SU1149603A1 - Polypeptide possessing hypoglycemic activity - Google Patents

Abstract

A polypeptide of the general formula, 1 R-, where R is the insulin molecule residue, containing from 1 to 29 amino acids; E is the rest of the insulin molecule, containing from 31 to 51 amino acids; , 30 V - L-serine, possessing hypoglycemic activity. (L

Description

This invention relates to a novel biologically active compound with a hypoglycemic activity polypeptide which can be used in medicine. Currently, a human insulin substitute (insulin containing L-TpeoHHH hydroxyamino acid residue) is used in medical practice - insulin I, which differs from human insulin in pigs, is because L-alanine occupies a position in the e molecule. The purpose of the invention is to search in a series of polypeptides with hypoglycemic activity of polypeptides that are closer in structure to human insulin. The goal is achieved by the described polypeptide of the general formula R - R ° - R 1l. o K. J where R is the residue of an insulin molecule containing from 1 to 29 amine acids; R is a residue of an insulin molecule containing from 31 to 51 amine acids; B b serine with hypoglycemic activity. The following shows the structural differences in position B between the described polypeptide (I), natural pig insulin (II) and human insulin (III). /// // UJLJLM ////// 1 sn-сГ cH-cf Ц ОН т ОН, I ОШ Ш The described polypeptide is obtained by enzymatic-chemical transformation of readily available and produced on an industrial scale pig insulin according to the two-stage scheme The stage consists in trypsin-catalyzed transformation of porcine insulin, which occurs when the latter reacts with L-serine ester. The second stage of the process consists in chemical demasking of the ester derivative under the action of trifluoroacetic acid in the presence of anisole as a rotor. Example. 140 mg (1.14 mmol) of tert-butyl tert-butyl ester hydrochloride 0-t ret-butyl-L-serine are dissolved in a mixture of 300 µl of dimethylformamide and 200 µl of tris buffer pH 5.7, by addition of 5 n. NaOH was adjusted to pH 8, O, the mixture was kept for 10 minutes at 20 ° C, and 100 mg (17.5 µmol) of porcine insulin, previously released from zinc ions, was added to it. By adding 50 µl of concentrated hydrochloric acid, the pH is adjusted to 6.3 and 6 mg of trypsin and 5 µl of a 0.5 M aqueous solution of calcium chloride are added. The mixture is kept for 24 hours at 24 ° C, then acidified to pH 2.5 with 50% acetic acid and subjected to gel filtration to a column (2.8 x 100 cm) with Sephadex G-50 using 10% as a solvent. acetic acid. After lyophilization of the eluate, 70 mg of an ester derivative of the target polypeptide (I) is obtained, which is purified by ion exchange chromatography on a column (1.7 x 16 cm) with DEAE Sephadex A-25, using 0.03 M tris buffer with a pH of 8, 2, containing 0.05% EDTA and 50% isopropyl alcohol. After the eluate is concentrated in vacuum, the solution (5 ml) is desalted by passing it through a column (3.5 x 60 cm) with Sephadex G-50, previously equilibrated with a 0.05 M solution of ammonium bicarbonate. The substance obtained after lyophilization is dissolved in 0.4 ml of trifluoroacetic acid containing 0.02 ml of anisole, the mixture is held for 1 hour at 20 ° C, cooled to –10 ° C, 20 ml of cooled diethyl ether is added, the mixture is kept for 2 hours at -, the precipitated substance is separated by centrifugation, dissolved in a minimum volume of 10% acetic acid, and the resulting solution is passed for final desalting through a column of 2.2 x 38 cm with Sephadex G-25, using as eluent 10% acetic acid. After lyophilization, 15 mg of the target polypeptide (I) with an electrophoretic mobility of 1.35 is obtained (paper electrophoresis Whatman No. 1, pH 1.9, 450 V, 7 mA, the reference standard is pig insulin B-8-sulfonate) .

Amino Acid Analysis: Asp 3.00 (3), Thr 1.80 (2), Ser 3.80 (4), Gld 7.06 (7), Pro 1.00 (1), Gly 4.00 (4) Ala 1.02 (1), Cys 4.60 (6), Val 3.60 (4), Jle 1.80 (2), Leu 6.00 (6), Tyr 3.50 (4), Phe 2 , 90 (3), His 2.00 (2) Lys 1.00 (1), Arg 0.95 (1).

Results of determination of C-terminal amino acids: Ser 0.98 (1), Asn .0.97 (O.

Biological tests of the described polypeptide have been carried out.

Tests were performed on mice (self) weighing 20-22 g after a 20-hour fast. Animals are divided into 6 groups of 10 animals each. The animals of the first three groups were subcutaneously injected with 1, 0.75, and 0.5 µg of insulin-stai, darth, respectively; in mice of the 4th, 5th and 6th groups, the same doses of polypeptide (I), the test preparations are administered in 0.2 ml of saline. The animals are placed in a temperature-controlled chamber divided into separate cells with and monitored for 90 minutes. In each group, the number of animals that develop seizures is counted.

The research results are presented in the table.

Thus, the biological activity of the described polypeptide is fully consistent with the activity of human insulin, taken as a standard (twa. The described polypeptide has 100% insulin activity).

The determination of the acute toxicity of the described polypiptide.

The acute toxicity of the described compound was tested in mice — cai-rts weighing 20-22 g. The test polypeptide was administered once subcutaneously in doses of 0.5, 0.75 and 1.0 µg / mouse (2550 µg / kg body weight), which exceeds the range of active therapeutic doses (720 mcg / kg). Observations on animals placed in isolated boxes at a temperature of 36 ° C were continued for 1 hour. The convulsions that developed during this time were stopped by subcutaneous administration of a 20% glucose solution and continued monitoring of animals for another day. Any deviations in behavior

The experimental and control rpyn were not observed. At the indicated doses, the drug did not produce a local irritant effect. When animals were slaughtered, after compaction (I) was injected, there were no seals, irritations, infiltrates in the injection sites. Changes in organs and tissues are not detected.

The toxic effect of insulins is associated with their main biological effect, the hypoglycemic effect. With an overdose of the drug, hypoglycemic convulsions and hypoglycemic coma develop.

The toxic effects are absolutely identical for human, pig, and cattle insulin.

The described polypeptide in comparison with the pig polypeptide is a closer structural analogue of human insulin.

Compared with the described insipepe human insulin is more difficult and more expensive than the 1st drug, because for obtaining human insulin it is necessary to use L-threonine hydroxyamic acid, which contains two asymmetric centers (for S) - vi.j carbon atoms); when preparing the described polypeptide, the starting material is the hydroxy amino acid L-serine, which contains only one asymmetric center (only at the (/ carbon atom).

These structural differences reduce in two pars the racemization side effects in the preparation of the described polypeptide compared to the production of human insulin.

Comparison of the cost of hydroxy-amino acids L-serine and L-threonine shows that the described polypeptide is cheaper than human insulin by more than 5 times.

The preparation of the described polypeptide having hypoglycemic 100% insulin activity is based on the enzymatic conversion of porcine insulin, which is an affordable commercial product.

Testing for convulsive effects in menpay.

Claims (2)

The polypeptide of the General formula 10 I k - wk; where K is the rest of the insulin molecule, containing from 1 to 29 amino acids; P' K - the residue of the insulin molecule, containing from 31 to 51 amino acids; B - b-serine, possessing hypoglycemic activity. ABOUT ω with one 1149603 2