning it into an airborne mixture. Perhaps the use of a composition made in the form of dry fine-dispersed powder or composition, which is immobilized in fine granules or nanoparticles with a diameter of from 0.1 to 7000 nm (for example, Esenaliev, 2000), or compositions immobilized liposomes (for example, was called D.W.et al., 1991).

In the treatment of skin lesions composition is applied in a thin layer and/or make application on the site of the lesion. Methods of delivery of the composition to the site of inflammation epithelial tissues listed in this description do not restrict the application of other known methods of delivery of biologically active polypeptides to the site of inflammation or lesions.

Peroxiredoxin Prx VI and dihydrolipoic acid as a radioprotector can be used as a component of drug and pharmaceutical compositions for prevention or treatment is a wide range of diseases. Diseases can be caused by: a) ionizing radiation (primarily with radiotherapy, and prevention research, when working personnel with radiation sources); b) cosmic radiation affecting primarily on astronauts and pilots;) radionucleotides contamination due to receiving infected radionucleotide food, water or air; d) exposure to non-ionizing radiation, for example, when CT; e) exposure to sources of ultraviolet radiation (sunlight, welding, pulsed light sources, displays). To prevent or reduce the effect of hyperproductive free radicals may apply antidotes that contain peroxiredoxin. The composition of antidotes should be focused on the type of exogenous exposure that exists in the area in which you want to work on repair of the equipment, or elimination of accidents or mitigate the consequences of disasters.

For the prevention or treatment of diseases associated with ionizing or non-ionizing effects on the body of a mammal, an effective amount peroxiredoxin Prx VI and dihydrolipoic acid is selected depending on the intensity of ionizing or non-ionizing exposure, given the parameters of a mammal (weight, age, state org the mechanism). In the treatment or prevention of a selected composition is administered one or more times before exposure, during exposure or after exposure of the organism to radiation or non-ionizing radiation.

For antioxidant protection of cells, tissues and the whole body from the effects of several factors (e.g., ionizing radiation and/or thermal and/or chemical burns, and/or infection of the body with radionuclides through food, water or air), using a combined method of applying the compositions, which consists of intravenous injection and additional applications on the location of the injury, which reduces toxicitiy of the body and to prevent the uncontrolled cell death.

The pharmaceutical composition

Known methods for producing natural and recombinant peroxiredoxin. In the framework of the present invention natural Prx VI was isolated from organs of mammals (Pesenko I.V. et al., 1998). Recombinant protein can be obtained using various expression systems (Sang W.. 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, see the materials and methods (examples 1-2).

Primary structure of Prx VI rats (Andreeva YEAR and others, 1998) contains 223 amino acids with raschetnosberegatelnaya mass 24630 Yes. (EMBL/GenBank Y17295). The gene encoding Prx VI of man (T. Nagase et al., 1995), was isolated from the cells of myeloblast (EMB/GenBank D1 4662). According to electrophoresis in the presence of sodium dodecyl sulfate molecular weight is defined as a 28 kDa.

Synthesized recombinant Prx VI (see example 2) was compared with the natural Prx VI rats, which was isolated from the olfactory epithelium in rats Wistar rats (see example 1).

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

In pharmaceutical compositions it is possible to use other types of peroxiredoxins (Prx I-Prx IV) since the distribution of concentrations of different types of peroxiredoxins varies depending on the type of tissue or organ (Knoops C. et al., 1999). It is preferable to use a recombinant human peroxiredoxin type Prx VI, which has a stronger antioxidant characteristics in comparison with the I-V types peroxiredoxins.

For the treatment of inflammatory processes of the minimum concentration peroxiredoxin Prx VI in the composition is determined, firstly, based on the effective concentration peroxiredoxin Prx VI in the protection of biomacromolecule from reactive oxygen species in vitro and, secondly, on the basis of concentration peroxiredoxin Prx VI in tissue nor the E.

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

For example, in the case of disorders of the respiratory system in rats with a solution of lipopolysaccharide and in the case of chemical burn empirically derived concentration peroxiredoxin Prx VI in the composition amounted to 0.5-1.0 mg/ml during the application of 20-50 ál its solution directly into the trachea, which corresponded to 10 to 50 µg peroxiredoxin Prx VI in the rat. Given the surface area of the rat trachea, this corresponds to the application of 5-10 µg peroxiredoxin Prx VI on 1 cm2the affected tissue. This number peroxiredoxin Prx VI can be used for application on the affected tissue to any mammal, including humans.

In the case of the use peroxiredoxin Prx VI and dihydrolipoic acid as a radioprotector number peroxiredoxin Prx VI, introduced into the body to radiation, can be selected from 1 to 10 mg/kg of body weight of the animal and depends on the radiation power. In the case of sublethal doses of gamma-irradiation empirically obtained number peroxiredoxin Prx VI was 2-5 mg/kg of body weight of the animal when injected 1 ml of its solution into the vein. This number peroxiredoxin Prx VI can be used to bring the AI to any mammal, including humans.

As peroxiredoxin Prx VI is a highly soluble protein, as solvents for the compositions can be used aqueous solutions, physiologic solution, ringer's solution and other balanced salt solutions (R. Dawson, 1986), as well as solutions based on mono - or polysaccharides, such as glucose, and/or containing vitamins. These solvents can be used to create compositions in droplet form, for example in the form of spray.

During experiments on simulation of exogenous and endogenous influences on the organism of animals were studied various types of activators. Classical activator peroxiredoxins is dithiothreitol (S.V. Novoselov et al., 1999). However, due to its toxicity its use in dosage forms is eliminated. One of the natural activators peroxiredoxin VI is dihydrolipoic acid, which is non-toxic and can be obtained by the reduction of S-S relation is widely used in medicine alpha lipoic acid (1,2-ditiolan-3-pentane acid) (Sang W.. et al., 1998). Dihydrolipoic acid as activator peroxiredoxin investigated in the work (Peshenko I.V. Shichi H., 2001) in in vitro experiments. The synergistic effect from the use of dihydrolipoic acid as activator peroxiredoxin in vivo is demonstrated in the example is 5.

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

The liquid form of the composition:

a) for intravenous injection 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 active substance peroxiredoxin and dihydrolipoic acid;

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

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

When creating liquid media using solvent selected from the group: a) balanced salt solution, (b) balanced salt solution and activator peroxiredoxin in a concentration of from 0.01% to 1.0% (w/w).

Ointment form or shape for suppositoria introduction (suppositories for rectal or intravaginal injection) contains from 0.1 to 1.0 wt.% the main active substances peroxiredoxin and dihydrolipoic acid.

Concentration peroxiredoxin in the composition of the powder is from 0.1 to 90 wt.%. In pill form, which is used sublingually, the concentration of peroxy ledoxina may be from 0.01 to 1.0 wt.%.

When creating a lyophilised form as stabilizers use one or more mono - or polysaccharides or sugar alcohols, amino acids, low molecular weight proteins, which are used for stabilization and subsequent freeze-drying of the composition.

Lyophilized forms can be used for cooking liquid forms, composition and powders selected from the group of powder for preparation of injection, infusion solutions, powders for inhalation, powder for use in ointments, gels, suspensions, powders for the preparation of tabloids. The concentration of active principle peroxiredoxin in lyophilised forms can range from 10.0 to 90.0 wt.%.

Perhaps the use of a composition comprising peroxiredoxin and activator, together with at least one therapeutic agent with a wide spectrum of action.

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

C) low molecular weight compounds, providing a further reduction in the level of free radicals within the cell (tocopherol of gluta the ion, ubiquinone); d) products used for transplantation or cryopreservation of organs; d) biologically active proteins, such as insulin.

therapeutic agent used in conjunction with peroxiredoxins, should not inhibit the biological activity peroxiredoxin as enzyme.

Generalization of the inflammatory process (for example, when the occurrence of sepsis) with involvement of multiple organs and/or systems recommended combination therapy and the use of antibiotics and/or corticosteroids in combination with the use of peroxiredoxin and activator.

Presents forms of compositions and methods of its application do not limit other options, which are known from the field of medicine or veterinary medicine with the use of antioxidant therapy. Variations arising from this invention and obvious to every specialist, with the average level of knowledge in this area should be considered in the framework of the invention.

1. Materials and methods to obtain peroxiredoxin and study its properties.

There are several ways to obtain peroxiredoxins, In the framework of the present invention peroxiredoxin was isolated from organs of mammals (Pesenko I.V. et al. 1998), or received recombinant protein using various expression systems (Sang Won Kang et al. 1998, J.W. hen et al. 2000).

Example 1. The natural Prx VI peroxiredoxin rats

The natural Prx VI peroxiredoxin rats was performed according to the following procedure. Natural Prx VI peroxiredoxin rats was isolated from the olfactory epithelium of rats of Wistar line, which contains the maximum amount of this protein. Isolated olfactory epithelium washed twice in saline and homogenized in it. The homogenate was centrifuged 5 min at 500g, the supernatant re-centrifuged twice at 20000g and were dialyzed for 12 hours against the solution, which includes: 12 mm Tris/HCl, pH 7.8, 1 mm MgCl2, 1 mm dithiothreitol. After dialysis, the extract was applied to a chromatographic column (305×12.5 mm), gel filled, DEAE-separate (DEAE-Sepharose, Pharmacia) and pre-equilibrated with a solution of A. Proteins were suirable at the linear change of the gradient of NaCl from 0 mm to 500 mm in the solution And with 17°C. the solution Volume was 750 ml, the rate of elution of 0.7 ml/min Fractions were analyzed for antioxidant activity. The fractions containing Prx VI peroxiredoxin, concentrated and then chromatographically on a column filled with a gel Sephacryl S-200 (820×16 mm). The column was suirable buffer B: 25 mm Tris/HCl, pH 7.8, 100 mm NaCl, 1 mm MgCl2, 1 mm dithiothreitol. The rate of elution - 0.6 ml/min at 4°C. the Fractions containing Prx VI, collected, concentrated, were dialyzed against physiological saline and used in further experiments.

Example 2. Receiving human recombinant Prx VI peroxiredoxin

Human recombinant Prx VI peroxiredoxin was prepared as follows. Complementary DNA encoding human Prx VI peroxiredoxin, was isolated from clone NA (Nagase T. et al., 1995) (GenBank, D 14662) known methods. Then DNA was cloned in the expression plasmid pet-23a (+) firm Novagen no restriction sites NdeI and EcoRI, using restriction enzymes company MBI Fermentas. The obtained plasmid was used for transformation of E. coli cells (strain BL 21 (DE3), from the library of strains IBPM RAS) to obtain recombinant human Prx VI peroxiredoxin. Cells of E. coli expressing peroxiredoxin, was destroyed by the ultrasound generator of USDN-2 at the frequency of 22 kHz for five minutes at 0°C. the Homogenate was centrifuged 5 min at 500 g, the supernatant re-centrifuged twice at 20000 g and were dialyzed for 12 hours against solution A (see example 2). Recombinant Prx VI peroxiredoxin was isolated according to the method of allocation of natural rat Prx VI peroxiredoxin (see example 1).

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

Recombinant human peroxiredoxin Prx VI (GenBank, D 14662) compare the factor of antioxidant activity with natural mouse-peroxiredoxin Prx VI (EMBL/GenBank, Y 17295). To determine the activity peroxiredoxin RGH VI use its ability to protect glutamylcysteine against DTT/Fe+3/About2the oxidative system. The incubation mixture by volume of 60 μl, containing 5 g of glutamine synthase, 3 mm DTT, 3 μm, Fe3+, 50 mm HEPES, pH 7.3, incubated 10 min at 37°C. as activator Prx VI peroxiredoxin use dihydrolipoic acid at a concentration of 3 mm. The residual activity of glutamine synthase determine pribivaniem to the sample 200 ál of the reaction mixture, which contains: ADP - 0.4 mm glutamine - 150 mm K-AsO4- 10 mm NH2OH - 20 mm MnCl2- 0.4 mm, HEPES - 100 mm pH 7.4. After incubation for 10 min at 37°C, the sample is poured 100 μl of the dye. In the dye composition includes: 5.5 g FeCl3·6N2Oh, 2 g THU, 2.1 ml conc. HCl (38%) in 100 ml of N2O. the Degree of activity peroxiredoxin determined by the concentration of the protein, which was observed in 50% of the saving activity of glutamine synthase. Recombinant peroxiredoxin Prx VI has antioxidant characteristics close to the natural rat peroxiredoxin Prx VI. In the future, human recombinant peroxiredoxin Prx VI and the use for practicing the methods of treatment or as a component of pharmaceutical compositions.

Example 4. Determination of cytotoxicity peroxiredoxin Prx VI

Cytotoxicity of peroxiredoxins determine the influence of Prx VI peroxiredoxin on the proliferation rate of the cell line L929 of lymphoblastoma human T-lymphocytes from the spleen of mice NRRI stimulated concanavalin A.

Cells of two lines with a concentration of 104cells/ml of medium were cultured in medium RPMI 1640 containing 5% bovine fetal serum. Stimulation of T-lymphocytes was performed at a concentration of concanavalin And 0.1 µg/ml In the case of T-lymphocyte proliferation rate was determined by the inclusion of3H-thymidine. The number of live cell line L929 was determined in 96-well plate using dye trendovogo with subsequent scanning multichannel photometer MultiScan (LKB, Sweden).

Natural rat and recombinant human Prx VI peroxiredoxin (in a concentration of 0.1-10 mg/ml) had no effect on the number of live cell line L929.

In the case of T-lymphocytes stimulated by concanavalin And the level of cell proliferation was increased approximately 2-fold in the presence of natural or recombinant rat human Prx VI peroxiredoxin (at a concentration of 0.1-1.0 mg/ml) compared with just stimulated concanavalin And cells (see figure 1). Thus, we can conclude that the test is consistent compounds are of low toxicity.

Example 5. Comparative characteristics of activators peroxiredoxin.

To determine the effectiveness of activation Prx VI dithiothreitol and dihydrolipoic acid (a natural activator) was used ability peroxiredoxin to protect glutamylcysteine from inactivation caused by metal-catalyzed oxidation system. The incubation mixture by volume of 60 μl contained 5 g of glutamine synthase, 50 µg Prx VI, 3 mm ascorbic acid, 3 μm, Fe+3, 50 mm HEPES, pH 7.3 and dithiothreitol or dihydrolipoic acid of different concentrations were incubated 10 min at 37°C. the Residual activity of glutamine synthase was determined by pribivaniem to the sample 200 ál of reaction mixture (ADP - 0.4 mm, gloamin - 150 mm K-AsO4- 10 mm NH2OH - 20 mm MnCl2- 0.4 mm, HEPES 100 mm, pH 7.4). After incubation for 10 min at 37°C, the sample was added 100 μl of the dye. The composition of the dye is 5.5 g FeCl3·6N2Oh, 2 g THU, 2.1 ml conc. HCl (38%) in 100 ml of N2O. the Degree of activity peroxiredoxin was determined by the concentration of the protein, which was observed in 50% of the saving activity of glutamine synthase.

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

the example 6. Distribution peroxiredoxin Prx VI in the body of the animal after its injection.

To identify the distribution ekzogennogo peroxiredoxin Prx VI in various tissues of the body in a vein of the rats were injected with recombinant human Prx VI, labeled fluoresceinisothiocyanate in the amount of 10 mg per animal. After a certain period of time the animals were killed, were isolated tissue of various organs and spent fluorescent analysis of the samples. The obtained data showed that 15 minutes after the injection of labeled peroxiredoxin Prx VI, the latter is evenly distributed to all organs of the rat, including the brain. The exception is the bone marrow, where the number of exogenous peroxiredoxin Prx VI was small compared with other tissues. Thus, the method of introducing peroxiredoxin Prx VI by injection into a vein can improve its content in almost all tissues of the body.

2. Materials and methods to determine peroxiredoxin in the sampling and analysis of the properties peroxiredoxin in vitro and in vivo.

Example 7. Protection of oxyhemoglobin from the effects of ionizing radiation using peroxiredoxin Prx VI.

The experiments showed that after exposure to mammalian lethal dose cell opportunities for the synthesis of antioxidant enzymes is limited and the concentration is not enough, even with pre-stimulation of cell synthesis peroxiredoxin.

To test the possibility of protection peroxiredoxins of biomacromolecule from the destruction caused by ionizing radiation was investigated characteristics of the irradiated oxyhemoglobin in the control and in the presence of peroxiredoxin Prx VI. The control solution was used oxyhemoglobin (0.16 D at 540 nm) in 0.1 M Tris-HCl (pH 7.0), which were irradiated in the installation of the LIP with a total dose of 10 Gy. As a radioprotector used peroxiredoxin Prx VI at various concentrations (0.005-0.1 mg/ml) in the presence of 50 μm of dithiothreitol as activator peroxiredoxin. The formation of oxidized forms of oxygenated hemoglobin (methemoglobin) were recorded at 635 nm, and the aggregation of methemoglobin was recorded at 700 nm.

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

Adding Prx VI in the solution of hemoglobin in a concentration of about 0.05 mg/ml to irradiation leads to a significant reduction in the formation of the oxidized form of hemoglobin, the concentration of which was reduced almost to zero.

Example 8. Definition peroxiredoxin Prx VI in a biological sample.

Polyclonal rabbit antibodies against rekombinationsreaktionen received in a standard way by combining two immunization of the animal with recombinant human peroxiredoxin. The titer of the obtained antibodies by ELISA was 1:10000, immunoblotting of 1:2000. The antibody specificity was verified by immunoblotting using water-soluble extract of the trachea person and immunohistochemical methods using paraffin sections of human lungs, which showed a high degree of specificity of the resulting antibodies.

Purified rabbit antibodies were obtained from serum by precipitation with ammonium sulfate followed by chromatography on DEAE cellulose. Conjugates of antibodies with sewn horseradish peroxidase was plocale standard method periodate oxidation according to the method of Wilson and Kang with a molar ratio of antibody to peroxidase, equal to 1:3.

The titer of the conjugate for human peroxiredoxin was as follows: ELISA 1:1,000 and for immunoblotting and immunohistochemistry of 1:500. The specificity of the conjugate match the specificity of the serum, which was used to produce IgG.

The Protocol immunoassay determination peroxiredoxin Prx VI in biological samples:

1. In wells dies pour 200 μl of a solution of immunoglobulin G against human recombinant peroxiredoxin (dilution 1:1000) and incubated for 30 min at room temperature.

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

3. In lunch is primed with 200 µl sample samples and incubated for 1 hour at room temperature.

4. Plates are washed with saline containing 0.1% tween 40 (3×5 min).

5. In wells dies pour 200 μl of conjugate solution (dilution 1:500) and incubated for 30 min at room temperature.

6. Plates are washed with saline containing 0.1% tween 40 (3×5 min).

7. In the hole, pour the ABTS solution (25 mg ABTS in 25 ml of citrate buffer, pH 4.0 + 40 μl of 3% peroxide).

8. Die scan on Multiscale at 405 nm.

The sensitivity of the system is tens of nanograms in a milliliter of sample.

Example 9. Immunohistochemical detection peroxiredoxin Prx VI in human tissues.

Selected tissue were 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 Fabric was rinsed in saline solution 4 times in an hour. Fabric dehydrational serial wiring through ethanol with increasing concentrations (40-100%). The fabric was washed with a solution of xylene-alcohol (1:1) for one hour, xylene and xylene-paraffin (1:1) for 12 hours. The tissue was placed in paraffin and received 5 MK slices on a microtome. Slices were deparaffinization in xylene and decreasing concentrations of alcohol. The sections were washed with saline, blocked with 5% fetal serum, endogenous peroxidase inhibited with 0.3% hydrogen peroxide and incubated with the conjugate for h is sa, thrice washed with saline and stained with diaminobenzidine (6 mg per 10 ml of 50 mm Tris-HCl buffer, pH 7.6 for plus 200 μl of 3% hydrogen peroxide).

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

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

In General, the technology of preparation of the compositions consists of the following operations: preparation of premises for work, training equipment for filling, sterilization of utensils, preparation of the main components of the composition, packaging and control.

3.1. Preparation of dry forms of composition

3.1.1. Dried product peroxiredoxin

Technology of production of dry lyophilized includes the following operations: natural selection or synthesis of recombinant peroxiredoxin; cleaning substance with known methods of purification of biologically active polypeptides and proteins, the introduction of at least one stabilizer, subsequent lyophilization of the solution peroxiredoxin and stabilizer.

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

Stabilizers necessary to maintain activity peroxiredoxin for a longer period, not less than one year, and in more accessible terms, at a temperature from +4 to -20°depending on the retention period.

Example 10. Getting lyophilized peroxiredoxin for the preparation of solid, liquid and ointment pharmaceutical compositions.

The fractions containing peroxiredoxin VI (see examples 1, 2), concentrated to a concentration of 5-10 mg/ml using hubs Ultrafree-15 10K by centrifuge (15 min, 2000 g) and were dialyzed against deionized water. In the solution peroxiredoxin enter at least one stabilizer and lyophilizer at -20°before the formation of a dry powder.

Dried product Packed in sterilized in an autoclave at 120°vials (depending on the application at doses from 1 to 100 mg and stored at -20°).

Get the finished product of the following composition:

Dried product for the preparation of solid, liquid and ointment pharmaceutical is omposite using peroxiredoxin and stabilizers (wt.%):

Peroxiredoxin - 90;

The stabilizer is about 10.

3.1.2. The drug peroxiredoxin in powder form

Technology of production of dry powder, for example, intended for inhalation or making other forms of composition, includes the following operations: natural selection or synthesis of recombinant peroxiredoxin; cleaning substance with known methods of purification of biologically active polypeptides and proteins, introduction to solution peroxiredoxin at least one stabilizer, the inclusion in the solution peroxiredoxin and stabilizer additives in the form of nanoparticles (such as microcrystalline cellulose, hydroxypropyl methylcellulose, lactose monohydrate), beads or liposomes, followed by lyophilization or drying the obtained product.

For the manufacture of aerosol products using known methods of dispensing a biologically active polypeptides in the form of powders, aqueous solutions or with the use of the liquefied gas (Extrem and other patent RU 2175866, 2001). Specialist secondary qualifications in this field can easily determine without undue experimentation, the appropriate dosages of these aerosol formulations.

Example 11. Getting peroxiredoxin in powder form for the preparation of pharmaceutical compositions using additives in the form of nanocat the CI. granules or liposomes

The fractions containing peroxiredoxin VI (see examples 1, 2) concentrate to the value of 5-10 mg/ml using hubs Ultrafree-15 10K and using a centrifuge (15 min, 2000 g). Fraction cialiswhat against deionized water. In the solution peroxiredoxin enter stabilizers and additives in the form of nanoparticles, pellets or liposomes and then lyophilizer at -20°before the formation of a dry powder. Dried product Packed in sterilized in an autoclave at 120°vials (depending on the application at doses from 1 to 100 mg and stored at -20°).

The drug in powder form for the preparation of pharmaceutical compositions using additives in the form of nanoparticles, pellets or liposomes (wt.%):

Peroxiredoxin of 0.1 to 90.0;

The stabilizer is from 0.01 to 10;

Additive - rest.

3.1.3. The drug peroxiredoxin in pill form

The technology of reception of a tablet form peroxiredoxin includes the following operations: natural selection or synthesis of recombinant peroxiredoxin; cleaning substance with known methods of purification of biologically active polypeptides and proteins; introduction to the solution, at least one stabilizer; lyophilization or drying of the drug; the stage of mixing the current start with the basic fillers; wet and dry pellet, the Finance; the dusting of the dry granulate and tableting.

As filler, you can use lactose monohydrate, corn starch, talcum powder, for dusting, you can use magnesium stearate.

Example 12. Tableted form peroxiredoxin for the preparation of pharmaceutical compositions.

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

Peroxiredoxin - 0,1-1,0;

Stabilizer - 0,01-0,1;

Additives and fillers rest.

3.2. Preparation of liquid composition

Technology for production of liquid forms based on the use of dry lyophilized forms of composition.

Example 13. The solution is to create a liquid form of the composition for intravenous, inhalation, and irrigation on the basis of peroxiredoxin.

Prepare medication dry form of the drug peroxiredoxin in accordance with example 10. Immediately prior to use dry powder lyophilized peroxiredoxin dissolved in distilled water or saline to the desired concentration. Solutions used immediately or stored before use at +4°C. To prevent the damaging effect of ionizing irradiation choose the composition of the following composition (wt.%):

Peroxiredoxin 0,1;

The stabilizer is about 0.01;

Saline - rest.

Por the measures 14-16.

Repeat the process described in example 13, but using in the following table 1 mass quantities of ingredients instead of the relevant quantities used in this example:

Table 1.

Content peroxiredoxin in liquid form compositions
ExamplePeroxiredoxin (wt.%)Stabilizer (wt.%)Saline (wt.%)
140,010,00199,99
150,50,0599,45
161,00,1the 98.9

Example 17. Solution for intravenous, inhalation, and irrigation using dihydrolipoic acid

Prepare the drug powder peroxiredoxin in accordance with example 10. Immediately prior to use dried peroxiredoxin dissolved in saline solution in the desired concentration. Prepare a solution of dihydrolipoic acid. Solutions used immediately or stored before use at +4°C. Immediately before use, mix the solutions peroxiredoxin and dihydrolipoic acid. For the treatment of small wounds, select the following composition with the composition (wt.%):

peroxiredoxin 0,05;

the stabilizer 0,005;

dihydrolipoic acid 0,01;

saline - rest.

Examples 18-24.

For preparation of compositions with other objects in the treatment or prophylaxis repeat the process described in example 17, but using are listed in the following table 2 mass quantities of ingredients instead of the relevant quantities used in this example:

Table 2.

Content peroxiredoxin and dihydrolipoic acid in liquid form compositions
ExamplePeroxiredoxin (wt.%)Stabilizer (wt.%)Dihydrolipoic acid (wt.%)Saline (wt.%)
180,010,0010,01of 99.98
190,50,050,598,95
201,00,11,097,9
210,50,050,0299,43
221,00,10,0498,86
230,50,050,0199,44
241,0 0,10,0298,88

Example 25. The solution is to create a pharmaceutical composition for intravenous injection, inhalation or irrigation with application peroxiredoxin and therapeutic agent.

Prepare the drug powder peroxiredoxin in accordance with example 10. Immediately prior to use dried peroxiredoxin dissolved in saline solution in the desired concentration. Prepare a solution of interferon. Solutions used immediately or stored before use at +4°C. Mixing mortars peroxiredoxin and the solution of interferon (wt.%):

peroxiredoxin 0,5;

the stabilizer 0,05;

interferon ME 100000;

Saline - rest.

For preparation of compositions with other objects in the treatment or prophylaxis repeat the process described in example 25, but using are listed in the following table 3 mass quantities of ingredients instead of the relevant quantities used in this example:

Table 3.

Content peroxiredoxin and interferon in liquid form compositions
ExamplePeroxiredoxin (wt.%)Stabilizer (wt.%)interferon (ME)Saline(wt.%)
260,010,001100000of 99.98
271,00,1100000the 98.9

Example 28. Solution for intravenous injection, inhalation or irrigation with application peroxiredoxin, dihydrolipoic acid and a therapeutic agent

Prepare the drug powder peroxiredoxin in accordance with example 10. Immediately prior to use dried peroxiredoxin dissolved in saline solution in the desired concentration. Prepare a solution of interferon. Prepare a solution of dihydrolipoic acid. Solutions used immediately or stored before use at +4°C. Mixing mortars peroxiredoxin, dihydrolipoic acid and a solution of interferon (wt.%):

peroxiredoxin 0,5;

the stabilizer 0,05;

dihydrolipoic acid 0,05;

interferon ME 200000;

Saline - rest.

For the preparation of other compositions, repeat the process described in example 28, but using are listed in the following table 4 mass quantities of ingredients instead of the relevant quantities used in this example:

Table 4.

Content peroxiredoxin, digitrip the eve acid and interferon in liquid form compositions
ExamplePeroxiredoxin (wt.%)Stabilizer(wt.%)Dihydrolipoic acid (wt.%)Interferon (ME)Saline (wt.%)
290,010,0010,01100000of 99.98
300,10,010,0110000099,88
311,00,10,550000098,4

Example 32 Cosmetic

It is known that antioxidant supplements introduced in the cream prevent oxidative reactions, protects the skin from adverse external influences (e.g. UV rays), are anti-inflammatory, antibacterial activity, enhance immunity. In addition, they prevent peroxidation contained in the cream or emulsion oils. The following example relates to a composition consisting of a water base, which was dissolved active principle compositions containing peroxiredoxin and superoxide dismutase (SOD), a fatty basis for improving the quality of the cream and auxiliary substances, which contain bioactive and antioxidant substances, such as the oil of wheat germ, grape seed oil and Supplement the sustained fashion contain low molecular weight antioxidants such as tocopherol in the following ratio of ingredients (wt.%):

fat basis 6-10;

peroxiredoxin 0,1-0,5;

superoxide dismutase 0,01-0,05;

excipients 2-6;

water base rest.

Interaction cheap low molecular weight antioxidants, reducing or preventing oxidation of high-molecular antioxidants during storage, allows to reduce the concentration of expensive and highly effective antioxidants type peroxiredoxin or superoxide dismutase and to achieve the maximum protective effect of the cream.

The examples should not be considered in the context of the limitations of the applications of the present invention. Variations arising from this invention and obvious to every specialist, with the average level of knowledge in this area should be considered in the framework of the proposed invention.

Example 33. Protective effect peroxiredoxin Prx VI in affecting the action of ionizing radiation.

Simultaneous irradiation of all animals (rats Wistar rats weighing 200 g) was carried out on the installation of GUBA. The radiation dose of 6 Gy. Rats were divided into two groups. The first group of animals (n=6) were used for control. Animals of the second group (n=6) for 30-60 minutes before irradiation was intravenously injected 1 ml of saline solution with a concentration of peroxiredoxin 1 mg/ml After exposure, animals were kept in a shared space with d is insufficient quantity of food, lighting and ventilation. On histological examination of selected animals from each group with signs of deteriorating General condition.

Results

Data on mortality mammals after exposure are presented in table 5, which shows the number of surviving rats in each group at a specified time.

Table 5.

Survival of rats in the introduction peroxiredoxin Prx VI before irradiation.
The time of the experiment3 months6 months12 months15 months
Survival controlnow group (pieces)4321
Survival in the group, whose tread was used Prx VI (pieces)6544

As can be seen from the table, the mortality of rats that before irradiation was introduced peroxiredoxin Prx VI, significantly lower than in the control group. The General condition of the surviving animals a year after irradiation was significantly different in favor of the second group compared with the control group (figb). Animals from the first group compared with the animals of the second group notes: large weight loss (cachexia) (weight 180-200 g), scarce is polozenie body, low motor activity, the presence of tumor process.

Example 34. Therapeutic and protective properties peroxiredoxin Prx VI in the treatment of upper respiratory tract of the rat after chemical burns

Therapeutic properties peroxiredoxin Prx VI in the treatment of upper respiratory tract of the rat through the day after burn tested on a group of 4 rats Wistar rats weighing 150-200 grams.

Chemical burns of the upper respiratory tract.

Rats were analgesically analgin (100 mg/kg of body weight) and placed in a desiccator saturated vapors of hydrochloric acid for 10 min (volume of desiccator - 10 liters) (only used 20 animals). After a chemical burn rats kept for a certain time (40 min, 6 h, 1 day, 2 days, 14 days, 30 days), sleep geksenalom (300 mg/kg of body weight), and provided a sample of epithelial tissue of the trachea. For histochemical studies highlighted the sample was placed in a fixative containing 2% formaldehyde and 0.5% glutaraldehyde. Fabric dehydrational serial wiring through ethanol with increasing concentrations (40-100%). The tissue was embedded in resin LR white resin. Polymerization of the resin was carried out at room temperature. The slice thickness of 0.5 μm was obtained on ultratome-LKB, Sweden, using glass knives. Sections were stained with a mixture of hematoxylin-eosin were examined on a light microscope.

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

In the first phase investigated the dynamics of changes in the level of neutrophils and enzymes-antioxidants in cell epithelium of the trachea of rats after burn pairs of hydrochloric acid. Watched two period, a sharp increase in the number of active neutrophils: 40 minutes and 6 hours after burn. A sharp increase in the number of active neutrophils exacerbates the extent of the lesion, as is the release of hydrogen peroxide by neutrophils.

Biochemical studies (see example 9) showed that the level of activity peroxiredoxin in epithelial cells increases approximately two times immediately after the burn. At the same time, the activity of superoxide dismutase, catalase and glutathione peroxidase, which are the main enzymes-antioxidants in the body, decreases in the mucus of the trachea is approximately 30%. One day observed a decrease in the level of activity of all these enzymes, including peroxiredoxin.

Histochemical studies (see example 10) showed that 40 minutes after burn in the epithelium of the trachea and bronchi begin the processes of cell death ciliarnogo epithelium, which later are avalanche-like character. The maximum loss of the epithelial cells of the mucosa of the upper respiratory tract is nabludaetsa day after the burn. When this occurs almost complete cell death of ciliated epithelium engaged in mucociliary transport in the trachea and bronchi. This state of the epithelium was taken as a control for comparative assessment of the degree of burn and the effectiveness of the treatment.

Without treatment, partial restoration of the epithelium of the mucosa of the trachea after a chemical burn occurs after 30 days.

On figb shows a slice of the epithelium of the rat trachea day after the burn, 7 - 14 days after burn. Maximum destruction of the epithelium is observed a day after the burn, with ciliated cells, ensuring the flow of mucus in the trachea, are practically absent. Two weeks later, there is partial recovery ciliated cells.

Protective and therapeutic properties peroxiredoxin

The application solution composition was started one hour after the burn (protective effect peroxiredoxin Prx VI) or a day after chemical burn (the time of maximum destruction of the epithelium of the mucosa of the trachea, therapeutic effect peroxiredoxin Prx VI). In the latter case, the application was performed once a day for 5 days. Rats pre-sleep through intraperitoneal administration geksenala (70 mg/kg), animals were fixed on the operating table and under visual control using binocula the Noah magnifier through the catheter with a diameter of 1 mm were introduced into the trachea solution peroxiredoxin volume from 20 to 100 ál. The concentration peroxiredoxin was chosen in the range from 0.5 to 5.0 mg/ml In the solvent used 0.9% NaCl solution.

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

Results

As one of the treatment options mammals after a chemical burn was used variant applications of the upper respiratory tract of the rat solution peroxiredoxin. Figure 5 shows the slice of the epithelium of the trachea in rats 14 days after burn during daily 5-fold application peroxiredoxin. There is an almost fully intact epithelium of the mucosa of the trachea, which indicates almost complete regeneration of ciliated epithelial cells. The presence of elements of mucous secretions, which are determined by phagocytes, indicates the presence of residual inflammation in the trachea.

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

Therapeutic properties peroxiredoxin Prx VI in the treatment of upper respiratory tract of the rat immediately after the application of LPS tested is as a group of 4 rats Wistar rats weighing 150-200 grams.

Application of lipopolysaccharide in the rat trachea.

Rats were anestesiologi geksenalom and through catheter were administered 100 μl of a solution of lipopolysaccharide (concentration of lipopolysaccharide 10-9 - 10-11 mg/ml, only used 20 animals). After application of lipopolysaccharide kept for a certain time (1 hour, 3 hours, 6 hours, 7 hours, 9 hours), sleep geksenalom (300 mg/kg of body weight), and provided a sample of epithelial tissue of the trachea. For histochemical studies highlighted the sample was placed in a fixative containing 2% formaldehyde and 0.5% glutaraldehyde. Fabric dehydrational serial wiring through ethanol with increasing concentrations (40-100%). The tissue was embedded in resin LR white resin. Polymerization of the resin was carried out at room temperature. The slice thickness of 0.5 μm was obtained on ultratome-LKB, Sweden, using glass knives. Sections were stained with a mixture of hematoxylin-eosin were examined on a light microscope.

After application of lipopolysaccharide, causing an acute inflammatory reaction in the trachea, investigated the dynamics of changes in the level of neutrophils, TNF-α and peroxiredoxin Prx VI in cell epithelium of the trachea of rats. To determine the characteristics of the treatment conducted study of the dynamics of neutrophil counts and antioxidants, as well as biochemical and histochemical studies of the impact of vos is alternova process on the body of a mammal.

Observed a sharp increase in the number of active neutrophils through hour after administration of LPS. A sharp increase in the number of active neutrophils in turn exacerbates the extent of the lesion, because the emission of N2O2neutrophils.

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

Histological examination (see example 10) showed that in the case of LPS concentration equal to 10-7mg/animal, was marked by a massive accumulation of neutrophils in the wall of the trachea, the development of edema with subsequent detachment 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 epithelium of the trachea after an hour there is a massive loss of cells secreting peroxiredoxin Prx VI, which resulted in a lack of peroxiredoxin Prx VI in the mucus covering the area of the trachea. This state of the epithelium was taken as control to compare the degree of inflammatory reaction and the effectiveness of the treatment.

On figb shows the slices of the epithelium of the trachea of the rat after 3 hours after application of lipopolysaccharide. Histological studies have shown that mo is in the case of application of the FSC 10 -7mg/animal of the drug was observed massive accumulation of neutrophils in the wall of the trachea, the development of edema with subsequent detachment of the mucosa into the lumen of the trachea and cell death after 3 hours.

Immunohistochemical studies showed that in many areas of the epithelium of the trachea after an hour there is a massive loss of cells secreting peroxiredoxin Prx VI, which resulted in a lack of peroxiredoxin Prx VI in the mucus covering the area of the trachea.

For biochemical studies scraped the epithelium of the trachea with a thin spatula, scraping washed with saline and centrifuged at 10,000 g for 15 minutes the Concentration of hydrogen peroxide was determined by the method of luminescence. Dynamics of activities peroxiredoxin Prx VI, TNF-α and respiratory burst of neutrophils after application of LPS in the rat trachea showed that the maximum secretion peroxiredoxin was observed one hour after application of LPS, while the maximum expression of TNF-α was observed after 3 hours and respiratory burst of neutrophils after 6 hours.

Therapeutic properties peroxiredoxin Prx VI in the treatment of LPS

After LPS conducted the application solution composition based on peroxiredoxin Prx VI in the trachea. Pre rats were euthanized using intraperitoneal administration geksenala (70 mg/kg). Animals were fixed on operations the main objective of the Desk and under visual control with the aid of a binocular through the catheter with a diameter of 1 mm were introduced into the trachea solution peroxiredoxin (20-100 µl). Concentration peroxiredoxin - 0.5 to 5.0 mg/ml) as a solvent was used with 0.9% NaCl. The application solution composition was performed only once immediately after the application of LPS. As control was used applique clean solvent solutions of serum albumin (10 mg/ml) and glutathione (10 mm).

The next day after the application of LPS was performed histological examination of the trachea. Histochemical studies were performed by the method described in example 3.

Results

Direct single applique human peroxiredoxin Prx VI in the trachea of rats immediately after application of lipopolysaccharide resulted in almost complete restoration of epithelial tissue after 2 weeks (see Fig.7), while animals in the control group who did not receive treatment after application of LPS, the ciliated epithelium of almost not recovered (see example 3, 6).

Introduction peroxiredoxin in the affected area of epithelial cells prevents the development of secondary alternative violations, thus limiting the amount of pathological changes. Neutralization of reactive oxygen restores the proliferative cellular processes, promotes rapid regeneration of damaged epithelium, reduces the risk of infectious complications in the lesion.

The use of other the antioxidants, for example, glutathione, gave no significant effect (see table 6).

Table 6.

The integrity of the epithelium of the trachea during the application of various antioxidants after lesions of the epithelium of the trachea (according to histological data).
Time after exposure to LPSGlutathionePrx VI
1 hour30%90%
3 hours20%80%
6 hours10%65%
Treatment a day for 5 days and integrity of the epithelium after 2 weeks20%80%

7 shows a slice of the epithelium of the trachea of rats one day after application of LPS with applique peroxiredoxin immediately after LPS. There is complete safety of the epithelium. The only difference from the intact trachea is that the increased number of macrophages and mast cells. As you can see from the pictures, was observed almost complete preservation or restoration of the epithelium of the trachea.

Example 36. Therapeutic properties peroxiredoxin RGH VI and dihydrolipoic acid in the treatment of wounds.

A) Pre rats were euthanized using intraperitoneal administration geksenala (70 mg/kg). Animals were fixed on operations the table and stabbed with the blade cuts the skin of both feet depth of 2-3 mm and a length of 1 see Control cuts were washed with saline and put a gauze bandage.

B) Made solutions peroxiredoxin in example 14, but used are listed in the following table 7 mass quantities of ingredients instead of the relevant quantities used in this example:

Table 7.

Content peroxiredoxin in liquid form solution
ExamplePeroxiredoxin (wt.%)Stabilizer (wt.%)Saline (wt.%)
370,010,00199,99
380,020,002of 99.98
390,10,0199.89 per

C) Prepared solutions dihydrolipoic acid, was used in the following table 8 mass quantities of ingredients.

/tr>
Table 8.

The content of dihydrolipoic acid in liquid form solution
ExampleDihydrolipoic acid (wt.%)Stabilizer (wt.%)Saline (wt.%)
400,010,00199,99
410,050,005at 99.95
420,10,0199.89 per

G) Prepared solutions peroxiredoxin and dihydrolipoic acid according to example No. 17, was used are listed in the following table 9 mass quantities of ingredients.

Table 9.

Content peroxiredoxin and dihydrolipoic acid in liquid form compositions
ExamplePeroxiredoxin (wt.%)Stabilizer (wt.%)Dihydrolipoic acid (wt.%)The ratio peroxiredoxin and dihydrolipoic acidSaline (wt.%)
430,10,010,052:199,84
440,10,010,025:198,87
450,10,010,00520:199,885

The wounds were washed with solutions prepared according to examples 37-45 and impose gauze bandage soaked in the same solution. The dressing was changed every day.

Results

Effects of solutions: a) peroxiredoxin Prx VI, b) dihydrolipoic the acid, in) peroxiredoxin PrxVI and dihydrolipoic acid on wound healing through one day is shown in figures 9-11. On figa, 10A, 11a presents the control.

Figure 9 presents the variation effects on wound solution peroxiredoxin. On figb, 9b, 9g - options presented experiments in which the concentration peroxiredoxin in solutions are: 1,0; 0,1; 0,2 mg/ml, respectively.

Effect on wound solution dihydrolipoic acid are presented in figure 10. On figb, 10B, 10g - options presented experiments in which the concentration of dihydrolipoic acid in solution, respectively: 1,0; 0,5; 0.1 mg/ml

Synergistic effects on wound peroxiredoxin and dihydrolipoic acid are presented figure 11. On figb, 11b, 11g variants of compositions in which the ratio between the constant concentration peroxiredoxin (1.0 mg/ml) and a variable concentration of dihydrolipoic acid is: 20:1; 2:1; 5:1 respectively.

In appearance the Russian Academy of Sciences, which was affected by dihydrolipoic acid (see figure 10), shows that although dihydrolipoic acid and an antioxidant, in its pure form it prevents healing and in high concentrations irritating to the wound.

Peroxiredoxin promotes the healing of wounds (see Fig.9), however, the best results were obtained when processing R the n compositions, composed of peroxiredoxin and dihydrolipoic acid (see 11).

External signs of wounds and shorter healing time can not talk about additive, and synergistic effects peroxiredoxin and dihydrolipoic acid in the intercellular space of the site of damage to the skin. Histological examination also showed significantly less scarring when applied peroxiredoxin Prx VI and dihydrolipoic acid.

Almost complete wound healing in control occurs within 7 days. Wound healing when applied purchaseretin happens for 5 days. When applying the composition on the basis of peroxiredoxin and dihydrolipoic acid complete healing occurs in 3 days.

Industrial applicability

Pharmacological composition containing at least one type peroxiredoxin can find a prophylactic and/or therapeutic use for many diseases, which are accompanied by the overproduction of free radicals. Higher efficacy of compositions based on peroxiredoxin compared to izvestnimi antioxidants can improve the efficiency and reduce the time of treatment.

Pharmacological composition has no toxic and biocompatible with mammalian organism, including man, as it as the e of the main element is used recombinant human peroxiredoxin. Low toxicity composition improves the efficiency of treatment by simultaneously combined impact on the local region of the body and the whole body through applications and intravenous administration of the composition. This is especially important when the treatment of multifactorial influences on the body, such as radiation, thermal, chemical burns, wounds, bruises occur during disasters and fires. The composition has high solubility and fast penetrate into the intercellular space of the body, which allows to treat diseases of almost all organs. The composition is compatible with any pharmaceutically suitable carriers, does not reduce the biological activity of the enzyme.

Sources of information

1. J.G. Babish, Howell T. Compositions containing carotenoids and tocotrienols and having synergistic antioxidant effect. PCT appl. WO 0230419 (2002-04-18).

2. H.Z. Chae, K. Robison, L.B. Poole, Church G.. G. Storz, and S.G. Rhee (1994) Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc. Natl. Acad. Sci. USA.91, pp.7017-7021.

3. McGonigle, S., Dalton, J.P., and James E.R. (1998) Peroxidoxins: a new antioxidant family. Parasitology Today.14, 139-145.

4. Prosperi M.T., Ferbus d, D. Rouillard, and Goubin G. (1993) A human cDNA corresponding to a gene over expressed during cell proliferation encodes a product sharing homology with amoebic and bacterial proteins. J. Biol. Chem. 268, 11050-11056.

5. Pesenko I.V., Novoselov V.I., Evdokimov VA, Nikolaev Ju.V., Kamzalov S.S., Shuvaeva T.M., Lipkin V.M., Fesenko E.E. (1998) Identification of a 28 kDa secretory protein from rat humans epthelium as a thiol-specific antioxidant. Free Rad. Biol. Med., 25, 654-659.

6. S.V. Novoselov, Peshenko I.V., Popov V.V., Novoselov V.I., Bystrova M.F., Evdokimov VA, Kamzalov S.S., Merkulova M.I., Shuvaeva T.M., Lipkin V.M., Fesenko E.E. (1999) Localization of the 28-kDa peroxiredoxin in rat epithelial tissues and its antioxidant properties. Cell. Tissue. Res. 298, 471-480.

7. L.H. Butterfield From cytoprotection to tumor suppression: the is multifactorial role of peroxiredoxins. Antioxid Redox Signal 1999 Winter; 1(4): 385-402.

8. Chung YM, Yoo YD, Park JK, Kim YT, Kim HJ. Increased expression of peroxiredoxin II confers resistance to cisplatin. Anticancer Res 2001 Mar-Apr; 21(2A): 1129-33.

9. Kinnula V.L. et al. Overexpression of peroxiredoxins I, II, III, V, and VI in malignant mesothelioma. J Pathol 2002 Mar; 196(3): 316-23.

10. Kim S.H. et al. Protein levels of human peroxiredoxin subtypes in brains of patients with Alzheimer's disease and Down syndrome. J Neural Transm Suppl 2001; (61): 223-35.

11. Das K.C. et al. Induction of peroxiredoxin gene expression by oxygen in lungs of newborn primates. Am J Respir Cell Mol Biol 2001 Aug; 25(2): 226-32.

12. Kim H.S. et al. Regulation of 1-cys peroxiredoxin expression in lung epithelial cells. Am J Respir Cell Mol Biol 2002 Aug; 27(2): 227-33.

13. Fujii T. Augmented expression of peroxiredoxin VI in rat lung and kidney after birth implies an antioxidative role. Eur J Biochem 2001 Jan; 268(2): 218-25.

14. Lee SC. et al. Peroxiredoxin is ubiquitously expressed in rat skin: isotype-specific expression in the epidermis and hair follicle. J Invest Dermatol 2000 Dec; 115(6): 1108-14.

15. Park, S.H. et al. Antisense of human peroxiredoxin II enhances radiation-induced cell death. Clin Cancer Res 2000 Dec; 6(12): 4915-20.

16. Chandrashekar R., Tsuji N. Dirofilaria and brugia thioredoxin buffer type-2 proteins and uses thereof. US Patent 6,352,836 March 5, 2002.

17. Novoselov V.I., Amelina S.E., Kravchenko I.N., S.V. Novoselov, Sadovnikov V.B., Fesenko E.E. Application of peroxiredoxine in the healing of lung. Problems of biological and ecological safety international conference. Obolensk p.203 (22-25 May 2000)

18. Walker, B.D., Lynn R.G. Peroxiredoxin drugs for treatment of HIV-1 infection and methods of use thereof. PCT appl. WO 02077294 (03.10.2002)

19. Andreeva YEAR and others (1998) Structural studies of the 28 kDa secretory protein from the rat's olfactory epithelium. Bioorganic chemistry, 24, N11 s-821

20. Sang Won Kang, Bains, I.C., Rhee S.G. (1998) Characterization of a mammalian peroxiredoxin contains one conserved cystein. JBC, 273,11, pp.6303-6311.

21. Chen J.W., C. Dodia, Feinstein, S.I., Mahendra K.J., Fisher A.B. (2000) 1-Cys Peroxiredoxin, a bifunctional enzyme with glutathione buffer and phospholipase A activities. JBC, 275, 37, pp.28421-28427.

22. Peshenko I.V., Shichi H. Oxidation of active center cysteine of bovine 1-Cys peroxiredoxin to the cysteine sulfenic acid form by peroxide and peroxynitrite. Free It Biol Med 2001 Aug 1; 31(3): 292-303.

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24. Knoops Century, Clippe A, C. Bogard, Arsalane K, Wattiez R., Hermans C., Duconseiulle E., Falmagne, P., Bernard, A. (1999) Clonning and characterization of AOEB166, a novel mammalian antioxidant enzyme of the peroxiredoxin family. J. Biol. Chem. 274, pp.30451-30458.

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29. Novoselov V.I., Amelina S.E., Kravchenko I.N., S.V. Novoselov, Sadovnikov V.B., Fesenko E.E. Application of peroxiredoxine in the healing of lung. Problems of biological and ecological safety international conference. Obolensk p.203 (22-25 May 2000).

1. Pharmaceutical composition for the antioxidant defenses of the cell, tissues and the whole body from hyperproductive free radicals in acute inflammation, chemical, thermal and radiation lesions containing peroxiredoxin Prx VI, characterized in that it contains additionally dihydrolipoic acid and pharmaceutically acceptable additives, and contains peroxiredoxin Prx VI and dihydrolipoic acid in an effective amount, and the ratio between peroxiredoxin Prx VI and dihydrolipoic acid is in the interval (ww) from 1:1 to 50:1.

2. The pharmaceutical composition according to claim 1, characterized in that the specified peroxiredoxin Prx VI is recombinant human peroxiredoxin Prx VI.

3. The way to increase antioxidant protection of mammals, characterized by the fact that the delivery of the pharmaceutical composition according to any one of claims 1 or 2 carried out in the extracellular space of the tissue, organ or whole organism of a mammal.

4. The method according to claim 3, characterized in that the delivery in the extracellular space of the tissue, organ or organism as a whole, is realized by means of passive or active diffusion in the application or spraying, by parentrule or endolyumbalno administration by injection, by means parentrule injection, by infusion, inhalation, drainage, via sublingual, vaginal, or rectal administration, p is the tool drops in the nose or eyes.

5. The method according to claim 3, characterized in that the delivery takes place simultaneously with the use of another therapeutic agent.

6. The method according to claim 6, characterized in that therapeutic agent is interferon.



 

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10 cl, 2 tbl, 1 ex

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40 cl, 1 tbl, 20 ex

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1 ex, 1 tbl

FIELD: organic synthesis.

SUBSTANCE: invention provides compounds of general formula I:

, where R1 represents -CO-Ra, -SO2-Rb, or aryl optionally substituted by lower alkoxy, wherein Ra represents cycloalkyl, cycloalkyl(lower)alkyl, cycloalkyloxy, aryl, aryloxy, aryl(lower)alkyl, aryl(lower)alkoxy, aryloxy(lower)alkyl, aryl-S-(lower)alkyl, aryl(lower)alkenyl, provided that aryl group can be optionally substituted by halogen, lower alkyl, hydroxy, nitro, cyano, lower alkoxy, phenyl, CF3, cyano(lower)alkyl, lower alkyl-C(O)NH, lower alkyl-CO, and lower alkyl-S; heteroaryl, heteroaryl(lower)alkyl, or heteroaryl(lower)alkoxy, provided that heteroaryl group is 5- or 6-membered ring or bicyclic aromatic group constituted by two 5- or 6-membered rings including 1-3 heteroatoms selected from oxygen, nitrogen, and sulfur and that heteroaryl group can be optionally substituted by lower alkoxy; Rb represents aryl, aryl(lower)alkyl, or heteroaryl, aryl group optionally substituted by halogen, cyano, or lower alkyl-C(O)NH; R2 and R3 represent hydrogen atoms; R4 representshydrogen or lower alkyl; R5 represents hydrogen, lower alkyl, cycloalkyl, benzodioxyl, or aryl optionally substituted by lower alkyl, halogen, lower alkoxy, hydroxy, or (lower)alkyl-C(O)O; n is 1 or 2; and pharmaceutically acceptable salts thereof and/or pharmaceutically acceptable esters thereof. Invention also provides a pharmaceutical composition exhibiting inhibitory activity with regard to cysteine proteases of the cathepsin family, which composition comprises compound of formula I, pharmaceutically acceptable recipient, and/or adjuvant.

EFFECT: increased choice of cysteine protease inhibitors.

34 cl, 1 tbl, 13 ex

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