Metabolic process modulator and method for making thereof

FIELD: pharmacology.

SUBSTANCE: invention refers to medicine, veterinary science and agriculture. There is presented exogenous modulator of metabolic and energy interchange processes between a cell and environment representing a polypeptide metabolic process modulator of molecular weight 15 to 10 KDa made of human placenta by chemical modification of placental biobased products with reactions of chlorination, oxidation, hydrolysis, and containing covalently bound chlorine atoms with nitrogen atoms of peptide bond in amount 0.5 to 40% of weight of an end product. There is also offered method for making said modulator.

EFFECT: correction and regulation of metabolic processes to ensure maximum viability of human body, animals and plants.

7 cl, 8 tbl, 10 ex

 

References: EN 2036650 C1, 09.06.1995, EN 2036651 C1, 09.06.1995, EN 223550 C2, 10.09.2004, EN 2215005 C2, 10.07.2003, EN 2223290 C2, 27.07.2004, EN 233290 C2, 16.07.1999. Remarry, Granner, Plies, W. Rodwell. Biochemistry of human rights. - M.: Mir, 2004. Grey et al. Sci. Am., 261: 38-46, 1989. Ipharmacy. The prospects for practical application of regulatory peptides and some basic research. // The. the honey. chem. - 1984 - THHH No. 3. Shitov, New approaches to the creation of medicines chondroprotective properties. // Vestnik Ross. Acad. the honey. Sciences. - 1992, No. 5, p.26-30.

The invention relates to biology and can be used in medicine, veterinary and plant for correction and regulation of metabolic processes in order to ensure maximum viability of the human body, animals and plants and, accordingly, maintain the normal physiological state.

Adverse Exo - and andfactory, the list of which is extensively described in the literature, cause changes in the properties of biomolecules, resulting in the disturbance of natural metabolism in metabolism and energy between the environment and the cell and the emergence of pathological conditions of various organs and systems (illness). Therefore, the fact that all without exception pathology in any organism is the result of metabolic disorders, is a common position and at the same time recognizing tsunorase the ima problems in medicine and biology.

The present invention aims to eliminate this problem and provides one possible practical solution.

Despite the complexity of the regulatory system, all Central metabolic pathways to the present time fully established, but the mechanisms of their regulation remain still unexplored. This, in turn, is caused by the absence of the necessary information about the regulation of metabolism in the cells of living organisms at the molecular level, and therefore require more time for further research (Remarry, Granner, Plies, Rodwell. Biochemistry of human rights. - M.: Mir, 2004).

By generalization, systematization and scientific interpretation of the results of studies of the mechanisms of natural regulation of enzymatic reactions in the cells of bacteria, defines the basic principle on which the process is carried out is the change in catalytic activity of one or more key enzymes of metabolic pathways. Biochemical compounds that increase the catalytic activity, is called positive modulators (Remarry etc. Biochemistry man. - M.: Mir, 2004) or effectors (Biochemistry. / Under obscured Essively. - M.: Publishing House. GEOTAR Media, 2000).

It should be borne in mind that the biochemical processes associated with changes in catalic the political activity of enzymes are endogenous in nature, i.e. proceed without the participation and the will of man. Biochemical products involved in such processes, are metabolites of cellular metabolism, the structure and the structure of which remain unexplained.

Information in the scientific and patent literature on the existence of exogenous bioregulators are missing. According to the author of the present invention is related not only to the fact that metabolism and its regulation in mammals extraordinarily complex, but with insufficient scientifically proven ideas about regulatory processes, especially about their mechanisms taking place in the cells of tissues of a living organism. In connection with this extremely difficult even hypothetically justify the chemical structure of the modulator with which it would be possible to influence by exogenous in the course of metabolic processes.

On the other hand it is also clear that in the practical use of any product protein nature based on natural fragments of amino acids, the body's response to his introduction is unequivocal: "Foreign", i.e. proteolysis and use as a building material with possible cytoxicity reactions.

The immune system protects individuals from foreign antigens and when meeting with them responds with activation of specific cells, such as T - and b-is Infocity, and production of soluble factors, such as interleukins, antibodies and complement factors. The antigen to which the immune system reacts, undergo degradation with antigen - presenting cells (APCs), and a fragment of the antigen associated with the glycoprotein major histocompatibility complex (MHC) class II is expressed on the cell surface. Complex MHC - glycoprotein - fragment antigen is presented to T cells via their T-cell receptors "learn" a fragment of the antigen together with protein MHC class II, with which they are associated. T-cell becomes activated, i.e. proliferate and/or produces interleukins, which leads to the expansion of activated lymphocytes to the antigen against which the immune attack is aimed (Grey et al. Sci. Am., 261: 38-46, 1989). Thus, the creation of modulators of metabolic processes on the basis of natural fragments of a protein nature is theoretically possible, but practically unlikely because of the high immunological defenses. In this regard, a pilot study with the aim of creating long-acting regulatory peptides remain inconclusive. This is because it is impossible to predict in advance immunological and allergic properties of the target products due to the presence of different species and Chennai specificity (species, Stadio-, organ - and organicosilicon). A significant obstacle to the creation of regulatory peptides is their rapid decay, regardless of method of administration, and the inability oral administration (Ipharmacy. The prospects for practical application of regulatory peptides and some basic research. // The. the honey. chem. - 1984 - THHH No. 3, pp.2-7). This fact proves the necessity of using special methods of their creation.

One of the promising areas targeted search of such substances is the synthesis of polypeptides of inducers of tolerance of the immune system to various antigens, which cause certain diseases (HIV, malignant tumor and other) by establishing a structure of peptides corresponding to the receptor and subsequent synthesis with an identical amino acid sequence (EN 2215005 C2, SK 14/47, AK 38/17, OR 35/00, 31/18 from 27.10.2003; EN 2233290 C2, SK 7/06, 14/47, AK 38/17 from 27.07.2004; EN 2199548 C2, SK 7/08, AK 38/17, AR 19/02).

Despite positive results in clinical trials patented peptides in individual patients, volunteers, remained outstanding question about their metabolism, because in spite of their "relationship", they remain for the body unrecognized antigens, therefore, according to the author of the claimed invention, they should quickly inaktivera Atisa. The rate of inactivation, as well as adaptation of the organism to such inductors is determined not only by the level of viability of the organism, but also by a variety of factors, including inevitably leaking hydrolysis by endo - and ectopeptidases. However, the main limiting factor for wide application in clinical practice is unknown metabolism such modulators, and therefore, there is the unpredictability of long-term effects because the nature of the "trick" is impossible. Therefore, the observed regulation of metabolism are private and cannot be viewed by the General method of correction of the disturbed natural metabolism, judging from the presence of more than 10 thousand items of nosological units (species) pathology and their clinical varieties.

No doubt the failure of the application of these peptides under these conditions, as "irreversible pathology", "degeneration", "atrophy", etc.

By the present invention the solution of this problem is achieved by the practical implementation of a theoretically founded by the author of the concept of creation of medicines for the regulation and correction of metabolic processes, published in the journal "Vestnik Ross. Acad. the honey. Sciences", 1992, No. 5, p.26-30.

The main provisions of the concept with separate specifications below.

In accordance with the priority provisions of the Soviet physiologist Pukina (Anokhin P.K. Central issues of theory of functional systems. - M., 1980) any organism is considered as a functional system, the main principle of which is self-regulation. The principle of self-regulation is that any deviation of the activity of the functional system level, which determines the normal (natural) metabolism or other side of the normal functioning of the body based on the feedback immediately selectively mobilizes various mechanisms for the system to return this result to optimal metabolism level. However, under certain environmental influences returns the system to the optimum level does not occur, resulting in a violation of metabolism leads to various pathological conditions.

Despite the complexity of the regulatory system, all Central metabolic pathways by now almost completely set, but the mechanisms of their regulation are still unknown (Newsholme E., Start K. Regulation of metabolism. - M.: Mir, 1977). Thus, the key to solving the problem of creating a highly effective medicines any pharmacotherapeutic group in unraveling the mechanism of regulation of metabolism. Since e is and the problem is almost difficult to solve, need an alternative or, more precisely, technological approach to solving this problem, based on the following well-known concepts and principles of biochemistry, biotechnology and pharmacy.

1. The drug should have a protein, for all biological processes, including metabolism and its regulation, is performed by means of proteins.

2. It is necessary to provide the maximum possible specific compatibility of the raw materials, i.e. the closer the nature of the protein substances to the human body, the greater the possibility of achieving the goal (the best option - the use of human organs and tissues such as the placenta).

3. The rate of biochemical reactions is determined not by the law of mass action, and the activity of key enzymes.

4. Use for the regulation of the metabolism of natural biochemical products of animal origin derived from organs and tissues by extraction and separation due to species incompatibility (antigenicity) and constantly changing composition and structure may not provide a high treatment efficiency and reproducibility as technology and pharmacological activity.

5. The desired effect of drug regulation (correction) metabolism can be achieved only in the case if the children are provided with the necessary stability of the active substance (substances) as outside the body, and in the system. This is one of the very important biochemical and technological conditions, because even if it is determined the structure of any natural regulator and implemented its synthesis, the introduction of it in the body will inevitably lead to deactivation.

One of the important ways of changing the activity of enzymes or respectively the rate of biochemical reactions is their interaction with certain signal biochemical products. This enables you to recognize a variety of signals and integrate the information received, thanks to him, the metabolic pathway can be activated or deactivated. In the case of pathological conditions of the "violent" introduction to dynamic self-regulating system, selectively combining different agencies and levels of nervous and humoral regulation, any substance or amount of substance of the same nature - controller only able to enable, not disable metabolic pathway, and simultaneously to increase the speed slow biochemical reactions, will restore partially or completely lost the physiological function of the tissue system.

Such products-regulators can be substances of protein nature - fragments of macromolecules containing in its composition of oxidized functional groups, missing, usually in the natural the governmental conditions under normal physiological condition. This is the essence of the concept of purposeful search for the next generation of drugs, which previously formulated on the basis of systematization, generalization of literature data and results of own research (Shitov, etc. // Chem.-Pharm. prom-St. Survey information. - M., 1989 - issue 3). Prerequisites, which formed the basis for the formation of this concept, discussed below.

1. thermodynamic driving force of any biochemical process is determined by the amount of energy released by the hydrolysis of special substances-donors are in high oxidation States. For example, such a substance is a carrier of energy is adenosine triphosphate (ATP), as it contains 2 phosphorus-angeilna connection corresponding to the maximum oxidation state of phosphorus. According to (Klotz I. Energetics of biochemical reactions. - M., 1979) actual released during the hydrolysis of energy in the conditions existing in the cell is normal, is about 12 kcal/mol. The energy release during hydrolysis of ATP provides the flow thermodynamically unfavorable processes and shift the equilibrium in coupled reactions about 108time. Similarly, thermodynamic driving force of the process of transfer-acceptance of electrons in the oxidation of fuel molecules is the energy determined by the degree of oxidation Niko is insidegamingmachinima (NADP) and dinucleotide (NAD).

When using this pattern of metabolism for drugs becomes obvious that the medicinal substance should have enough expressed thermodynamic activity, i.e. to be in the oxidized state, and be to some extent a source of energy for the flow energetically unfavorable reactions with enzymes. The translation of the protein molecule or the polypeptide in a higher degree of oxidation can be achieved by introducing additional oxygen atoms (hydroxyl or carbonyl groups, sulfones, and so on).

2. Oxidation (hydroxylation) of amino acids in the body is, apparently, a special protective measure, as evidenced by numerous publications about the increase in the content of hydroxyproline and hydroxylysine in the serum or urine in many pathological conditions (Mosquitoes FI etc. // Klin. the honey. - 1977, No. 11. - Pp.62-67). In this case we are talking about how to interpret these data. The increase in the content of hydroxyproline is a fact of metabolic disorders (pathological tissue destruction) or, on the contrary, the special safeguard measure? In favor of the latter said not only the facts of his increase in almost all types of pathology or normal pregnancy, and special studies. If you type labeled14C-g is roxiprin or 14S-hydroxylysine the rat, the newly synthesized protein of connective tissue (collagen) radioactivity not detected, while when the input14With-Proline collagen is radioactive (Cardinale G. // Enzymol., 1974. Vol.41, p.245-300). At the same time, it is unclear why the content of oxidized amino acids in pathology in the blood or urine increases if the Proline cannot be progeressive outside of the biopolymer. Therefore, proteins of the connective tissue or other protective proteins in pathological condition optionally oxidized, in "thrown out" of their fragments, which can be viewed as andregulatory pathological process. The above facts indicate that amino acids in higher oxidation States play a special role in increasing the body's resistance to harmful factors and suggest that the inclusion in the composition of the protein or polypeptide of hydroxylated acids can lead to reduced immune deficiency, and to accelerate biochemical reactions aimed at the correction of metabolism.

3. One of the essential prerequisites for the formation of concepts are the results of a study of chemical structure of fibrillar proteins (collagen)found in the skin, bones, tendons, cartilage, teeth, etc. Collagen region which gives an unusual composition and an unusual amino acid sequence. Amount of Proline in collagen significantly higher than in most other proteins, which facilitates hydroxylation. In addition, the collagen comprises oxidized amino acids, for example in the basal membranes of 6%, and in hyaline cartilage of 2.0-2.5% (Strayer L. Biochemistry. - 3 So - M: 1985). Under irradiation and thermal effects of the content of hydroxylated Proline and lysine in collagen significantly increased, and therefore the adjustment process of oxidation of amino acids flowing under the influence of the enzyme prolylhydroxylase (Strayer L. Biochemistry. - 3 So - M: 1985), not only collagen, but other protective proteins, is crucial for increasing the resilience of the body.

4. Of particular interest in theoretical terms is a question about the metabolism of biopolymers in the oxidized form. The metabolism of individual drug substances, xenobiotics studied in detail, and proved in some cases harmful effects of metabolites on the protective system, especially with prolonged use. In other cases, the metabolites are the main active ingredient. Getting natural biopolymer in a higher oxidation degree means nothing, as his transfer in metabolic state. However, unlike xenobiotics he will not be displayed in this form of the organism, as this status is e, is a form of existence of protein Ph. Further metabolism of oxidized biopolymer, as will be shown further proceeds, in all probability, as andregulation, i.e., through hydrolysis into smaller fragments. The resulting products of enzymatic hydrolysis and are the main active substance.

Thus, the proposed concept of purposeful search of medicines can be considered as the concept of drug metabolism regulation in the immune and other pathological conditions.

For the practical implementation of this direction was needed substantiation of technological techniques and methods by which it would be possible to carry out the process of chemical modification of biopolymers using previously described conditions.

Choice as the main source of biopolymers placental tissues based on the fact that they are "ideal" object to achieve goals. To date, published numerous works on the use of the placenta to obtain preparations of a different orientation. Latitude pharmacological spectrum of action and high impact open up almost unlimited possibilities for creation of medicines, including those which allow you to regulate (correction) bre the frame of metabolism.

Based on the presented study, a prerequisite for the creation of modulators of metabolic processes is the use of biopolymers in species trait through a special chemical process them. For example, for mammals - relevant biopolymers contained in the placental tissues, plant biopolymers vegetable origin, etc.

In accordance with the above concept of the target product after chemical modification of biopolymers must have the following physical-chemical and biochemical properties:

1) the presence of peptide bonds;

2) high bioavailability and biocompatibility;

3) the absence of antigenic properties;

4) the highest oxidation state.

Use as feedstock for bioplastics in species trait determines high bioavailability, biocompatibility and allows saving of peptide bonds. However, all of the biopolymers in the native form regardless of the degree of homogenization, temperature of incubation, separation into fractions, etc. are immunogenic properties, therefore, to obtain the target products with no antigenic properties you want to retrieve them in the oxidized, i.e. metabolic condition which is typical for natural andregulation, containing in their structure one is or more amino acids in oxidized form type hydroxyproline, oxylipin and others In natural conditions andregulatory after performing their functions undergo proteolytic cleavage, resulting in examination not used as "building material" and therefore are excreted with the urine.

Oxidation of biopolymer significantly alter the physical-chemical and biochemical properties of the target product by increasing the free energy, which, in turn, leads to increased reactivity of the modulator when interacting with enzymes. Thus, accession to the oxidized enzyme fragment biopolymer (modulator) with a large supply of free energy would mean not that other, as the increase in the catalytic activity of the key enzyme in the metabolic process. On the other hand, the oxidized state of the modulator reduces the speed of its further metabolism and therefore increases the time of biological action.

For the practical implementation of this idea by the author of the claimed invention with the staff conducted a study of the process of chemical modification of various biopolymers using acid chloride, known as the reagent having a high selectivity in the hydrolysis of peptide-glycosidic linkages and the oxidation of individual functional groups in biomolecules. In the high selectivity and the selectivity of the acid chloride due to its instability:

9HClO2→3HCl+3H2O+3ClO2+1,5Cl2

Education in the reaction mixture of chlorine dioxide and chlorine provides simultaneously the oxidation of certain amino acids and quantitative substitution of hydrogen atoms by chlorine in the peptide bond and the amide groups of arginine, asparagine, histidine and glutamine. The formation of the covalent bond to the nitrogen - chlorine leads to mate regard, the nitrogen - carbon, which determines its high resistance to proteolysis, which increases due to the impossibility of the formation of the enzyme-substrate complex.

The study was able to determine that in the polypeptide chain are subjected to oxidation only serine, threonin, methionine, cystine, cysteine. All amido and amino compounds amino acids undergo chemical transformation due to the formation of covalent bonds, nitrogen - chlorine. Thus in the target product, obtained by chemical modification of biopolymer remain intact glycine, alanine and phenylalanine, which were identified by amino acid analyzer. In addition, we found that the serine in the composition of the polypeptide chain is oxidized to α-aminomalonate acid, tryptophan and terazin peterpaul full destruction, that is, the polypeptide chain is broken at the place of their location, as evidenced by the literature on nimi (see Protein: 4 so edited Pairote and Calli. - M: Il, vol. 1. Chemistry of proteins, s-135, 173, 186, 281-290).

Sulfur-containing amino acids (cystine and cysteine) are easily oxidized hipogalactiei to acid cysteine, and methionine to the corresponding sulfide (MR, Felwort. Titrimetric methods of analysis of organic compounds. Methods of direct titration. / Lane. from English. - M.: Chemistry, 1968, s.219-223). The presence of the polypeptide of sulfo characterizing cysteine acid, we have proved the IR spectra by the presence of stretching vibrations in the region 1670 cm-1and quantitative determination method (Kullbom S.D., H.F. Smith // Analytical Chemistry. - 1963, 35, p.1005).

The process of chemical modification of biopolymers with the polypeptide chain can be expressed by the following reaction equations:

(a) chlorination of the peptide bond

/Polypeptide chain/

b) oxidation of amino acids in the biopolymer

Amino acid composition of the obtained modified biopolymer is characterized by the indicators shown in table 1.

The amino acid composition of the modulator

The content of chlorine per 1 mol of the modulator is mA the maximum possible value 37,45%, according to the claimed invention provides an upper limit of 40%, lower to 0.5%.

The content of oxidized amino acids per 1 mol of the modulator is from 2 to 5 residues, the claimed invention provides from 1 to 20.

Using ion-exchange chromatography of the reaction products of biopolymers with chloride acid selected two polypeptide fraction with an average molecular weight 13800 and 14200 E determined by the method of Lowry. The polypeptide chain consists of 82 and 86 amino acid residues (respectively). The first fraction according to IR-spectroscopy (Kullbom S.D., H.F. Smith //Analytical Chemistry - 1963, 35, C) contains sulfopropyl in terms of sulphur about 0.6% by weight of the polypeptide, whereas the second fraction in the infrared spectrum stretching vibrations in the region 1670 cm-1no. The chlorine content in the polypeptides, respectively 35,6 and 36.5%.

Thus, the target product of the present invention is a chlorinated this highly oxidised polypeptide, in which the combustible part (carbon and hydrogen) is only 32% of the total mass. The accession of chlorine atoms to the polymer should be seen as a translation of it even to a higher degree of oxidation.

Each chlorine atom gives the polymer to the electron, which leads to the greatest extent possible to create free energy. Such chemical modification of biopolymer which determines special, previously unknown physical-chemical, biochemical and pharmacological properties, which are characterized by the following indicators:

1) the Polypeptide has an extremely high resistance to acid and alkaline hydrolysis. After heating the drug in a sealed vial at 100°C in a solution of hydrochloric acid for 24 hours can only identify glycine, phenylalanine and alanine.

2) the Drug has a high resistance to enzymatic hydrolysis when taking it through the gastrointestinal tract, since the pharmacological activity is practically identical with the injectable dosage form.

High resistance to proteolysis due to the presence of the covalent bond to the nitrogen - chlorine, which leads to a coupling connection, the nitrogen - carbon.

3) freezing point of a 1% aqueous solution of the drug - -70°C, due to the high surface activity.

4) Sterile aqueous solution in vials (no spores) observation retains its physico-chemical and pharmacological properties over 10 years (when stored without exposure to sunlight). This property is due to the high degree of oxidation. The oxygen contained in the aqueous solution is not able to oxidize the drug, just as water does not cause its hydrolysis regardless of the time of impact.

5) No that is classical properties. Conditionally therapeutic human dose of 0.07 mg/kg, with the introduction of the drug to animals 100 times greater than conventionally-therapeutic dose within 6 months of the death of animals does not occur.

6) Has a previously unknown high adaptogenic action in relation to thermal, physical strain and the effects of poisonous and toxic substances, including insect bites, snakes, etc.

7) the Presence of a polyfunctional pharmacological action (immunotropic, reparative, anti-inflammatory, resolving, lactogenic, antioxidant, chondroprotective, anti-stress, anti-mutagenic, antishock, anticoagulant, fertile, including in the treatment of erectile dysfunction and prevention of miscarriages).

8) the Presence of previously unknown high biological activity when used as a prophylactic for most known diseases, including increasing life expectancy and exceptions premature aging of the skin (the lifespan of mice with the drug at a dose of 1 mg/kg for 30 days increased on average 1.5 times).

9) Long-term pharmacological effect after treatment (duration of preservation of the pharmacotherapeutic 5-6 years).

10) Lack of adaptation of the organism to the drug with long-term use.

11) Paul is Chen high effect of the drug as a regulator of growth and development of plants (the yield of most crops increased in 1,5-2 times, moreover, increases the resistance of plants and their fruits to harmful environmental factors).

12) Set the high activity of the drug in beekeeping, mushroom cultivation, sericulture, animal husbandry, fisheries, etc.

These pharmacological properties and the degree of effectiveness for each species was determined on the basis of the results of preclinical and clinical studies conducted at leading research institutes and medical institutions of Ukraine and Russia.

High pharmacotherapeutic efficiency of patentable modulator (hereinafter "Product") found in studies on laboratory animals (mice, rats, rabbits, dogs), during clinical trials in humans and in postclinical application. Below are the results of preclinical, clinical and postclinical of the proposed drug in medicine, veterinary medicine and agriculture.

Immunotropic action.

Evaluation of immunotropic activity of the proposed drug is determined by the standard methods recommended by the world health organization (WHO). Specific pharmacological (immunological) activity was tested in model systems, which quantitatively characterize the effect of tested drug on the basic parts of the IMM is nitya. Studies were conducted on Guinea pigs, rats, Wistar rats, outbred mice and mice CBA, W (SWAHS)G1. Verified the impact of the proposed drug on cellular and humoral immune response (phagocytic activity, influence on the generalized Pseudomonas infection, complement system, on the cooperation of T - and b-lymphocytes, graft vs. host reaction of hypersensitivity of the delayed type, the humoral response). As Comparators used known therapeutic agents such as levamisole, T-activin, cyclophosphamide, nucleat sodium, amniotsen, pyrogenes.

Test results of the inventive tool has superior activity of these drugs comparison and only in some tests detect the same activity as the T-activin. The most typical indicator of immunotropic (together with immunomodulating activity of any medication is the assessment of its impact on the hypersensitive reaction of the delayed type (GST). In studies on the induction of GST sublethal irradiated mice (ionizing radiation raises the immunological competence of the body, suppressing the manifestation GST) claimed the drug was administered subcutaneously 5 times in a total dose of 0.4 and 0.6 mg/kg, starting from after the ith day after irradiation. As can be seen from the table below, treatment of animals at a dose of 0.4 mg/kg increased hypersensitivity reactions by 30% compared to irradiated mice of the control group. At a dose of 6.0 mg/kg index of reaction almost reaches the level of the group of non-irradiated animals, which proves the ability to protect the body during radiation exposure.

Table No. 2
The impact of the proposed drug reaction GST
GroupMedicationDose, mg/kgWR% (M±m)P
1 control (intact)--20,0±2,46-
2 control (irradiated)--6,1±1,21-
3 irradiated with drug useThe claimed preparation0,47,9±1,28<0,05
0,617,8±2,94<0,01
R - relative to the control groups

Antioxidant effect.

Antioxidant effect of the proposed drug is installed in the peroxide oxidation of lipids, whose efficiency is higher than that of ascorbic acid is almost 10 times. This fact is important in the practical use of the proposed drug not only in dermatology, and Oncology.

Fertile action.

In the research process of the proposed drug and mutagenic properties (on the model of dominant lethal mutations) revealed an increase in the fertility and fecundity of experimental animals compared to control. At the stage of Mature spermatozoa observed a significant reduction in postimplantation loss, which is in good agreement with literature data on antimutagenic action of drugs from the placenta.

Adaptogenic effect.

This type of action in the process of creating new drugs is one of the most actual directions of search, because it can give the opportunity to influence physiological processes in the physical, thermal, chemical overload, especially to prevent shipments, burns, increase your endurance, etc. In which the process of the research claimed the drug is established, that pre-treatment of animals (mice) for 7 days in conventionally-therapeutic dose (0.7 mg/kg) prevents the death of animals from toxic doses of toxic substances (strychnine), lethal irradiation, infitsirovanija by Escherichia coli. The influence of the drug on the lifespan of mice after administration of the drug at a dose of 1 mg/kg for 30 days. It is established that such prophylactic treatment of mice their life expectancy increases on average 1.5 times.

Anti-stress effect.

Anti-stress effect of the proposed drug is studied on the basis of multivariate methods of assessment of different components of resistance to emotional stress in rats (behavioral and visceral components). As a result of these studies found that the claimed product has the ability to increase resistance to emotional stress, that is, to prevent the development of functional disorders of higher nervous activity, immunity and metabolic disorders of cardiac activity. The most effective was the dose of 30 mcg per 100 g

The lactogenic effect.

During practical use of the proposed drug in veterinary medicine found that when agalactia in sows claimed the drug leads to the restoration of lactation process.

And decoagulant action.

In the research process, the degree of influence of the proposed drug in the blood is found that at a dose of 0.15 mg/kg by month introduction found anticoagulant effect, which disappears after 30 days after the end of injection. This fact may have important practical implications for the treatment of preinfarction and post-mi States.

Chondroprotective effect.

As Comparators for the assessment of activity of the proposed drug for its ability to rebuild cartilage tissue were selected known chondroprotective drugs, such as rumalon, macartan and arteparon. Assessment chondroprotective properties produced by the standard model of post-traumatic arthrosis of the hip joint in rats, which develops after application of the defect in the form of round holes about 2 mm in diameter, passing through the articular cartilage into the subchondral region. The application of this defect leads to the development of posttraumatic osteoarthritis. To test the activity of the proposed drug and Comparators were selected dose: 0,01; 0,03; 0,06; 0,1; 0,3; 0,5; 1,0 mg/kg animal mass. Assessment of the effectiveness of chondroprotective action was carried out on a specially designed scale (in points). The maximum possible score is 12 points, which corresponds to the complete resorption of connective tissue and is to restore cartilage. Results scoring:

Rumalon - 7,6

Muqarrin - 8,4

Arteparon - 8,9

The claimed drug - 11,8.

Anti-inflammatory effect.

This type of validity is tested in experimental granuloma in rats. As comparative drugs were used medicinal product derived from human placenta - PDS (placenta denatured suspended) and amniotsen. The drugs were administered subcutaneously at the same time with the operation. The test results presented in table No. 3.

Table No. 3
The results of the study impact of the proposed drug on the proliferation and exudation
Drug
tool
The number of animalsDose, mg/kgThe decrease in proliferation, %% controlThe decrease in exudation, %% control
The claimed preparation101,038,7±10,828,0±10,9
Amniotsen1110 26,2±6,314,2±3,6
PDS85033,5±6,725,3±4,8

Resolving action.

Resolving action was studied on the model of adhesions that occur in non-pregnant female rats in the dosage trauma uterine horns. The results of the experiments are presented in table No. 4.

Table No. 4
The results of the study impact of the proposed drug adhesions in rats.
MedicationThe number of animalsDose, mg/kgAdhesive mass, % of control
3 days10 days21 days
None (control)15-57,4±4,3141,6±37,836,5±5,8
The claimed preparation5 0,518,5±3,616,8±2,83,2±0,9
-!-151,014,8±3,712,5±3,71,2±0,8
Amniotsen152019,2±2,330,5±6,27,0±1,1
-!-155014,0±1,921,6±5,25,3±0,9

Reparative action.

This type of pharmacological action set on the standard model mesh with the skin defect in mice that were injected claimed the drug in doses of 0.5-1.0 mg/kg Under the influence of the drug the healing period compared to the control is reduced from 17 days to 10. A similar effect has a drug from the placenta Amniotsen only at a dose of 75 mg/kg While a significant advantage of the proposed drug is no scarring in the healing process.

The claimed preparation in full investigated in acute and chronic toxicity according to methods recommended by BOAZ.

Stra toxicity.

Validation of the proposed drug on the acute toxicity was carried out on mice by a gradual (twice) of increasing doses of the drug, starting with 1 mg/kg animal mass to 100 mg/kg To maximum dose (100 mg per 1 kg of weight) animals do not die, which proves the absolute harmlessness of the drug. In high doses, which exceed conditionally therapeutic 5000 times, of course, the animals find physical inactivity, apathy, lethargy, but restore its activity after 3-4 days without signs of poisoning.

Chronic toxicity.

The study of chronic toxicity claimed the drug was carried out according to the recommendations of WHO on three groups of animals (rats, rabbits and dogs) during the 6-month daily administration of the drug in doses that exceed the conventionally-therapeutic in 10, 20, 50 and 100 times. After 6 months of injection 50% of the animals were scored to assess the degree of influence of the proposed drug in organs and tissues, and other animals rehabilitated for 1 month and then scored for comparative evaluation of the pathological effects of the drug.

The study of the internal organs of animals after 6 months of administration in the above doses, none of the animals died. Identified changes in the lungs, brain and kidneys after a month of rehabilitati is recovered entirely, that confirms the efficacy of the proposed drug (high bioavailability and biocompatibility).

The results of clinical trials.

Clinical testing of the proposed drug was carried out in three stages. The first phase of volunteers (30 people), which was administered intramuscularly solution of the proposed drug in water containing 1 mg of active ingredient, for 10 days. Toxic effect and other side effects not established. The second phase (90 people) - the definition of a therapeutic dose of drug use as anti-inflammatory and analgesic in osteoarthritis (as one of the most promising areas of biological action of the drug).

The third phase of the study, conducted in 3 hospitals of Moscow with the total number of patients 156 people (men and women aged 18 to 78 years) with a diagnosis of osteoarthritis of different etiology and severity. The main clinical manifestation of osteoarthritis is pain, evaluation is performed on a specially designed scale scores on a 5-point scale as before treatment and after treatment. The claimed drugs were administered parenteral and intra-articular dose of 2 mg of the active substance, first through the day (10 injections), then every day (20 injections). Each patient received a total of 60 mg of the drug. Sub is active assessment of effectiveness showed that 58 people pain decreased significantly (37%), 42 people (27%) had completely disappeared, 33 people (21%) pain disappeared completely in the knee joints and decreased significantly in the hip joints. All patients had increased range of motion, including when walking on the stairs. In 12 patients who had been admitted to the hospital on crutches, came the ability to move independently. Side effects in the process of testing is not installed.

Postconcussive testing of the proposed drug has shown good effect in many pathological deviations that proves it binormality action. Below are examples of such tests.

Example 1.

Patient L., 33 years. Diagnosis - the consequences of cerebral palsy (spasm of the right hand and entire arm, motor and physical inactivity). After treatment of the claimed drug for one year in combination with physiotherapy methods, also with the use of the proposed drug, achieved almost complete recovery. The patient began to write, peel potatoes, and perform other small movements with high accuracy. He started to run, to swim. Application of known therapeutic agents over the last 30 years has not yielded positive results.

Example 2. The use of the proposed drug in child and adolescent psychiatric practice is the IR.

Therapeutic correction of mental and neurological syndromes, which are formed due to early organic lesions of the Central nervous system, closed craniocerebral injury and neuroinfections in children and adolescents, remained until the application of the proposed drug problem, which is hard to be solved. When used for these purposes, the proposed drug for 25-30 days in combination with physical therapy practices in the Ukrainian NGI health of children and adolescents has high stable therapeutic effect (95.3 per cent). The other patients showed significant improvement in health status.

Example 3. The use of the proposed drug with toxoplasmosis.

Toxoplasmosis - zooropa parasitic disease, which is characterized by a polymorphic clinical picture, latent, acute or chronic course with acquired or congenital infection. Toxoplasmosis usually not amenable to effective treatment, and the disease takes a protracted chronic. The use of the proposed drug for the treatment of toxoplasmosis according to the company "Bacterin" in Dnipropetrovsk medical Academy of proven high pharmacotherapeutic efficiency, which is characterized in most cases, full recovery.

Example 4. The use of the inventive PR the drugs for chronic disorders of cerebral circulation.

According to the results of treatment of patients with a diagnosis of stroke, regardless of the duration obtained in the polyclinic №3 Kharkov, installed complete resorption of hematoma of the brain, adhesions that have formed, and other pathological defects. Ultimately restore lost motor function and speech. The duration of treatment depended on the severity and duration of the pathological state.

Example 5. The effectiveness of treatment of urogenital infections.

The use of the proposed drug in urological practice in the treatment of diseases such as chlamydia, ureaplasmas and others, especially in their combination gives a high therapeutic effect. According to the results obtained in the urological clinic at the Kharkov medical University, a positive effect was achieved in 94.7% of cases.

Example 6. The use of the proposed drug in cardiology.

The polyclinic №6 of Kharkov were treated more than 200 patients with cardiovascular insufficiency (post condition, angina, myocarditis and other). The absolute majority of the patients received stable therapeutic effect, especially angina and post-infarction state.

Example 7. The use of the proposed drug for the treatment of injuries and diseases of the musculoskeletal system.

According to the results of treatment of patients with what deseases musculoskeletal different etiology, conducted on the basis of polyclinic №6 Kharkov, there was a positive effect in almost 100% of cases.

Example 8. The use of the proposed drug in gynecological practice.

High efficiency treatment of gynecological diseases such as endometritis, diffuse fibromyoma, adnexitis, polycystic, inflammation of the appendages, etc. shown in the results of its application in gynecological institutions of Kharkov.

High effectiveness of the proposed drug is established also in Oncology, ophthalmology, surgery and other medical fields. A drug called "Bioglobin" approved for medical use (Registration certificate № UA / 3273/01/01 from 24.06.2005, Ukraine).

Example 9. The use of the proposed drug in crop production.

In biocatalytic action of the modulator on the seed and foliar parts of plants increases the rate of biochemical reactions in photosynthesis as in the development, flowering, and at plodoobrazovanie. This factor leads to an increase in the length and thickness of the above-ground parts of plants, number of stems, inflorescences and area of leaves, and thus to increase the yield of the treated cultures. This effect covers nearly all kinds of plants, including room, weeds, and fruit trees. Such a wide range of actions which I caused by the unity of wildlife (plants) and modulator, ensuring its existence.

The presence of adaptogenic and immunotropic activity of the claimed modulator provides the resistance of plants to frost on the ground and drought. As shown by the results of observations embedded in individual private farms, the processing by modulator fallen and blackened by frost stems of tomatoes leads to the resumption of growth, development and full plodoobrazovanie. A similar effect is observed when drought. The increase in yield compared to the control 25 to 200% depending on the agricultural background.

Due to the adaptogenic and immunotropic activity of the claimed modulator when it is achieved by increasing the resistance of the fruit to infection both during growth and during storage.

One of the significant advantages of the claimed modulator of growth and development of plants in comparison with the known is the provision of ecological purity of the finished product and the elimination of the negative effects on the environment due to the unity of the biochemical nature of plants and modulator.

Practical application of the claimed modulator is accomplished by pre-treatment of seeds of vegetables or tubers before planting of 0.0005-0.005% of a solution of the drug (current substance), which is prepared at the rate of 2 ml trademark Myoglobin (1 tea lo the SC) per 10 liters of water. During the pre-treatment of a small number of (private sector) the treatment is carried out with a solution of the trademark drug of calculation 5-10 drops to 1 Cup of water. For presowing treatment of seeds for 24 hours, and tubers before planting 10-15 minutes.

Foliar treatment of plants or fruit trees is carried out by spraying a solution of the commodity Myoglobin according to the above concentration sprayers in seedlings, during budding and flowering.

The results verify the effectiveness of the claimed modulator as growth and development of plants, held in conditions of field research-based NIDS vegetable and melon crops UAAS, Kharkiv vegetable factory and garden plots of individuals presented in table No. 5 - 9.

Table No. 8
Performance and its constituents of sweet pepper under different treatment options
The grade nameTreatment optionThe performance of the plant, gThe average number is ladow, piecesAverage fruit weight, g
Hope1678,009,1773,95
21355,0016,0085,05
3422,008,9044,81
1852,009,7987,00
Gift Moldova2has 650,5010,5161,72
3590,508,5569,11
HIP0538,920,785,94

The study of the effect of the proposed drug on the development of plants beet (sort Dios), radish (sort Xenia), parsley (sort Kharkov), carrots (grade Bright) conducted in field crop rotation. Seeds Zama is ivali 24 hours in the solution, containing 0.5 mg/l of the drug.

The test was carried out according to such variants:

I dry the seeds,

II-soaking in water

III - soaking in a solution of the modulator.

Particularly noteworthy is the regulatory effect of the proposed drug on the formation of biometric measurements cabbage 1 year of life. In plants treated with the drug, indicated a much greater increase in the length of the stem, and the surface area of the second and third true leaves. The best results in reducing the number of ledogorov noted when administration of the drug in the highest concentration (5 mg/l) - 80.3 per cent for grade Kharkov winter and 78.6% for grade Yaroslavna. Watch - 43,9% and 37.9%, respectively. It should be noted that meristem material cabbage bald, like any other vegetable crops, when moving from aseptic cultivation conditions is always observed low survival rate. In this respect it should be noted the positive role of the proposed drug as a stimulant adaptogenic actions in vitro plants to growing conditions in vivo.

During the growing period of the plant cabbage varieties Kharkov winter in conditions of glass greenhouses marked the best performance increase leaf surface is 1.6 times in comparison with control, with 95% strung heads to the crucibles of commercial maturity, control of 56.7%. Similar figures are reported for grades Yaroslavna - an increase of 1.33 times and 98% of heads of cabbage had marketable maturity (control - 46,1%). It should be noted that the cultivation of cabbage in greenhouses throughout the summer period was marked by high daytime temperatures (up to 35-40°C), which is not a typical factor in the cultivation of this vegetable crops in the open ground. In this respect it should be noted the positive role of the proposed drug, as regulator, which increases the resistance of plants to this biotechnolo stress.

Provided in the tables the results of the effectiveness of the claimed regulator obtained on fields with weak agricultural background. In the presence of high agricultural conditions the yield of potatoes, cabbage and other agricultural crops in the processing of the modulator is increased more than 2 times. As shown by the results of applying the proposed modulator at Kharkov vegetable factory, the yield of cucumbers and tomatoes with full processing (in the period of germination, budding, flowering) increased with each 1 m22-2,5 kg

Installed high efficacy of the proposed drug for the treatment of grapes during budding, flowering and early education peas. The yield of grapes, with appropriate feeding increases more than 4 times. T is the train high effect of the proposed drug is due to the above specific physical-chemical and biochemical properties (reproductive effects) of the target product. Moreover, when used as a feedstock for bioplastics plant origin, such as the application instead of the placenta soy flour.

The closest analogue of the claimed invention is "Drug binarymessage action and method thereof" (EN 2235550 AC 35/50, publ. 10.09.2004,) by which the target product is allocated after chemical treatment of the tissues of the placenta chloride acid is a complex mixture of biochemical substances, in which the main active ingredient is not identified.

Main indicators of the quality of the finished product obtained by the prototype is the biological activity and the content of the polypeptides. Therapeutic dose of the drug that is installed in the testing process is 2 ml of a solution containing 40 mg of dissolved solids, including not less than 1 mg of the polypeptide. The biological activity is measured by the ability of the drug to increase the activity of lysosomal membrane enzymes of neutrophils in the blood and increase energy and oxidative metabolism. The definition of manufacturer for the standard method recommended by the who, i.e. the response of neutrophils heparinised blood of mice, manifested in the spontaneous recovery narasinga tetrazole (NBT-test).

The biological activity of the preparation is the same received the prototype is not less than 50,000 units per 1 ml of the drug.

Despite the high pharmaco-therapeutic effects of the drug received by the prototype, patented method has some significant drawbacks due to its technical shortcomings. The main disadvantages are the following:

- the complexity of the composition, most of the components are the ballast impurities, which, although they do not exhibit toxic properties, but can have a negative effect on the principle of inhibiting the activity of the main active substances;

- the finished product from the point of view of the novelty is characterized by the presence in the composition of polypeptides, amino acids, hexuronate acids and aminosaccharides in the oxidized form, while not defined qualitative and quantitative characteristics, including the degree of oxidation;

- not installed the main structure of the active substance and not defined its main features that determine the achievement of a positive effect;

- patented method of obtaining funds cannot provide the main active substance in high content, which leads to reduction of biological activity in General, especially its reactivity as a modulator of metabolic processes.

Marked is installed shortcomings the author by conducting additional research and the development of a new method of production, the hallmark of which is used for chemical modification of biopolymers oxidation-hareruya and hydrolysing the mixture allocated after processing the chloride salts of the acid, sulfonic cation exchanger in the H form and ion exchange technologies

R So3H+Na ClO2→ R So3Na+HClO2.

Under the action of the sulfonic cation exchanger in chloride acid undergoes a chemical transformation

9HClO2→ 3HCl+3H2O+3ClO2+1,5Cl2.

The main obstacle to the selection of the active substance from the reaction mixture in the process of getting a drug prototype is incomplete desorption of the target product at ion exchange. This obstacle has been removed by pre-fine grinding sylvopastoral of the cation type KU-2-8, Lewatit, Dewax and others in the protonated form on the cage of desi type II ML 1F (Estonia), provided with two toothed wheels, with oppositely directed rotation speed 16000 rpm particle Size after grinding no more than 1-2 microns. Below are examples of specific performance of the proposed method.

Example 10.

In a titanium reactor equipped with a stirrer and a coil for heating or cooling load minced in a meat grinder and washed from the blood to the placenta of humans or livestock, add pre-cooked mixture of e is vivalentny the ratio of sodium salts of amino acids (preferably lysine) and cobalt chloride at the rate of 0.001-0.1% of cobalt salt of lysine on dry residue powdered placenta. Then the reactor was quantitatively transferred oxidation-glorious mixture, prepared by treatment with 5% solution of sodium chloride salt of the acid, sulfonic cation exchanger in the H form within 24 hours based on 1 part dry placenta 0.1 to 1.5 parts of the sodium chloride salt of the acid, taken on the sorption and incubated for 1-45 days at a temperature of 18-60°C to the desired degree of chemical modification.

The degree of completeness of chemical treatment is determined by the percentage of chlorine in the water soluble reaction products, which should be in the range of 0.5-40%. Upon reaching the desired degree of chemical processing exposure ceased, the reaction mixture representing a heterogeneous system divide, the liquid phase is directed to ion-exchange purification to obtain the dosage forms of the drug for medical purposes (solution or liofilizovannye powder in capsules).

To obtain the target product for use in crop production, the reaction mixture after the desired degree of chemical modification is neutralized with an alkaline agent to form a slurry Packed in consumer packaging.

The liquid phase after treated with activated charcoal and passed through an ion exchange column filled with shredded resin. To reduce the resistance of the resin in the process Sorb the AI and desorption powdered resin in H-form, mixed with unground cation in the Na-form in a ratio of 1:1 or 1:2, depending on the degree of grinding.

Target product elute 1 N sodium hydroxide solution are selected fraction at pH 11.5 before "breakthrough" alkali. For finer separation of the target product sampling fractions produced after each increase in pH=0.5 units. Selected fractions analyzed qualitatively IR spectrophotometric by well-known methods (Kullbom S.D., H.F. Smith // Anal. Chem. - 1963, 35. - S). According to the results of the analysis carried out combining fractions of the target product on the content of sulfo and chlorine (method Lanigera).

Selected fraction at pH of 11,5 14,0, neutralized with hydrochloric acid to pH=7,0, determine the biological activity of a solution of the target product by NBT-test and diluted with distilled water at the rate of a therapeutic dose, appropriate 50000 UE 1 ml from the neutralized solution. The diluted solution is subjected to sterilizing filtration and transferred to dosage injectable form (solution in ampoules or lyophilized powder in a known manner.

1. The modulator of metabolism on the basis of polypeptides with a molecular mass of from 15 to 10 kDa, derived from human placenta by chemical modification of bio placenta using chlorination reactions, oxidation, hydrolysis, characterized in that the target is, the product contains covalently bound chlorine atoms with nitrogen atoms of the peptide bond in the amount of from 0.5 to 40% by weight of the target product.

2. The modulator according to claim 1, characterized in that the target product contains in its composition methanesulfonic, α-aminomalonate, α-aminoazotoluene and cysteine amino acids in an amount of from 1 to 20 amino acid residues on 1 mol of the target product, introduced into the composition of the polypeptide by oxidation of methionine, serine, threonine, cystine and cysteine in the original bioproduct.

3. A method of obtaining a modulator according to claims 1 and 2, by chemical modification of tissue from the placenta of humans or farm animals chlorine compounds in the presence of the modifying additive salts of cobalt, followed by the separation of the resulting precipitate from the solution of the target product, wherein the chemical modification using oxidation-glorious and gidrolizuemye mixture allocated after processing the chloride salts of the acid, sulfonic cation exchanger in the static mode.

4. The method according to claim 3, characterized in that the process of chemical modification of bioproducts placental oxidative-hareruya and hydrolysing the mixture is carried out at a ratio by weight of organic food products, calculated on the dry matter: salt chloride acid, taken on ion exchange, 1:(0,1-1,5).

5. The method according to claim 3, characterized in that the process of chemical modification is carried out at a temperature of 18-60°C.

6. The method according to claim 3, characterized in that the target product is separated from p the promotional mix by sorption sulfoxylate cation exchange resin in H-form, pre-grinded to fine condition, with subsequent fractional desorption solution of caustic soda with the control process according to the content of the target product of chlorine and sulfo, and translation in the dosage form in a known manner.

7. The method according to claim 3, characterized in that the target product for crop production is transferred to a working form in suspension by neutralizing the reaction mixture after chemical modification of the alkaline agent.



 

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12 cl, 4 tbl, 4 ex

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6 tbl, 2 ex

FIELD: medicine; veterinary science.

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FIELD: medicine.

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FIELD: medicine.

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2 cl, 9 ex

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2 cl

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3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: according to the first version, method includes extraction of allogenic mesenchymal stem cells culture from umbilical cord of newborn after normal delivery for their parenteral introduction in patient according to specified technology. According to the second version, method includes extraction of allogenic mesenchymal stem cells culture from placenta of newborn after normal delivery for their parenteral introduction in patient according to the same technology. The first and second preparations contain the following amount of allogenic mesenchymal stem cells in sterile physiological solution - in the range from 1 to 5 million cells in 5 ml of solution.

EFFECT: increase of therapeutical effect.

6 cl, 2 ex

FIELD: medicine, pharmacology.

SUBSTANCE: invention concerns chemical-pharmaceutical industry, namely to creation of the agent possessing selective radio- and chemoprotective action on damaged normal tissues. The agent contains a denaturated, emulsified, disharmonic placenta, i.e. without an admixture of hormones, and also hyaluronic acid, natural honey, natural olive oil and starch potato in the parities providing expressed selective therapeutic effect. The agent does not cause toxic reactions, possesses ability to stimulate hemopoiesis by augmentation of a pool of precursor cells.

EFFECT: creation of agent possessing selective radio- and chemoprotective action on damaged normal tissues which possesses ability to stimulate hemopoiesis by augmentation of pool of precursor cells.

4 ex, 5 tbl

FIELD: medicine, veterinary science, cosmetics.

SUBSTANCE: bioconcentrate is prepared from placenta by treatment of intact placenta of native structure with hydrophilic organic solvent at temperature 0-25oC for 3-8 h at M = 5-30. Bioconcentrate represents extract containing lipoprotein complex enriched with water-soluble and lipid-soluble vitamins and a solid residue containing glycolipoprotein including protein, polysaccharide, nucleoprotein and lipid components. Invention provides the development of the wasteless manufacturing bioconcentrate eliciting high stability at storage and high biological activity that allow expanding region in applying bioconcentrate and to develop the broad spectrum of medicinal and cosmetic formulations used in medicine, veterinary science and cosmetics. Invention can be used as an agent normalizing the metabolism in different organs and tissues and eliciting the reparative and anti-inflammatory effect.

EFFECT: improved preparing method, valuable medicinal properties of bioconcentrate.

1 tbl, 8 ex

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