Tetrapeptide with geroprotective activity, pharmacological tool based on it and a method for its use

 

(57) Abstract:

The invention relates to medicine and can be used to prevent premature aging. Proposed biologically active compound tetrapeptide L-alanyl-L-glutamyl-L-aspergillosis General formula L-Ala-L-Glu-L-Asp-Gly and its use for making money with geroprotective activity. Pharmaceutical preparation containing as active principle an effective amount of tetrapeptide or its salts, is available for parenteral, intranasal, oral and local applications. The way to prevent premature aging include preventive and/or therapeutic administration to a patient a pharmaceutical in doses of 0.01 - 100 μg/kg of body weight at least once during the period required to achieve a therapeutic effect. 4 c. and 7 C.p. f-crystals, 17 PL.

The invention relates to medicine and can be used as a geroprotective means for preventing premature aging.

It is known that one of the leading mechanisms of aging are: accumulation of molecular damage caused by Svobodnyi functions epiphysis (1, 2, 3).

Since the proposed tetrapeptide responsible to the invention, exhibits biological activity, namely geroprotective activity of the analogues on the application should include a group of compounds with antioxidant properties. Add in food known inhibitor of radical processes of ionol (2,6-di - tert-butyl-4-METHYLPHENOL) contributed to the increase in the lifetime of mice with accelerated aging line LAF1(4). However, the drug on the basis of 2,6-di-tert-butyl-4-METHYLPHENOL (BHT) is produced in the form of liniment and is used in humans, mainly in Oncology in urological practice (5). Introduction to food antioxidant of idoxifene (Santorin) increased the lifespan of mice C3H (6). Increase life span of laboratory animals causes low water-soluble antioxidant hydrochloride 2-ethyl-6-methyl-3-oksipiridina, a structural analogue of vitamin B6(7, 8). A slight increase in life expectancy in the experiment gave 2-mercaptoethylamine, equivalent, cysteine, 3-hydroxypyridine, centrophenoxine, lactic and gluconic acid, glutathione (9, 10). However, these chemical with the s means. Increased life expectancy in the experiment vitamins A, C, E (11, 12, 13). However, the oversaturation of the body with these vitamins may adversely affect the functional state of organs and systems, leads to the rapid development of hypervitaminosis. Known drug-catechin, which includes vitamins and herbal substances with antioxidant activity (14). The use of this drug to mice with accelerated aging line SAM-P8 increased the survival of animals. However, the mechanisms of geroprotective activity of this combination drug is poorly understood, which limits its introduction into clinical practice. After a 7-month upkeep of mice SAM, prone to accelerated aging, a diet with increased content of carnosine (-Ala-His) decreased mortality (15). However, carnosine is not a medication, it geroprotective properties are not well understood. A slight increase in the average life expectancy of mice was caused by the hormone of the pineal gland - melatonin (16). Effects of melatonin are associated with its antioxidant properties (17). However, the use of melatonin in Drosophila melanogaster lines WPP, selectively at a high level embrio the Melatonin is not a drug, and produced as a biologically active food supplements.

As geroprotective tools used drug containing procaine, Gerovital (18). The disadvantage of this drug are possible negative effect on the function of the cardiovascular system, allergic action, sometimes the deterioration of sleep, cause feelings of anxiety, pain in muscles and joints.

The invention is aimed at obtaining new biologically active compounds peptide having geroprotective activity.

The claimed peptide compound - tetrapeptide - structural analogues has not.

According to the claimed invention, tetrapeptide L-alanyl-L - glutamyl-L-asparagi-glycine General formula: L-Ala-L-Glu-L-Asp-Gly.

Tetrapeptide L-alanyl-L-glutamyl-L-asparagi-glycine with the following amino acid sequence: L-Ala-L-Glu-L-Asp - Gly, responsive to the invention, exhibits biological activity, namely geroprotective activity, due to the stimulation indices of antioxidant defense system and process for the synthesis of melatonin in the structures of the diffuse neuroendocrine system.

Tetrapeptide get the class of the Glu-L-Asp-Gly revealed in his experimental study. The study of geroprotective activity conducted on Drosophila melanogaster, exploring the indicators of antioxidant protection, life expectancy and duration of the reproductive period, and rats, exploring the synthesis extremealex melatonin.

According to the invention, the pharmacological agent with geroprotective activity, contains as active principle an effective amount of tetrapeptide formula L-alanyl-L-glutamyl-L - asparagi-glycine (L-Ala-L-Glu-L-Asp-Gly) or its salts.

According to the invention, the pharmacological agent with geroprotective activity, may contain salt at the amino group (acetate, hydrochloride, oxalate) or carboxyl groups (salts of metals - sodium, potassium, calcium, lithium, zinc, magnesium, and other organic and inorganic cations - ammonium, triethylamine).

According to the invention, the pharmacological agent is intended for parenteral; intranasal, oral or local application.

The claimed pharmacological agent with geroprotective activity can stimulate the indicators of the antioxidant defense system and processes for the synthesis of melatonin in the structures of the life.

The concept of "geroprotective tool" used in this application means a means of slowing the aging process and prolong life by preventing premature aging, in which the necessary stimulation of the antioxidant defense system and regulating effect on the metabolic processes in the structures of the diffuse neuroendocrine system.

The term "pharmacological agent", as used in this application means the use of any pharmaceutical form containing tetrapeptide or its salt, which can find a prophylactic and/or therapeutic use in medicine as a geroprotective means of premature aging.

The term "effective amount", as used in this application involves the use of the number of active principle, which is in compliance with its quantitative indicators of activity and toxicity, as well as on the basis of knowledge of a specialist should be effective in the form of the drug.

The term "pharmaceutical composition", as used in this application means different dosage forms of the drug.

To obtain pharmaceutical compositions that meet the effective ingredient with a pharmaceutical carrier according to accepted pharmaceutical methods of compounding.

The carrier may take various forms, depending on the dosage form of the drug, desired for introduction into the body, for example, parenteral, intranasal or oral.

In the manufacture of compositions in the preferred dosage form for oral administration can be any known pharmaceutical components.

For parenteral (intranasal) administration, the media typically comprises sterile water, although there may be other ingredients that promote stability or to maintain sterility.

According to the invention the method comprises a prophylactic or therapeutic administration to a patient of the claimed pharmacological tools in doses of 0.01 - 100 μg/kg of body weight at least once a day during the period required to achieve a therapeutic effect - 10-40 days depending on the nature of the pathologic process.

According to the invention tetrapeptide active at its introduction in doses of 0.01-100 μg/kg of body weight, although you may be used and lower (higher) dose depending on the nature of the pathologic process.

The invention is illustrated in the example, C is oxyconti and biological activity of tetrapeptide (examples 2, 3, 4, 5, 6, 7, 8), as well as examples of the results of clinical application of tetrapeptide demonstrating its pharmacological properties and confirming the possibility to achieve a prophylactic and/or therapeutic effect (examples 9, 10).

Example 1. Synthesis of tetrapeptide L-Ala-L-Glu-L-Asp-Gly

1. Connection name: L-alanyl-L-glutamyl-L-aspartyl-glycine.

2. The structural formula is:

H-Ala-Glu-Asp-Gly-OH

< / BR>
3. Brutto-formula without counterion: C14H22N4O9.

4. Molecular weight without counterion: 390,35.

5. The counterion: acetate.

6. Appearance: white amorphous powder, odorless.

7. The method of synthesis: peptide obtained by the classical method of synthesis in solution according to the scheme presented in the end of the description.

The solvent used is N,N'-dimethylformamide, with the introduction of aspartic acid used protection-COOH groups by salt formation with triethylamine. The release of the BOC-protective group is carried out by solution triperoxonane acid (TFA), the Z-protective group by catalytic hydrogenolysis. Isolation and purification of the drug was carried out using preparative HPLC on a column with reversed phase.

Characteristics goth is HPLC, 220 nm);

TLC - individual, Rf= 0,73 (acetonitrile-acetic to-the-water 5:1:3);

moisture content: 5%;

the pH of 0.001% solution: 4,37;

specific optical rotation: []2D2: -32o(C=1, H2O).

Example of synthesis:

1) BOC-Glu(OBzl)-Asp(OBzl)-OH (I), N-tert.butyloxycarbonyl-(-benzyl)glutamyl-(-benzyl)aspartate.

N-oxysuccinimide ester of N-tert.butyloxycarbonyl- (- benzyl)glutamic acid BOC-Glu(OBzl)-OSu 4,34 g (0,0100 mol) was dissolved in 20 ml of dimethylformamide, add triethylamine 1,72 ml of 0.0125 mol) and bansilalpet 2,80 g (of 0.0125 mol). Stirred at room temperature for 24 hours. Product planted 0.5 n sulfuric acid solution (150 ml), extracted into ethyl acetate (CH ml), washed with 0.5 n sulfuric acid solution (2x20 ml), water, 5% sodium bicarbonate solution (g ml), water, 0.5 n sulfuric acid solution (2x20 ml), water and dried over anhydrous sodium sulfate. Ethyl acetate, filtered, evaporated in vacuum at 40oC, the residue is dried in vacuum over P2O5. Get the oil of 5.68 g (100%). Rf= 0,42 (benzene-acetone 2: 1, plate PTSH-P-UV Sorbfil, silica gel CTX-WE 8-12 μm, the manifestation of UV and chlorine/benzidine).

2)TFAH-Glu(OBzl)-Asp(OBzl)-OH(II),triptorelin(-benzyl)glutamyl-(-benzyl)aspartate.

N-t-triperoxonane acid (3: 1). After 2 hours the solvent is evaporated in vacuum at 40oC), evaporation is repeated with a new portion of dichloromethane (2x10 ml), the residue is dried in vacuum over NaOH. Receive oil 5,80 g (100%). Rf= 0,63 (n-butanol-pyridine-acetic acid-water, 15:10:3:12).

3) Z-Ala-Glu(OBzl)-Asp(OBzl)-OH (III), N-carbobenzoxy -(-benzyl) glutamyl-(-benzyl)aspartate.

Triptorelin (-benzyl) glutamyl-(-benzyl)aspartate (II) 5,65 g (0.01 mol) dissolved in 10 ml of dimethylformamide, add triethylamine 2,80 ml (0.02 mol) and N-oxysuccinimide ether N - carbobenzoxy to 4.14 g (0,013 mol). The mixture is stirred for 24 hours at room temperature. Product planted 0.5 n sulfuric acid solution (150 ml), extracted into ethyl acetate (CH ml), washed with 0.5 n sulfuric acid solution (2x20 ml), water, 5% sodium bicarbonate solution (g ml), water, 0.5 n sulfuric acid solution (2x20 ml), water and dried over anhydrous sodium sulfate. Ethyl acetate, filtered, evaporated in vacuum at 40oC, the remainder zakristallizuetsya in the system ethyl acetate/hexane. The product is filtered and dried in vacuum over P2O5. Exit 4, 10 g (66%). Tpl.= 154oC. Rf= 0,48 (benzene-acetone, 1:1), Rf= 0,72 (n-butanol-pyridine-acetic acid-water, 15:10:3:12).

4) Z-Ala-Ghi(OBzl)-AspsOH H-Gly-OBzl, toilet benzyl ester of glycine 1.01 g (3 mmol) are suspended in 15 ml of tetrahydrofuran, and with stirring, add triethylamine mmol), followed after 5 min. N-carbobenzoxy-(-benzyl) glutamyl-(- benzyl)aspartate (III) 1.28 g (2 mmol), N-oxybisethanol 0.27 g (2 mmol) and cool the mixture to 0oC. Then add cooled to 0oC a solution of N,N'- dicyclohexylcarbodiimide of 0.42 g (2 mmol) in 5 ml of tetrahydrofuran, stirred the mixture at this temperature for 2 hours and left overnight to mix at room temperature. Sediment dicyclohexylamine filtered off, the solvent evaporated in vacuum, the residue is dissolved in 30 ml ethyl acetate and washed with a solution of 1N hydrochloric acid, water, 5% sodium bicarbonate solution, water, 1N hydrochloric acid, water and dried over anhydrous sodium sulfate. The solvent is evaporated in vacuum and the product zakristallizuetsya in the system ethyl acetate/hexane. The output of 1.30 g (82%). Tpl.= 146-148oC. Rf= 0,75 (benzene-acetone, 2:1).

5) H-Ala-Glu-Asp-Gly-OH (IV), alanyl-glutamyl-aspartyl-glycine.

Benzyl ester of N-carbobenzoxy -(-benzyl) glutamyl-(-benzyl)aspiringicon (III) of 1.25 g was first made in the system methanol-water-acetic acid (3: 1: 1) on the I acid-water (5:1:3). Upon completion of the reaction the catalyst is filtered off, the filtrate is evaporated in vacuo and the residue zakristallizuetsya in the system water/methanol. The product is dried in vacuum over KOH. Yield 520 mg (95%). Rf= 0,73 (acetonitrile-acetic acid-water 5:1:3). For cleaning 390 mg of drug was dissolved in 4 ml of 0.01% triperoxonane acid and subjected to high performance liquid chromatography on a column of reversed phase h mm Diasorb-130-C16T, 7 mkm. Chromatograph Beckman System Gold 126 Solvent Module, 168 Diode Array Detector Module. Chromatograph conditions were as follows A: 0,1% TFA; B: 50% MeCN/0.1% of TFA, gradient 0 ---> 5% for 80 minutes the Volume of the sample 5 ml, detection at 215 nm, scanning 190-600 nm, flow rate 10 ml/min was Selected fraction 54,0-66,0 minutes the Solvent was evaporated in vacuum at a temperature not exceeding 40oC), evaporation repeatedly (5 times) was repeated with 10 ml of 10% solution of acetic acid. Finally the residue was dissolved in 20 ml of deionized water and liofilizovane. Received 290 mg of the pure product in the form of an amorphous white powder, odorless. The obtained peptide in the form of acetate translate into a free form by treatment with anion exchange resin IRA or similar in (OH-)-form. Next, get the salt at the amino group, adding the equivalent of the appropriate acid (hydrochloric or oxalic). Received podnyavshim groups to free tetrapeptide add the calculated amount of an aqueous solution of a suitable metal hydroxide (NaOH, KOHN, ZnOH2, LiOH, CaOH2, MgOH2, NH4OH). To obtain triethylammonium salt treatment is carried out in a similar way, using it as a base triethylamine.

6) Analysis of the finished product.

The basic substance content was determined by HPLC on a column of Supeico LC-18-DB 4,h mm, gard. LC-18-DB. A: 0,1% TFA; B: 50% MeCN/0.1%of TFA; grad. 0 ---> 20% in 30 min flow Rate 1 ml/min, Detection at 220 nm, scanning 190-600 nm, the sample 20 ál. The content of the main substance of its 98.45%.

Amino acid analysis was performed on the analyzer AAA"T-339" Prague. Hydrolysis in 6N HCl at 125oC 24 hours.

Ghr 1,02; Asp 1,00; Ala Is 1.01; Gly 1.00 Each.

TCX: individual, Rf= 0,73 (acetonitrile-acetic acid-water 5:1:3 plates PTSH-P-UV Sorbfil, silica gel CTX-WE 8-12 μm, the manifestation of chlorine/benzidine).

Moisture content: 5% (gravimetrically by the weight loss at drying 20 mg under 100oC).

the pH of 0.001% solution: 4,37 (potentiometric).

Specific optical rotation: []2D2-32o(C=1, H2O), "Polamat A" Carl Zeiss Jena.

Example 2. Study of toxicity of tetrapeptide L-Ala-L-Glu-L-Asp-Gly

Study of General toxic effect of tetrapeptide L-Ala-L-Glu-L-Asp-Gly was conducted in accordance with Priviredliviy portable toxic doses of the drug, assessing the extent and nature of pathological changes in various organs and systems of the body and reveal a dependence of the toxic effects of dose and duration of use of the drug.

Determination of acute toxicity of tetrapeptide L-Ala-L-Glu-L-Asp-Gly was carried out according to the method of Cerberus. The study was conducted on 66 outbred mice-males weighing 20-23 g contained in standard mode and receiving a standard diet in vivarium conditions. Animals were divided randomly divided into 6 equal groups of 11 mice in each. The drug was administered to the animals once intramuscularly in a volume of 0.25 ml at doses of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg (several thousand times therapeutic dose recommended for clinical studies). Animals of the control group in the same volume was injected with saline.

Within 72 hours, and after 14 days in either group of animals of the death of the mice was not detected. There were no changes in General condition, behavior, motor activity, hair and skin, physiological shipments of animals.

Thus, tetrapeptide L-Ala-L-Glu-L-Asp-Gly in doses exceeding therapeutic recommended for linearities the breadth of the drug.

The study of subacute toxicity of tetrapeptide L-Ala-L-Glu-L-Asp-Gly conducted on 60 outbred rats weighing 150-250 mg once Daily, experimental animals were injected drug intramuscularly within 90 days at doses of 1 mg/kg, 0.3 mg/kg, 3 mg/kg in 0.5 ml saline. Animals of the control group were injected in the same volume of saline.

Throughout the study period the animals were monitored on a daily basis. Noted the behavior of animals, feed and water intake, the condition of the scalp and mucous membranes. Held weekly weighing of animals. Before drug administration, at 30, 60 and 90 days after the start of a drug in animals have investigated the morphological structure and properties of peripheral blood. At the end of the experiment investigated the biochemical reagents and coagulation of the blood.

The chronic toxicity of tetrapeptide L-Ala-L-Glu-L-Asp-Gly, obtained by the claimed method, studied the long introduction to his rats weighing 150-250 mg Animals daily was administered intramuscularly in doses of 1 μg/kg, 0.1 mg/kg, 1 mg/kg in 0.5 ml saline for 6 months. Noted animal behaviour, feed intake is s 3 months of the experiment, then 1 time per month. 3 months after the beginning of the introduction and at the end of the experiment conducted hematological and biochemical studies. Assessed the function of the cardiovascular system, liver, pancreas, kidneys and adrenal glands. After the injection the animals were subjected to pathological study to assess the status of the various departments of the brain and spinal cord, heart, aorta, lungs, liver, kidneys, endocrine and immune systems.

When assessing the General condition of the animals, morphological and biochemical indices of peripheral blood, the morphological status of the internal organs, the state of the cardiovascular and respiratory systems, liver and kidney pathological changes in the organism is not detected.

The study of subacute and chronic toxicity of tetrapeptide L-Ala-L-Glu-L-Asp-Gly indicates the absence of side effects with long-term use of the drug in doses exceeding therapeutic 100-1000 times.

Example 3. The influence of tetrapeptide L-Ala-L-Glu-L-Asp-Gly at the rate of aging and free radical processes in Drosophila melanogaster lines WPP, otsilindrovannoy high embryonic mortality

Tetrapeptide L-Ala-L-Glu-L-Asp-Gly was added in the amount within 0.00001 percent by weight of the nutrient medium that is extremely low dose. In the literature on the influence of different substances on the Drosophila typically used doses from 0.001 to 0.01%. Smaller doses usually cause no effect.

Biochemical parameters were studied in flies at the age of 14 days. Analyzed the activity of catalase and the intensity of chemiluminescence tissues (21, 22, 23). Catalase is a major antioxidant enzymes. On the level of its activity can be judged on the body's ability to resist oxidative stress. Induced hydrogen peroxide Lomonosovskaya chemiluminescence reflects the level of active forms of oxygen in the tissues. Thus, the decrease in the intensity of chemiluminescence reflects increasing the body's ability to resist oxidative stress.

The results are presented in TMAC and males are shown separately.

From the presented data it follows that tetrapeptide L-Ala-L-Glu-L-Asp-Gly significantly increase catalase activity in female and male flies compared to control.

Total antioxidant activity of tissues, characterized by the level of chemiluminescence increases under the action of tetrapeptide L-Ala-L-Glu-L-Asp-Gly-only for females.

Table 2 presents the results of the analysis of life expectancy in different experimental groups.

As can be seen from table 2, tetrapeptide L-Ala-L-Glu-L-Asp-Gly has a strong geroprotective effect. In females it significantly increases life expectancy (P<0.001) and reduces the rate of aging (R) (P<0,001). Males having slightly reduces the rate of aging (P<0,05).

Example 4. The influence of tetrapeptide L-Ala-L-Glu-L-Asp--Gly on the reproductive functions of Drosophila melanogaster lines WPP, otsilindrovannoy high embryonic mortality

The impact of tetrapeptide L-Ala-L-Glu-L-Asp-Gly conducted at the stage of larva 2-3rd age, adding the drug in the culture medium on the 5 pairs in a test tube and through every 3-6 days threw them in a fresh environment.

Take into account:

The proportion of tubes (from the start), which was attended by live females (table 3 - proportion of live cultures).

The share of the tubes (the number of live cultures), which appeared in the offspring (table 3 - proportion of fertile crops). In each experimental variant was analyzed 75 parental pairs. Differences between the fertility rates were determined using Fisher's exact test for 2x2 contingency tables (24).

The results of the experiment are presented in table 3. Given the proportion of living and fertile cultures for each experimental variant. As can be seen from the table, starting with the 19th day of the proportion of fertile cultures after treatment with tetrapeptides higher than in the control. It is shown that the proportion of fertile crops in a variant of tetrapeptide L-Ala-L-Glu-L-Asp-Gly significantly (P<0,05) higher compared to control at the age of 32 days.

Linear regression coefficients are presented in table 4. As can be seen from the table, the coefficients of the control of the regression line significantly differ from the experienced. The coefficient "a" (slope of a line) in the control version 1.8 times higher regression coefficient of experimental variation.

Thus it is shown that the share of L-Asp-Gly. The duration of the reproductive period was 30 days for cultures not exposed to processing, and 47 days for cultures treated with tetrapeptides. It is established that tetrapeptide L-Ala-L-Glu-L-Asp-Gly increases the duration of the reproductive period 1.5 times.

Similarly analysed data on life expectancy. The data of regression analysis are presented in table 5.

As follows from the table, the share of live cultures (i.e., cultures in which there are living females) decreased in the control on average 1.5 times faster in comparison with the test option. The estimated median lifetime (i.e. the time by which 50% die from individuals) and actually achieved the maximum life expectancy for different variants of the experiment are shown in table 6.

As can be seen from the table, in addition to the increase in the median life expectancy in the variant treated with tetrapeptide L-Ala-L-Glu-L-Asp-Gly, reached a record maximum lifespan (for a rapidly aging lines Drosophila) - 100 days.

Example 5. The influence of tetrapeptide L-Ala-L-Glu-L-Asp-Gly at the rate of aging and free radical processes in Drosophila melanogaster lines HA, otsilindrovannoy low palavecino oxidation of lipids (Pol), life expectancy and the rate of aging has been studied in Drosophila melanogaster lines HA.

Flies at the age of 1 day were placed in tubes of 10 animals of the same sex. After 2 days they threw in a fresh environment, given the number of dead individuals. Were used to determine average and maximum lifespan. The rate of aging was determined by calculating the parameters of the Gompertz equation.

Tetrapeptide L-Ala-L-Glu-L-Asp-Gly influenced larvae of the 3rd age in a dose 0,00001% plumb nutrient medium, which is approximately 0.015 mg/100 ml In the homogenates of adult flies at the age of 14 days according to standard techniques determined the activity of catalase. The test was carried out in two replications.

The primary method of statistical processing was regression and analysis of variance. The significance of differences was determined by the method of least significant difference.

It is established that tetrapeptide L-Ala-L-Glu-L-Asp-Gly females does not change ALE, but increases NRM. However, significantly increases the mean life span of males. Analysis of survival curves and genersously graphs shows that tetrapeptide L-Ala-L-Glu-L-Asp-Gly females acts as a geroprotector of the first type (ALE and NRM increased, but the rate of aging is not manaedjaedje and thus tends to increase the rate of aging). At the same time survival analysis flies without regard to gender differences shows that the NRM increases significantly under the influence of tetrapeptide L-Ala-L-Glu-L-Asp-Gly.

When exposed to tetrapeptide L-Ala-L-Glu-L-Asp-Gly males increased catalase activity, while females have significantly reduced lipid peroxidation. The leveling of gender differences shows high antioxidant activity of tetrapeptide L-Ala-L-Glu-L-Asp-Gly.

The results of the study are presented in tables 7-9.

Example 6. The influence of tetrapeptide L-Ala-L-Glu-L-Asp-Gly at the rate of aging and free radical processes in Drosophila melanogaster lines BA, otsilindrovannoy low adaptation to external conditions

In Drosophila melanogaster lines BSA studied the effect of tetrapeptide L-Ala-L-Glu-L-Asp-Gly on catalase activity, chemiluminescence tissues and life expectancy.

Flies at the age of 1 day were placed in tubes of 10 animals of the same sex. Every 5-7 days they threw in a fresh environment, given the number of dead individuals. Were used to determine average and maximum lifespan. The rate of aging was determined by calculating the parameters of the Gompertz equation.

Tetrapeptide L-Ala-L-Glu-L-Asp-Gly influenced larvae 3-is in libraries flies at the age of 7 days according to standard techniques determined the activity of catalase and the intensity of the chemiluminescence. The test was carried out in two replications.

The primary method of statistical processing was regression and analysis of variance. The significance of differences was determined by the method of least significant difference.

The results of the study are presented in tables 10-13.

It is established that tetrapeptide L-Ala-L-Glu-L-Asp-Gly affects Lomonosovsky the chemiluminescence (PL. 14). The decrease of this indicator favors the activation of the antioxidant defense system in tissues of flies. It is likely that the increase in tissue antioxidant defense in this case is associated with activation of low molecular weight endogenous antioxidants such as glutathione, tocopherol and other

The increase in average and maximal lifespan was observed only in conditions of low viability and accelerated aging of the control variant. As can be seen from the presented data, in this case study tetrapeptide detects clear geroprotective effect.

Example 7. The influence of tetrapeptide L-Ala-L-Glu-L-Asp-Gly on the lifespan of Drosophila melanogaster "wild" line Canton-S

In this work, we used Drosophila melanogaster "wild" line Canton-S. Tetrapeptide L-Ala-L-Glu-L-Asp-Gly in VI is about stirred and poured into a test tube.

To ensure that the terms of the development control populations were adequate conditions for the development of experimental populations in nutrient medium for the control group contributed the same amount of physiologic saline, and when dissolved tetrapeptide L-Ala-L-Glu-L-Asp-Gly. The concentration of all substances result obtained with respect to the mass medium for reproduction.

Randomly selected 4-day females were crossed with males of one pair in vitro for 72 hours, after which the parent pair was removed from the tube. For the formation of each generation used the offspring 50-80 parent pairs.

Lifespan (PI) were studied in laboratory populations, consisting of 90 individuals of each sex. Directly after hatching flies randomly planted 10 individuals in a test tube. Replacement of the nutrient medium was performed three times per week.

As parameters of the distribution of life expectancy was calculated using the traditional method averages (ALE) and standard errors. Mean values were compared using t-student test.

Table 15 presents the results of measuring the characteristics of the location is.

For the experimental study chose 6 of the following concentrations tetrapeptide L-Ala-L-Glu-L-Asp-Gly: 0,01 x, 0,1, x, 1x, 5x, 7.5 x and 12,510-6% by weight of the nutrient medium.

For males the effect of tetrapeptide L-Ala-L-Glu-L-Asp-Gly in the four lowest concentrations (from 0.01 to 510 x-6%) had a reliable geroprotective action: ALE statistically significantly increased in the experimental groups. The relative increase in mean life span of males ranged from 3.3 to 10.8 per cent.

In experiments with females of tetrapeptide L-Ala-L-Glu-L-Asp-Gly showed geroprotective activity only at concentrations of 0.01 x and 0,110-6% significantly increased the mean life span by 13.4% (p<0.004) and 11,8% (p<0.03 in), respectively.

The uniqueness of the obtained data is the effectiveness of ultra-low concentrations used tetrapeptide L-Ala-L-Glu-L-Asp-Gly. It should be noted that the use of tetrapeptide L-Ala-L-Glu-L-Asp-Gly did not change the duration of developmental stages of Drosophila that in the integral form reflects the absence of genotoxic effects of the investigated substances.

Example 8. The influence of tetrapeptide L-Ala-L-Glu-L-Asp-Gly on extreniely synthesis of melatonin after epiphysectomy in rats

Work vesanen on male Wistar rats weighing 130 to 140, the Total number which the shaft 2 is 5 groups was removed epiphysis (EE - apisalome). Three weeks after the operation (21 days) animals 2 and 3 groups were subcutaneously injected with saline at 0.5 ml over the next 10 days. Animals 4 and 5 groups according to the same scheme was introduced tetrapeptide L-Ala-L-Glu-L-Asp-Gly in the dose of 0.5 μg.

The slaughter of animals and the allocation of bodies held in the morning from 10 to 12 hours in natural light under Nembutal anesthesia (50 mg/kg). Animals 1, 2, 4, groups were scored after 3 days after the final injection (33 days after surgery and the start of the experiment) and 3, 5 group - 12 days (42 days after epiphysectomy). The study has been the main bodies of the diffuse neuroendocrine system (DNES) (stomach, thyroid and pancreas).

Operation - apisalome was carried out under ether anesthesia and the developed technique. On the middle line of the head was done the skin incision length 1-1,5 cm After exposure of the skull roof above the confluence of the sinuses was visualimages hole with the help of specially made from a metal tube of a hollow drill with a diameter of 0.5 cm Through the hole in the skull epiphysis were extracted with an eye mode, and trepanation hole was closed bone fragmentation Buena for light microscopic studies and Karnofsky for electron microscopy. Dehydration of the material and fill in paraffin for light microscopy and the mixture apanov for ultrastructural studies were performed according to standard techniques. Paraffin sections (7 μm) were placed on glass slides, covered with a film of poly-L-lysine (Sigma). Ultrathin sections (100 nm), prepared by ultramicrotome LKB-7A (LKB) was contrasted by uranylacetate and lead citrate.

Histological and immunohistochemical studies were performed on microscope Jenamed-2 (Zeizz). Electron-microscopic study was carried out in the electron microscope JEM-100S (JEOL).

Applied color with hematoxylin-eosin. The General population of audiiton in pyloric and fundic stomach was revealed by histochemical method of silvering on Grimelius (25).

Immunohistochemical detection of EC-cells was performed using mouse monoclonal antibodies to serotonin (Dako, titer 1:15). Monoclonal antibodies identified avidin-Biotin-peroxidase (ALD) method (Vectastain kit) for detection of mouse immunoglobulins.

Quantitative studies were performed using a system of computer analysis of microscopic images (Imstar) with the use of software agents is a defective density enteroendocrine (&) (NEND/1 mm2) and serotonin-positive cells (Nser/1 mm2) was determined in 10 fields of view. The test area (S) was 5 mm2.

For statistical processing of results, we applied non-parametric U-test Mann-Whitney.

The results of studies of the functional morphology DNES pipesection rats under the action of tetrapeptide L-Ala-L-Glu-L-Asp-Gly demonstrate the stimulating effect of the drug on tissue and cellular metabolism.

It is established that the introduction of tetrapeptide L-Ala-L-Glu-L-Asp-Gly has a compensatory effect on the structural and functional organization of cells DNES pipesection animals. Its effect is manifested through 3 days after the injection full leveling effect epiphysectomy and stored in 12 days, i.e. until the end of the experiment with respect to all investigated organs.

Our data indicate that the main point of application actions tetrapeptide L-Ala-L-Glu-L-Asp-Gly are the EC cells of the stomach, in which under the influence of tetrapeptide increases extreniely the synthesis of serotonin and melatonin (PL. 16), which is essentially fully compensates Ala-L-Glu-L-Asp-Gly has no toxicity, normalizes the.

Identified in the preclinical experimental studies of the properties of tetrapeptide L-Ala-L-Glu-L-Asp-Gly allow us to consider its prophylactic and/or therapeutic use as geroprotective tools.

The following examples are the results of a clinical study of the proposed tetrapeptide demonstrate its pharmacological properties and confirm the possibility of carrying out the invention.

Example 9. Efficacy of pharmacological products containing tetrapeptide L-Ala-L-Glu-L-Asp-Gly in patients with impaired antioxidant defense system to prevent premature aging

Investigational pharmacological tool was used in 14 patients (34-69 years) with age-related pathology (hypertension, ischemic heart disease, chronic gastritis, non-insulin-dependent diabetes mellitus) and decreased antioxidant defense system in the blood.

The tool containing tetrapeptide L-Ala-L-Glu-L-Asp-Gly in the form of solution for injection was administered intramuscularly in a dose of 10 μg per injection once daily for 10 days.

On the background of the application tool was reliable surface is L. 17).

Example 10. Efficacy of pharmacological products containing tetrapeptide L-Ala-L-Glu-L-Asp-Gly in patients with impaired synthesis of melatonin for the prevention of premature aging

Pharmacological tool was used in 11 patients with aspirin-induced asthma aged from 39 to 63 years. The main symptom of this disease is the relationship of asthma with intolerance to acetylsalicylic acid and other nonsteroidal anti-inflammatory drugs. It is known that similar chemical structure with acetylsalicylic acid substance is N-acetyl-5-methoxykynuramine (N-AMK) is formed in the body during metabolism epiphyseal hormone melatonin. The synthesis of melatonin and, consequently, the level of endogenous N-AMK in patients with aspirin-induced asthma is significantly reduced, as evidenced by the low urinary excretion of the main metabolite of melatonin - 6-sulfatoxymelatonin

This was the rationale for the new pathogenetic approach to the treatment of aspirin-induced asthma by correcting the content of melatonin in the body of the patient pharmacological preparation containing tetrapeptide L-Ala-L-Glu-L-Asp-Gly.

Means in the form of a solution DLH">

Treatment was carried out in the phase of damped exacerbation or remission of the disease on the background of constant anti-asthma therapy, including inhaled and oral glucocorticoids.

Before, during treatment and then every month the patients subjectively assessed their health by the presence of symptoms such as cough, sputum production, shortness of breath, stuffy chest, wheezing, asthma, intolerance to exercise, cold, smells. The clinical effect of the drug was evaluated immediately after treatment and for 7 months on the basis of the analysis of reports of patients, which included information about the dynamics of well-being, health, psychological state, sleep disorders and daily doses used anti-asthmatic medications.

In all patients before treatment and immediately after the last dose of the drug was evaluated the function of external respiration on the basis of the following parameters: vital capacity (VC), residual volume (CV), total lung capacity (oil), forced vital capacity (FVC), peak flow rate of exhalation (POSvyd), the maximum volume exhalation rate at 50% and 75% of forced VC (MOSx from the predicted values. In addition, the measured conductivity of the bronchial tree (SGAW) in cm of water. Art. the Whole complex studies were repeated 15 minutes after inhalation beroteka. The dynamics of the VILLAGE, MOS50, MOS75after inhalation beroteka was evaluated as percentage of the initial values. All patients before treatment had a rate of FEV1less than 80% of the proper.

The content of the main metabolite of melatonin - 6-sulfatoxymelatonin were determined before treatment, immediately after and 10 days in the day (from 9.00 to 21.00) and night (from 21.00 till 9.00) portions of urine by ELISA using kits "DRG instruments GmbH, Marburg, Germany).

The results showed that the improvement in clinical status by the end of treatment was observed in the vast majority of patients receiving the investigational pharmacological tool. Was a decrease in the frequency of daytime asthma symptoms, intolerance to exercise, sharp smells and cold air, improved sleep. An objective examination of patients noted a decrease or disappearance of wheezing in the lungs - symptoms of bronchial obstruction. Study of the function of external respiration immediately after the end of treatment revealed no significant the General tetrapeptide L-Ala-L-Glu-L-Asp-Gly, there was an improvement in response Berotec at the level of the distal bronchi. In patients with increased excretion of 6-sulfatoxymelatonin with urine, indicating the increased production of melatonin, not only at night but in the daytime.

Sources of information

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3. Yu, B. P., Yang R. Critical evaluation of the free radical theory of aging: a proposal for the oxidative stress hypothesis // Ann. N. Y. Acad. Sci. - 1996. - Vol. 786. - P. 1-11.

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1. Tetrapeptide L-alanyl-L-glutamyl-L-asparagi-glycine General formula

L-Ala-L-Glu-L-Asp-Gly.

2. Tetrapeptide L-alanyl-L-glutamyl-L-asparagi-glycine General formula

L-Ala-L-Glu-L-Asp-Glu,

with geroprotective activity.

3. Pharmacological tool with geroprotective activity, containing the active principle and a pharmaceutically acceptable carrier, characterized in that the active agent contains an effective amount of tetrapeptide L-Ala-L-Glu-L-Asp-Gly or its salts.

4. Means on p. 3, characterized in that it contains salt aminogram by carboxyl groups, for example, salts of sodium, potassium, calcium, lithium, zinc, magnesium, or organic or inorganic cations, for example ammonium, triethylamine.

6. Means on p. 3, characterized in that it is intended for parenteral administration.

7. Means on p. 3, characterized in that it is intended for intranasal administration.

8. Means on p. 3, characterized in that it is intended for oral administration.

9. Means on p. 3, characterized in that it is intended for local administration.

10. The way to prevent premature aging, which consists in the introduction to the patient a pharmaceutical containing an effective amount of tetrapeptide L-Ala-L-Glu-L-Asp-Gly or its salts at the amino group, for example the acetate, hydrochloride, oxalate, or its salts at the carboxyl group, for example, salts of sodium, potassium, calcium, lithium, zinc, magnesium, and organic and inorganic cations, for example ammonium, triethylamine in the dose of 0.01 - 100 μg/kg of body weight at least once a day during the period required to achieve a therapeutic effect.

11. The method according to p. 10, characterized in that the tool is administered parenterally, intranasally,

 

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FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

FIELD: organic chemistry, medicine.

SUBSTANCE: invention represents ligands MC-4 and/or MC-3 of the formula (I): , wherein X means hydrogen atom, -OR1, -NR1R1' and -CHR1R1' wherein R1 and R1' are taken among the group: hydrogen atom, (C1-C6)-alkyl and acyl; (1) each R2 is taken independently among the group: hydrogen atom, (C1-C6)-alkyl; or (2) (a) R2 bound with carbon atom that is bound with X and Z1 and substitute R5 can be optionally bound to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; or (b) R2 bound with carbon atom that is bound with ring Ar can be bound with R7 to form ring condensed with ring Ar; each among Z1, Z2 and Z3 is taken independently from the following groups: -N(R3e)C(R3)(R3a)-, -C(R3)(R3a)N(R3e)-, -C(O)N(R3d)-, -N(R3d)C(O)-, -C(R3)(R3a)C(R3b)(R3c)-, -SO2N(R3d)- and -N(R3d)SO2- wherein each among R3, R3a, R3b and R3c, R3d, R3e when presents is taken independently among hydrogen atom and (C1-C6)-alkyl; p is a whole number from 0 to 5 wherein when p above 0 then R4 and R4' are taken among hydrogen atom, (C1-C6)-alkyl and aryl; R5 represents 5 substitutes in phenyl ring J wherein each R5 is taken among hydrogen atom, hydroxy-, halogen atom, thiol, -OR12, -N(R12)(R12'), (C1-C6)-alkyl, nitro-, aryl wherein R12 and R12' are taken among hydrogen atom and (C1-C6)-alkyl; or two substitutes R5 can be bound optionally to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; q = 0, 1, 2, 3, 4 or 5 wherein when q above 0 then R6 and R6' are taken among hydrogen atom and (C1-C6)-alkyl; Ar is taken among the group consisting of phenyl, thiophene, furan, oxazole, thiazole, pyrrole and pyridine; R7 are substitutes at ring Ar wherein each R7 is taken among hydrogen, halogen atom, -NR13R13', (C1-C6)-alkyl and nitro- wherein R13 and R13' are taken among hydrogen atom and (C1-C6)-alkyl; r is a whole number from 0 to 7 wherein when r is above 0 then R8 and R8' are taken among hydrogen atom and (C1-C6)-alkyl; B is taken among -N(R14)C(=NR15)NR16R17, -NR20R21, heteroaryl ring and heterocycloalkyl ring wherein R14-R17, R20 and R21 are taken independently among hydrogen atom and (C1-C6)-alkyl; s = 0, 1, 2, 3, 4 or 5 wherein when s is above 0 then R and R9' are taken among hydrogen atom and (C1-C6)-alkyl; R10 is taken among the group consisting of optionally substituted bicyclic aryl ring and optionally substituted bicyclic heteroaryl ring; D is taken among hydrogen atom, amino- and -C(O)R11 wherein R11 is taken among the following group: hydroxy-, alkoxy-, amino-, alkylamino-, -N(R19)CH2C(O)NH2 wherein R19 represents (C1-C6)-alkyl, -NHCH2CH2OH and -N(CH3)CH2CH2OH, or its isomers, salts, hydrates or biohydrolysable ester, amide or imide.

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EFFECT: higher efficiency and prophylaxis.

4 dwg, 5 ex

FIELD: medicine, chemistry of peptides, amino acids.

SUBSTANCE: invention relates to novel biologically active substances. Invention proposes the novel composition comprising peptides of the formula: H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys. The composition shows ability to inhibit proliferative activity of mononuclear cells, to induce suppressive activity and their ability for secretion of cytokines TNF-1β (tumor necrosis factor-1β) and IL-10 (interleukin-10 ).

EFFECT: simplified method for preparing composition, valuable medicinal properties of composition.

4 cl, 16 tbl, 9 ex

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