SU1011632A1 - Decapeptide having lipotronic effect - Google Patents

Abstract

Decapeptide form / w iH-u-Tsr-u-ige-u-Piro-v-Uss-i.-Geu-ti-Gien-Vi-x-vs-u-T r-u-ser-u-Phe it has lipotropic activity. i

Description

00E The invention relates to a novel biologically active compound, a decapeptide, having lipotropic activity, which can be used in medicine as; zhiromobioeuschego drug. Growth hormone is known to have lipolytic activity 1. The tetradecapeptide sequence 31 of the human omatotropic hormone of the formula HLPhe-L-C, eu-U-Greu-L-AEa-LT-yr-It-Dee-b-Pro-L-Lye-U-Qeu-U-Cieyi-L- is known LvS-b-TvH-Lc, ev-u-PV) eoH has high lipotropic activity. The disadvantages of this tetradec of peptide are the complex structure (1 amino acid residues;) and multi-step synthesis. The purpose of the invention is to expand the arsenal of agents that influence fat metabolism in animal tissues. This goal is achieved according to the invention by a decapeptide of the formula HLTvr-b-aee-L-Pro-b-Lvs-b-Qeu-b-Gevi-b) - Vi-Ser-L.-PlieOH with lipotropic activity. The proposed dec, apeptide is obtained by a solid phase synthesis method using chloromethylated styrene polymer with divinylbenzene as a polymeric carrier by stepwise growth of the peptide chain. The figure shows a diagram of the synthesis of decapeptide. Abbreviations: CP) is a polymeric carrier. Baugh - tert-butyloxycarbonyl, Z-benzyloxycarbonyl, BZ - benzyl, ONP - p-nitrophenyl ether. The following LS-amino acid derivatives (Reanal VNR company) were used in the synthesis: tert-butyloxycar bonide (CPB) -phenylalanine; N-BOC-O-ben zyl-serine, N-BOK-0-benzyl-tyrosine, N-BOK-N -carbobenzoxy-silin, N-BOC-glutamine p-nitrophenyl ester, N-BOC-glutamic acid y-benzyl ester; N-BOC-proline and N-BOC-isoleucine. A spherical chloromethylated styrene copolymer with 2% divinylbenzene (chlorine content 2 mmol / g polymer) was used as the insoluble carrier in the synthesis of decapeptide. The condensation reactions are carried out using dicyclohexylcarbodiimide (DCPG) as the condensing agent; the only exception is the addition of a glutamine residue, which is introduced into the peptide chain by the method of p-nitrophenyl esters. The removal of the peptide from the polymer carrier and its exhaustive release are carried out by the action of hydrogen bromide in trifluoroacetic acid in the presence of anisole. The crude product after removal from the carrier polymer is precipitated from methanol with ether, chromatographed on a column of cellulose in n-butanol-water-pyridine-acetic acid (15: 12: 10: 3), and lyophilized. The purity of the preparation obtained is controlled by paper chromatography (BH) on Whatman MM No. 3 and thin-layer chromatography (those) on Silufol plates (Czechoslovakia) in n-butanol-water-pyridine-acetic acid systems (15:12: 10: 3) (system A), ethyl acetate-pyridine-acetic acid-water (5: 5: 1 3) (system B), isoamyl alcohol-pyridine-water-acetic acid (7: 8: 6: 2) (system B), as well as N-terminal amino acids by the method of Edman and amino acid and elemental analyzes. The main steps in the synthesis of decapeptide 1 are as follows: the addition of a C-terminal amino acid to the polymer carrier; polypeptide chain buildup on a polymer; separating the decapeptide from the carrier polymer while simultaneously removing the protecting groups; purification of decapeptide with subsequent characterization of its physicochemical and lipotropic properties. The accession of the C-terminal amino acid to the polymer carrier. The mixture, 3 g (16 mmol) of BOC-phenylalanine, 8 g of the carrier polymer and 2.016 ml (13 mmol) of triethylamine in 5 ml of ethyl acetate are stirred for 18 hours while boiling. Next, the reaction mixture is cooled to, the polymer is filtered off, sequentially washed with ethyl acetate, ethanol, water, ethanol, dioxane, treated, 7 n. a solution of hydrogen chloride in dioxane, washed with dioxane and ether and dried in vacuum

above . To determine the amount of amino acid on the polymer, 18 mg of the obtained phenylalanyl polymer is hydrolyzed under a mixture of 12 n for 5 hours. HC and propionic acid (2 ml) and filtered. According to the amino acid analysis of the filtrate after evaporation, the amount of BOC-phenylalanine on the polymer carrier is mmol / ton.

Building a polypeptide chain on a polymeric carrier.

To construct a polypeptide chain on the polymer, BOC-phenylalanine-polymer (6.37 g; amino acid content 6.27 mmol) is sequentially subjected nine times to the next treatment cycle with the appropriate solvents and reagents (the amount of solvents for each treatment is 60 ml7: a) washing with dioxane , 2x3 min, b) processing C, 7 n. hydrogen chloride in dioxane 1 x 30 mi. c) washing with dioxane, 3 x 3 min; g) washing with methylene chloride, 2 X 3 min e) treatment with 10% solution of triethylamine in methylene chloride, 1 X 10 min; -e) washing with methylene chloride, - 4x3 min; g) adding 18 mmol of the corresponding BOC-amino acid, starting with N-BOC-O-benzyl-serine, in methylene chloride; h) adding 9 mmol of DCC to the reaction mixture (start of condensation); i) after 30 min, add another 9 mmol of DCC; j) after 60 minutes washing with methylene chloride, 3x3 minutes (end of condensation); processing mixture; 2 ml of acetic anhydride and 1 ml of three ethylamine in methylene chloride to block possible free amino groups that did not react to condensation, 1 X 30 min (then the cycle is repeated with a new BOC-amino acid).

When a glutamine residue is introduced into the peptide chain by the p-nitrophenyl esters method (duration, condensation 18 h), stage 6 uses dimethylformamide instead of methylene chloride, condensation is also carried out in dimethylformamide, and stage h) and) are excluded.

At the end of the peptide chain buildup, the resulting protected decapeptide-polymer is washed with ethanol, acetic acid and ether and dried in vacuo over.

Separating decapeptide from carrier polymer with simultaneous removal of protecting groups

Bubrost hydrogen is bubbled for 90 minutes through a suspension of 2 g of protected decapeptide polymer in 20 ml of trifluoroacetic acid, 20 ml of methylene chloride and 2 ml of anisole. The polymer is then separated by filtration, washed with trifluoroacetic acid (2 x 10 ml), and the combined filtrates are evaporated at. The residue is dissolved in a mixture of 20 ml of methanol and 5 ml of dimethylformamide and reprecipitated by the addition of kS ml of ether. The precipitate is separated by centrifugation, washed with ether, centrifuged again and dried. 1 g of crude product is obtained.

Cleaning up the decapeptide t. .

0.308 g of the crude product is dissolved in 2 ml of a mixture of n-butanol-acetic acid-water-pyridine (15: 3: 12: 10), and the solution is applied to the column (25 x 3 cm filled with cellulose suspension (Filtrak-KND) ( GDR) in the same solvent mixture. This solvent mixture is used further in the process of elution of the substance from the column; Separation is controlled by using those Silufol plates in system A. The fractions containing the base material are combined and lyophilized. 0.171 g of C is obtained, yield 50 in the calculation of the C-terminal amino acid) of the decapeptide in the form of a white solid mp 193-195 C (decomp., (O1) -73, from 0.37;. N .R $ 0.50 (BH in system A), 0, tl (TLC in system A, 0.51 STG.X in system B), 0.31 GHSS in system C), Elemental analysis. Found: C 53,5,1; H 7.07; M11.23

, DL CH, COOH.N2.0: Calculated, C 58.51; H 7.07;

: 11.11.

Amino acid analysis,% i P 1 0 S1.00) 1.00 (1.00); 1.63 2.0 (1) 1.8i (2.00); Pro 1.00: (1, 00); 31e 0.90 (1.00); Glu 2.17 (2.00) ...

 Biological tests of the proposed decapeptide are carried out.

The test aims to determine the effect of synthetic decapeptide,. on the rate of lipolysis in rabbits in vivo and in vitro and to compare the lipotropic effect of the described decapeptide and the known compounds, human somatotropin and synthetic tetradeapeptide. Study of the lipotropic action of decapeptide. In in vivo experiments, lipotropic activity is determined by increasing the concentration of non-esterified fatty acids (EFA) in the blood plasma of animals after administering pepaoic to them to rabbits (weight 2000–2500 g). After 2k fasting, the drug is intramuscularly administered in 0.5 ml of physiological saline . Control animals were injected with pure saline. Each group of animals consists of 6 rabbits. Blood samples are taken from the ear vein before administration and 30 minutes after administration of decapeptide and tetradekapepeptide or minutes after administration of somatotropin, since its lipotropic action requires a long latent period. The level of NLC in the blood is determined by the method of Falholt. The results of the effect of decapeptide on the rate of lipolysis in rabbit in vivo are given in Table. 1. faces. In experiments in vitro, rabbit pararenal adipose tissue weighing 80 µg is crushed with scissors and incubated in 2 ml of Cres-Ringer bicarbonate buffer containing 3% bovine serum albumin at 37 C. in the atmosphere of carbogen (95% 0 C0.2 ) for 120 min. The experiments on the control group consist of 5 rabbits. 0.1 ml of a physiological solution containing the tested doses of decapeptide or tetradekapepeptide are added to the test samples, and 0.1 ml of physiological solution is added to the control samples. At the end of the incubation, the content of the NLC was determined by the method in 5 µl of the incubation mixture. Falcolt with the help of calibro. palmitic acid curve. The results of the effect of decapeptide I on the lipolysis rate in koppek's adipose tissue in vitro are presented in Table. 2. Table2

Physiological plant. The concentration of the NA in the plasma of the cr is determined in experiments ff 2 and 30 minutes after the administration of the preparations, and in experiment No. k, the cut min. Lipotropic activity of In v | tro is assessed by stimulation of lipolysis in isolated rabbit pararenal adipose tissue incubated in the presence of a decapeptide. Lecapeptide, tetradecapeptide isomatotropin in in vivo experiments increase the level of NLC in the blood plasma of rabbits, respectively, by, and. Thus, under in vivo conditions, decapeptide and tetradecapeptide have approximately equal effect, significantly exceeding the lipotropic effect of somatotropin. and in vitro experiments, the decapeptide of formula I and the tetradecapeptide of formula II also have approximately equal lipotropic effects, increasing the concentration of NLC in the incubation medium, respectively, by and. The proposed decapeptide of formula I is lipotropic activity, commensurate with the activity of tetradekapepeptid, and significantly exceeds the activity of GH. However, in contrast. From the known tetradekapeptida, the proposed decapeptide has a simpler structure, since it is a shorter peptide and contains only 10 amino acid residues. The toxicity of the described decapeptide was tested. The toxicity of decapeptide is investigated in acute experiments f acute toxicity on three species of animals and in chronic experiences (chronic toxicity) on one species of animals. ; Determination of acute toxicity: The acute toxicity of a decapeptide is tested in rats, mice, and marine mammals. When tested on male Wistar rats weighing 110-120 g (10 rats). , and weighing .50 g (10 rats), the decapeptide is administered intraperitoneally once a dose of 20 mg / kg of body weight in 0.5 ml of saline. The indicated dose is significantly higher than the active dose in rats C 1 µg / kg of weight). In this dose, the drug does not. local irritant effect and does not change the external state and behavior of animals. When animals were slaughtered a day after the administration of the drug, no seals, infiltrates in the injection sites were revealed. No toxic changes were observed in organs and tissues. When tested in male mice weighing 20 g by 10 mnia, decapeptide was administered intraperitoneally once in a dose of 50 mg / kg body weight, in 0.5 ml of physiological solution. Animals Shoot down a day after administration of the drug. No toxic changes of irritation, seals, infiltrates in the administration of the administration, changes in the behavior and condition of the animals after administration of the preparation, as well as changes in the organs and tissues were not detected. In tests on guinea pigs weighing 180–200 g of tlO animals (2), decapeptide was administered intraperitoneally at a dose of 25 mg / kg of body weight in 0.5 ml of saline. Animals were slaughtered a day after the drug was injected, irritation, compaction or infiltrates were not detected at the drug administration sites, no changes in the behavior and condition of the animals after the drug administration were observed, toxic morphological changes in the organs and tissues were not detected. Definition of chronic toxicity. The test was conducted on male mice weighing 20–25 g (baseline). By the end of the experiment, the weight of the mice increased to 26–35 Ti. The capsule was administered intraperitoneally 15 times over 3 weeks in a total dose of 600 mg / kg body weight a single dose of kQ mg / kg of weight per day). Animals are slaughtered the day after the last administration of the drug under ether anesthesia. Intact mice of the same age served as controls. The number of mice in the control and experimental groups is 10. No irritations, seals, infiltrates were detected at the injection sites. The behavior and appearance of the experimental animals during E1L, the periment did not differ from those in the control mice of the same litter that did not receive the drug. No morphological changes were revealed in organs and tissues removed after autopsy. The data on the weight of individual organs and tissues of mice that received a decapeptide for 3 weeks are presented in Table 1. In the total dose, 40 times the amount of the active dose of the drug, with no introduction, there are no statistically significant differences in the weight of different organs and tissues of animals that received decapeptide, and control animals.

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Claims (1)

The decapeptide of the formula 'Hu-Tsr-u-iee-L-pro-v-L'ss-t.-qeu-b-cieh-b-vjs-u-Tsr-t-ser-u-Phe it> with lipotropic activity .