EA040166B1 - POLYPEPTIDE FOR THE TREATMENT OF TYPE 2 DIABETES MELLITUS AND ITS COMPLICATIONS - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to a new analogue of exenatide formula

H-His-Gly-GluGly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Gly-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-IleGlu-Trp-Leu- Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-D-Arg-D-Arg-DArg-D-Arg-D-Arg-D-Arg-D- Arg-D-Arg-Gly-OH, which can be used for the treatment and prevention of diabetes mellitus, as well as for the treatment and prevention of complications of type 2 diabetes mellitus such as diabetic neuropathy, muscular dystrophy and endotheliopathy.

The invention relates to the field of medicine and pharmacy and can be used as medicines for the prevention and treatment of type 2 diabetes mellitus, as well as its complications, such as diabetic neuropathy, muscular dystrophy and endotheliopathy.

Prior Art

Type 2 diabetes mellitus (DM 2 or non-insulin-dependent diabetes) is a chronic, multi-organ disease that manifests itself mainly as a violation of carbohydrate metabolism. As a result of pathological changes, hyperglycemia develops, in addition, there is dysfunction of specialized pancreatic cells responsible for insulin production. The main cause of mortality in patients with type 2 diabetes mellitus is, first of all, the development of macrovascular complications (damage to the coronary, cerebral and peripheral arteries). Also, DM 2 is one of the main components of the development of the metabolic syndrome (insulin resistance or syndrome X) [1]. Recently, there has been a growing number of observations that DM 2 is a pathological marker for the development of Alzheimer's disease [2].

According to the WHO, by the end of 2013, there were about 347 million people with diabetes worldwide [3]. In 2004, 3.4 million people died from the effects of high blood sugar [4]. In 2010, the number of deaths from complicated diabetes remained at the same level [5]. According to WHO forecasts, in 2030 diabetes will become the seventh leading cause of death [4]. It was assumed that type 1 diabetes in developed countries occurs in 10-15% of patients, and type 2 diabetes in 85-90%. But in recent years, the frequency of type 2 diabetes in developed countries has been growing very rapidly, and the number of patients with type 1 diabetes has changed little.

According to the latest WHO information, the ratio of type 1 and type 2 diabetes in the world today has changed towards an increase in the frequency of type 2 diabetes [5].

The earliest and most frequent complication of diabetes mellitus is diabetic neuropathy [6], which is characterized by a disorder of the nervous system associated with damage to small blood vessels (vasa vasorum, vasa nervorum). This complication not only leads to a decrease in working capacity, but is often the cause of the development of severe disabling lesions and death of patients. The pathological process affects all nerve fibers: sensory, motor and autonomic. According to different authors, diabetic neuropathy occurs in 90-100% of diabetic patients. The frequency of lesions of the nervous system in diabetes mellitus is directly proportional to the duration of the disease, and in some cases it precedes the appearance of the main clinical signs of diabetes. Thus, 5% of patients with manifestations of diabetes mellitus already have symptoms of damage to the nervous system, which increase with the duration of the disease, reaching 60% with diabetes for more than 25 years.

In diabetes mellitus, all parts of the nervous system are affected: the central nervous system (encephalopathy, myelopathy), the peripheral nervous system (poly- and mononeuropathies) and the peripheral autonomic nervous system (autonomic neuropathy).

Considering the foregoing, it is obvious that the development and introduction into everyday practice of a drug for the treatment of both type 2 diabetes mellitus itself and its complications seems promising.

Currently, the basis of drug treatment of type 2 diabetes mellitus and its complications are hypoglycemic drugs, which are grouped into several groups:

the first group includes two types of drugs - thiazolidinediones (rosiglitazone and pioglitazone), PPARY agonists, stimulators of nuclear gamma receptors, and biguanides (Metformin, Siofor, Avandamet, Bagomet, Glucophage, Metfogamma). The drugs of this group increase the sensitivity of cells to insulin, reduce insulin resistance and absorption of glucose by intestinal cells, so they are often prescribed to overweight people;

the second group of hypoglycemic drugs also consists of two types of drugs - sulfonylurea derivatives (Maninil, Diabeton, Amaryl, Glurenorm, Glibinez-retard), which stimulate the production of their own insulin, thereby increasing its effectiveness, and meglitinides (Repaglinide (Novonorm) and Nateglinide (Starlix )), which enhance the synthesis of insulin by the pancreas, and also reduce postprandial peaks (increased sugar after eating);

The third group of hypoglycemic drugs includes the drug Acarbose (Glucobay), which reduces the absorption of glucose by intestinal cells due to the fact that it blocks the alpha-glucosidase enzyme, which breaks down polysaccharides that come with food.

Direct incretin mimetics, glucagon-like peptide-1 (GLP-1) receptor agonists and indirect dipeptidyl peptidase type 4 blockers are a new direction in the treatment and prevention of complications of type 2 diabetes. Direct incretin mimetics include drugs such as exenatide (Byetta), liraglutide (Victoza), albiglutide, and dulaglutide. Indirect drugs include Sitagliptin (Januvia), Saxagliptin (Ongliza), Linagliptin (Trajenta) and Vildagliptin (Galvus).

Of particular interest is the group of incretin-like drugs.

Incretins such as GLP-1 improve beta cell function, suppress inappropriately increased glucagon secretion, and cause an increase in glucose-dependent insulin secretion. Exenatide (exendin-4), which is an incretin receptor mimetic (GLP-1R), is one of the drugs, the effectiveness of the treatment and prevention of its complications in non-insulin-dependent diabetes mellitus has been proven in human clinical trials.

Exenatide is a polypeptide originally known as exendin-4, whose isolation method and amino acid sequence (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG-PSSGAPPPS-NH2) from the venom of the lizard Heloderma suspectum were first published in April 1992 [7]. Exendin-4, being a member of the incretin peptide family, has a wide range of activities, such as a glucose-dependent increase in insulin secretion, increased glucose processing, appetite suppression, slowing down the movement of food from the stomach, etc.

In type 2 diabetic patients with hyperglycemia, the administration of exenatide suppresses excess glucagon secretion, while exenatide does not interfere with the normal glucagon response to hypoglycemia.

Therapy with exenatide in combination with metformin and/or sulfonylurea drugs results in a decrease in fasting blood glucose, postprandial blood glucose, and glycated hemoglobin (HbA _1C ), thereby improving glycemic control in these patients.

Known areas of application of exenatide: for example, in the US patent [8] discloses the use of exenatide to stimulate the release of insulin with an efficiency greater than that of the endogenous hormone GLP-1. In the description of the invention, the examples given confirm that exenatide is effective for the treatment of diabetes mellitus.

Subsequently, other therapeutic effects of exenatide were discovered: weight loss [9], decreased motility and slowing gastric emptying [10, 11], as well as the inotropic and diuretic effects of exenatide [12, 13, 14]. The use of exenatide for the treatment of polycystic ovaries [15, 16, 17], the treatment and prevention of nephropathy [18, 19], the prevention and treatment of cardiac arrhythmia [20], for the differentiation of bone marrow cells [21], and the treatment of diabetes mellitus in pregnant women [22 , 23], to modulate the level of triglycerides and treat dyslipidemia [24, 25], to suppress glucagon secretion [26, 27], to differentiate non-insulin-producing cells into insulin-producing ones [28]. Application [29] describes the use of exenatide, agonists and antagonists of exenatide to affect the central nervous system. Patents [30, 31] describe the use of exenatide and other incretin receptor agonists to reduce food absorption. Application [32] describes stabilized exenatide compounds. Patents [33, 34] describe novel uses for exenatide (exendin and exendin agonists).

All of the above indicates the high effectiveness of exenatide in the prevention and treatment of type 2 diabetes. However, such complications that occur in type 2 diabetes mellitus as diabetic neuropathy, muscular dystrophy and endotheliopathy are not corrected during therapy with both exenatide and all other hypoglycemic agents listed above.

Therefore, there is a challenge to develop new analogues of exenatide that are effective in the treatment of complications that occur in type 2 diabetes mellitus, such as diabetic neuropathy, muscular dystrophy and endotheliopathy.

The closest analogue of the claimed invention is US patent US 5424286 [8], selected as a prototype, in which common with the claimed invention is the use of incretin mimetics to stimulate the release of insulin.

The main disadvantage of the known invention is the lack of therapeutic effects in the claimed incretin mimetics (exenatides), which allow for the prevention and treatment of complications of diabetes mellitus such as diabetic neuropathy, muscular dystrophy and endotheliopathy.

The essence of the invention

The technical result of the claimed invention is the prevention and treatment of complications of diabetes mellitus such as diabetic neuropathy, muscular dystrophy and endotheliopathy.

The technical result in the claimed invention is achieved by using the compound of the formula:

- 2 040166

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Gly-Glu-Glu-Glu-AlaVal-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-ProSer-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg- D-Arg-D-Arg-Gly-OH.

In this regard, the present invention is directed to a polypeptide of the formula

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Gly-Glu-Glu-Glu-Ala-Val-ArgLeu-Phe-Ile-Glu-Trp-Leu- Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-D-ArgD-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D- Arg-D-Arg-Gly-OH effective in the treatment and prevention of type 2 diabetes mellitus and its complications selected from diabetic neuropathy, muscular dystrophy and endotheliopathy.

In one embodiment, the invention is directed to the use of a polypeptide of the formula

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Gly-Glu-Glu-Glu-Ala-ValArg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-DArg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg- D-Arg-D-Arg-Gly-OH for the treatment and prevention of type 2 diabetes mellitus and its complications, selected from diabetic neuropathy, muscular dystrophy and endotheliopathy.

List of figures of drawings and other materials

In FIG. 1 shows the effect of a polypeptide of the present invention on lowering blood glucose compared to other known hypoglycemic drugs.

In FIG. 2 shows the effect of the polypeptide of the present invention on tactile allodynia in rats with alcoholic neuropathy. Tactile allodynia was assessed before modeling alcoholic neuropathy and after treatment with the polypeptide of the present invention (1, 50 and 100 days).

In FIG. 3 shows the effect of the polypeptide of the present invention on the thickness of the intima of the mesenteric artery in comparison with other known hypoglycemic drugs.

Information confirming the possibility of carrying out the invention

The synthesis of the claimed compound is described in examples 1-5.

Example 1 Synthesis of Fmoc-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-OH

6.0 g of 2-chlorotrityl resin (capacity 1.5 mmol/g) was suspended in a reactor for solid phase synthesis in 45 ml of DCM, kept for 5 min, the resin was filtered off, washed with 2x30 ml of DCM. A solution of 2.95 g (9.9 mmol) of Fmoc-Gly-OH and 6 ml (36 mmol) of DIPEA in 30 ml of DCM was added to the resin, and the mixture was stirred for 60 min at room temperature. The resin was filtered off, washed with 2x30 ml DCM, treated with 2x30 ml DCM/methanol/DIPEA (17:2:1) for 10 min, washed with 2x30 ml DCM and 3x30 ml DMF. 30 ml of a 20% solution of diethylamine in DMF was loaded into the reactor, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of a 20% solution of diethylamine in DMF was added, kept for 20 min, filtered, washed with 5x30 ml of DMF.

A cooled (4°C) solution of 7.67 g (20.0 mmol) Fmoc-Ser(tBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.67 g (20.0 mmol) Fmoc-Ser(tBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°С) solution of 6.75 g (20.0 mmol) Fmoc-Pro-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 3.50 g (20.0 mmol) Fmoc-Gly-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered off, washed with 6x30 ml DMF, 3x30 ml DCM. The resin was then treated with 10x30 ml of 1% trifluoroacetic acid in DCM, the resulting solutions were combined in a flask containing 30 ml of 10% pyridine in methanol. The mixture was evaporated to a volume of ~50 ml, to the residue

- 3 040166 200 ml of water was added. The precipitate formed was filtered off, washed with water, and dried. Received 6.4 g of the product (91%) with HPLC purity of 97%.

Example 2 Synthesis

Fmoc-Glu(OtBu)-Glu(OtBu)-Glu(OtBu)-Ala-ValArg(CF3COOH)-Leu-Phe-Ile-Glu(OtBu)-Trp(Boc)-Leu-Lys(Boc)-Asn(Trt )-Gly-OH

6.0 g of 2-chlorotrityl resin (capacity 1.5 mmol/g) was suspended in a reactor for solid phase synthesis in 45 ml of DCM, kept for 5 min, the resin was filtered off, washed with 2x30 ml of DCM. A solution of 2.95 g (9.9 mmol) of Fmoc-Gly-OH and 6 ml (36 mmol) of DIPEA in 30 ml of DCM was added to the resin, and the mixture was stirred for 60 min at room temperature. The resin was filtered off, washed with 2x30 ml DCM, treated with 2x30 ml DCM/methanol)/DIPEA (17:2:1) for 10 min, washed with 2x30 ml DCM and 3x30 ml DMF. 30 ml of a 20% solution of diethylamine in DMF was loaded into the reactor, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of a 20% solution of diethylamine in DMF was added, kept for 20 min, filtered, washed with 5x30 ml of DMF.

A cooled (4°C) solution of 11.93 g (20.0 mmol) Fmoc-Asn(Trt)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 9.37 g (20.0 mmol) Fmoc-Lys(Boc)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.07 g (20.0 mmol) Fmoc-Leu-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 10.53 g (20.0 mmol) Fmoc-Trp(Boc)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 8.51 g (20.0 mmol) Fmoc-Glu(OtBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.07 g (20.0 mmol) Fmoc-Ile-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.75 g (20.0 mmol) Fmoc-Phe-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.07 g (20.0 mmol) Fmoc-Leu-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30

- 4 040166 ml DMF.

A cooled (4°C) solution of 8.66 g (20.0 mmol) Fmoc-Arg-OH-HCl, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 6.79 g (20.0 mmol) Fmoc-Val-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 6.23 g (20.0 mmol) Fmoc-Ala-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 8.51 g (20.0 mmol) Fmoc-Glu(OtBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 8.51 g (20.0 mmol) Fmoc-Glu(OtBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 8.51 g (20.0 mmol) Fmoc-Glu(OtBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered off, washed with 6x30 ml DMF, 3x30 ml DCM. The resin was then treated with 10x30 ml of 1% trifluoroacetic acid in DCM, the resulting solutions were combined in a flask containing 30 ml of 10% pyridine in methanol. The mixture was evaporated to a volume of ~50 ml, 200 ml of water was added to the residue. The precipitate formed was filtered off, washed with water, and dried. Received 21.8 g (80%) of the product with HPLC purity of 95%.

Example 3 Synthesis

Boc-His(Trt)-Gly-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)Ser(tBu)-Asp(OtBu)-Leu-Ser(tBu)-Lys(Boc)-Gln (Trt)-Gly-OH

6.0 g of 2-chlorotrityl resin (capacity 1.5 mmol/g) was suspended in a reactor for solid phase synthesis in 45 ml of DCM, kept for 5 min, the resin was filtered off, washed with 2x30 ml of DCM. A solution of 2.95 g (9.9 mmol) of Fmoc-Gly-OH and 6 ml (36 mmol) of DIPEA in 30 ml of DCM was added to the resin, and the mixture was stirred for 60 min at room temperature. The resin was filtered off, washed with 2x30 ml DCM, treated with 2x30 ml DCM/methanol/DIPEA (17:2:1) for 10 min, washed with 2x30 ml DCM and 3x30 ml DMF. 30 ml of a 20% solution of diethylamine in DMF was loaded into the reactor, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of a 20% solution of diethylamine in DMF was added, kept for 20 min, filtered, washed with 5x30 ml of DMF.

A cooled (4°С) solution of 12.21 g (20.0 mmol) Fmoc-Gln(Trt)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 9.37 g (20.0 mmol) Fmoc-Lys(Boc)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30

- 5 040166 ml DMF.

A cooled (4°C) solution of 7.67 g (20.0 mmol) Fmoc-Ser(tBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.07 g (20.0 mmol) Fmoc-Leu-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 8.23 g (20.0 mmol) Fmoc-Asp(OtBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.67 g (20.0 mmol) Fmoc-Ser(tBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.95 g (20.0 mmol) Fmoc-Thr(tBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.75 g (20.0 mmol) Fmoc-Phe-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 7.95 g (20.0 mmol) Fmoc-Thr(tBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 5.95 g (20.0 mmol) Fmoc-Gly-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 8.51 g (20.0 mmol) Fmoc-Glu(OtBu)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30 ml DMF.

A cooled (4°C) solution of 5.95 g (20.0 mmol) Fmoc-Gly-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature . The resin was filtered, washed with 6x30 ml DMF, 30 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x30 ml of DMF, 30 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x30

- 6 040166 ml DMF.

A cooled (4°C) solution of 9.95 g (20.0 mmol) Boc-His(Trt)-OH, 2.98 g (22.0 mmol) HOBt, and 3.42 ml (22.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 2 h at room temperature. The resin was filtered off, washed with 6x30 ml DMF, 3x30 ml DCM. The resin was then treated with 10x30 ml of 1% trifluoroacetic acid in DCM, the resulting solutions were combined in a flask containing 30 ml of 10% pyridine in methanol. The mixture was evaporated to a volume of ~50 ml, 200 ml of water was added to the residue. The precipitate formed was filtered off, washed with water, and dried. Received 20.9 g (85%) of the product with HPLC purity of 95%.

Example 4 Synthesis of 14-Glycine-Exendin-4 (Heloderma Sn8pctm)-(1-39)-neptidyl-octa-O-arginylglycine

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Gly-Glu-Glu-Glu-AlaVal-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro_ProSer-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D- Arg-D-Arg-Gly-OH

2.0 g of 2-chlorotrityl resin (capacity 1.5 mmol/g) was suspended in a reactor for solid phase synthesis in 15 ml of DCM, kept for 5 min, the resin was filtered off, washed with 2x10 ml of DCM. A solution of 0.98 g (3.3 mmol) of Fmoc-Gly-OH and 2 ml (12 mmol) of DIPEA in 10 ml of DCM was added to the resin, and the mixture was stirred for 60 min at room temperature. The resin was filtered off, washed with 2x10 ml DCM, treated with 2x10 ml OCM/methanol/O1PEA (17:2:1) for 10 min, washed with 2x10 ml DCM and 3x10 ml DMF. 10 ml of a 20% solution of diethylamine in DMF was loaded into the reactor, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of a 20% solution of diethylamine in DMF was added, kept for 20 min, filtered, washed with 5x10 ml of DMF.

A cooled (4°C) solution of 2.60 g (6.0 mmol) Fmoc-D-Arg-OH-HCl, 0.98 g (7.2 mmol) HOBt, and 1.12 ml (7.2 mmol) DIC in 10 ml DMF was loaded into the reactor; the mixture was stirred for 2 h at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 2.60 g (6.0 mmol) Fmoc-D-Arg-OH-HCl, 0.98 g (7.2 mmol) HOBt, and 1.12 ml (7.2 mmol) DIC in 10 ml DMF was loaded into the reactor; the mixture was stirred for 4 h at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 3.46 g (8.0 mmol) Fmoc-D-Arg-OH-HCl, 1.35 g (10.0 mmol) HOBt, and 1.56 ml (10.0 mmol) DIC in 10 ml DMF was loaded into the reactor; the mixture was stirred for 3 hour at room temperature. The resin was filtered off, washed with 6x10 ml of DMF, 10 ml of a 20% solution of diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3xx10 ml of DMF, 10 ml of a 20% solution of diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 3.46 g (8.0 mmol) Fmoc-D-Arg-OH-HCl, 1.35 g (10.0 mmol) HOBt, and 1.56 ml (10.0 mmol) DIC in 10 ml DMF was loaded into the reactor, the mixture was stirred for 4 h at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 3.46 g (8.0 mmol) Fmoc-D-Arg-OH-HCl, 1.35 g (10.0 mmol) HOBt, and 1.56 ml (10.0 mmol) DIC in 10 ml DMF was loaded into the reactor; the mixture was stirred for 10 hour at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 4.33 g (10.0 mmol) Fmoc-D-Arg-OH-HCl, 1.62 g (12.0 mmol) HOBt, and 1.88 ml (12.0 mmol) DIC in 10 ml DMF was loaded into the reactor; the mixture was stirred for 12 h at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 4.33 g (10.0 mmol) Fmoc-D-Arg-OH-HCl, 1.62 g (12.0 mmol) HOBt, and 1.88 ml (12.0 mmol) DIC in 10 ml DMF was loaded into the reactor; the mixture was stirred for 12 h at room temperature

-7 040166 temperature. The resin was filtered, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed

5x10 ml DMF.

A cooled (4°C) solution of 4.33 g (10.0 mmol) Fmoc-D-Arg-OH-HCl, 1.62 g (12.0 mmol) HOBt, and 1.88 ml (12.0 mmol) DIC in 10 ml DMF was loaded into the reactor; the mixture was stirred for 16 h at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°С) solution of 3.07 g (8.0 mmol) Fmoc-Ser(tBu)-OH, 1.35 g (10.0 mmol) HOBt, and 1.56 mL (10.0 mmol) DIC in 30 mL DMF was loaded into the reactor, the mixture was stirred for 12 h at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 2.70 g (8.0 mmol) Fmoc-Pro-OH, 1.35 g (10.0 mmol) HOBt, and 1.56 ml (10.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 12 h at room temperature . The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 2.70 g (8.0 mmol) Fmoc-Pro-OH, 1.35 g (10.0 mmol) HOBt, and 1.56 ml (10.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 12 h at room temperature . The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 2.70 g (8.0 mmol) Fmoc-Pro-OH, 1.35 g (10.0 mmol) HOBt, and 1.56 ml (10.0 mmol) DIC in 30 ml DMF was loaded into the reactor, the mixture was stirred for 12 h at room temperature . The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 2.49 g (8.0 mmol) Fmoc-Ala-OH, 1.35 g (10.0 mmol) HOBt, and 1.56 ml (10.0 mmol) DIC in 30 ml DMF was loaded into the reactor, and the mixture was stirred for 12 h at room temperature. . The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 5.90 g (8.0 mmol) FmocGlyProSer(tBu)Ser(tBu)Gly-OH (product of example 1), 1.62 g (12.0 mmol) HOBt, and 1.88 ml (12.0 mmol) DIC in 30 ml of DMF, stirred for 12 h at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°С) solution of 11.35 g (4.0 mmol) Fmoc-Glu(OtBu)-Glu(OtBu)Glu(OtBu)AlaValArg(HCl)LeuPheIleGlu(OtBu)Trp(Boc)Leu

Lys(Boc)Asn(Trt)Gly-OH (product of Example 2), 0.81 g (6.0 mmol) HOBt and 0.95 ml (6.0 mmol) DIC in 30 ml DMF were stirred for 24 h at room temperature. The resin was filtered off, washed with 6x10 ml DMF, 10 ml of 20% diethylamine in DMF was added, stirred for 5 min, filtered, washed with 3x10 ml of DMF, 10 ml of 20% diethylamine in DMF was added, kept for 20 min, filtered, washed 5x10 ml DMF.

A cooled (4°C) solution of 9.94 g (4.0 mmol) BocHis(Trt)Gly-Glu(OtBu)GlyThr(tBu)PheThr(tBu)Ser(tBu)Asp(OtBu)LeuSer(tBu)Lys(Boc) solution was loaded into the reactor Gln(Trt)G!y-OH (product of Example 3), 0.81 g (6.0 mmol) HOBt and 0.95 ml (6.0 mmol) DIC in 30 ml DMF were stirred for 24 h at room temperature. The resin was filtered off, washed with 6x20 ml DMF, 4x20 ml DCM, dried, 50 ml TFA/TIS/EDT/H2O (97:1:1:1) was added, kept for 4 h at room temperature, filtered, washed with 3x20 ml trifluoroacetic acid, the combined filtrates were evaporated to a volume of ~20 ml, 60 ml of dry ether was added to the residue. The precipitate that formed was filtered off, washed on the filter with ether, and dried. The resulting product was dissolved in 50 ml of water, the solution was frozen and lyophilized. The lyophilizate was dissolved in 40 ml of water and applied to a column with Amberlite IRA-400 ion exchange resin (Cl-form). The column was washed with water, the fractions containing the product were combined, evaporated to a volume of ~50 ml, and applied to a Waters X-Bridge C18 reverse phase column, 10 μm, 127 A, 50x250 mm. Elution was carried out at an eluent flow rate of 50 ml/min. Phase A: 0.1% HCl/H ₂ O, B: acetonitrile. Gradient: 0% (B)-70% (B) in 70 min. Fractions containing the main product were combined, evaporated to a volume of ~50 ml, frozen, and lyophilized. 3.9 g (20%) of the product was obtained with a purity (HPLC) of 97.5%. Mass spectrum: calculated for C ₂₃₁ H ₃₇₄ N ₈₂ O ₇₀ MH+ 5420.98, obtained MH+ 5420.80. Amino acid analysis: alanine 2.02 (2), arginine 9.0 (9), aspartic acid + asparagine 2.05 (2), glutamic acid + glutamine 5.80 (6), glycine 7.25 (7), histidine 1.02 (1), isoleucine 1.03 (1) , leucine 2.96 (3), lysine 2.07 (2), phenylalanine 2.05 (2), proline 4.10 (4), series 4.85 (5), threonine 1.90 (2), valine 1.02 (1).

Example 5 Synthesis

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-GlyGlu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-GlyAla-Pro-Pro-Pro-Ser-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg- D-Arg-D-Arg-Gly-OH using Corynebacterium acetoacidophilum strain

1.1. Construction of the pBSΔCg10278 vector to remove the Cg10278 gene encoding PBP1a.

The genomic sequence of C. acetoacidophilum ATCC 13032 and the nucleotide sequence of the Cg1278 gene encoding the PBP1a penicillin-binding protein are known (GenBank accession number BA000036 (version BA000036.3 GI: 42602314, locus_tag=NCg10274). Primers P1, P1, P1, were synthesized with reference to this sequence. P3 and P4 By PCR using as template chromosomal DNA of C. acetoacidophilum ATCC 13869 prepared in the usual way (Saito H. and Miura K. I., Biochim. Biophys. Acta, 1963, 72:619-629) and primers P1 and P2, and P3 and P4, a fragment (about 1 kb) from the 5' side and a fragment (about 1 kb) from the 3' side were obtained from Cg10278 encoding PBP1a, respectively Then, using PCR, using both obtained DNA fragments and primers P1 and P4 as a template, a DNA fragment (about 2 kb) was obtained, consisting of both fragments fused to each other. and P4, recognition sites for restriction enzymes BamH I and Xba I were introduced, respectively. But. For PCR, Pyrobest DNA polymerase (manufacturer Takara Bio) was used and conditions recommended by the manufacturer. This DNA fragment was digested with restriction enzymes BamH I and Xba I and introduced into the BamH I-Xba I site of pBS4 described in WO 2005/113744 to obtain the vector pBSACg10278 for the deletion of the Cg0278 gene. For ligation DNA Ligation Kit Ver. 2.1 (manufacturer Takara Bio) and conditions recommended by the manufacturer.

1.2. Construction of a PBP1a-deficient strain.

The C. acetoacidophilum YDK010 strain described in WO 2004/029254 was transformed with the constructed vector pBSACg10278. The C. acetoacidophilum YDK010 strain is a strain deficient in the PS2 cell surface protein of the C. acetoacidophilum strain AJ12036 (FERM BP-734) (WO 2004/029254). The strain was selected from the resulting transformants as described in WO 2005/113744 and WO 2006/057450 in order to obtain a YDKO1OAPBP1a strain deficient in the Cg1278 gene.

The examples below relate to pharmacological testing of the resulting peptide.

The aim of the study was to identify hypoglycemic activity and preventive and therapeutic effects of the claimed compound (hereinafter peptide D) in the development of complications in the model of chemical experimental diabetes induced by a single administration of streptozotocin (STZ) and nicotinamide to male rats [35].

The following were used as comparators:

1. Byetta® (as a reference drug for the prevention and treatment of complications of type 2 diabetes) - a clear solution for subcutaneous injection 250 μg / 1 ml: syringe pens 1.2 ml, BAXTER Pharmaceutical Solutions.

2. Synthetic substance exenatide.

3. Metformin (Siofor® 500) (as a classic hypoglycemic agent from the group of biguanides).

Used reagents and materials.

A set of reagents for determining the level of glycosylated hemoglobin (LLC Phosphosorb, Russia).

Used equipment:

1. Medical laboratory dispenser, 10-100 µl.

2. Medical laboratory dispenser, 100-1000 µl.

3. Microcentrifuge Z 216 MK (Hermle Labortechnik GmbH, Germany).

4. Glucometer OneTouch UltraEasy® (LifeScan, USA).

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5. OneTouch Ultra® test strips (LifeScan, USA).

6. Von Frey threads (Stoelting, USA).

Animals

Adaptation and selection of animals.

Laboratory animals prior to the start of the study were kept for 7 days for adaptation when group kept in cages. During this period, the animals were clinically monitored every day by visual inspection. Animals with abnormalities detected during the examination were not included in the experimental groups. Before the start of the study, animals that met the inclusion criteria in the experiment were divided into groups.

Distribution by groups.

Animals were selected using the modified block randomization method [36]. To do this, all animals received from the nursery were randomly placed in the cells of the randomization block (the number of cells in the randomization block is a multiple of the number of groups in the experiment). Further, using a random number generator (statistical program Statistica 6.0), a list of data was obtained containing the numbers of cells with animals and the corresponding numbers of groups where the animals were subsequently placed [36].

Animal identification.

Cell labeling encoded gender, number of animals, start date of the experiment, and group name. Each animal selected for the study was assigned an individual number by marking the tail.

The animals were kept under standard conditions in accordance with the rules approved by M3 of the USSR on July 6, 1973, on the arrangement, equipment and maintenance of experimental biological clinics (vivariums) and GOST R53434-2009.

Animals were kept in standard transparent plastic cages, in groups of 5, on bedding; cages are covered with steel lattice covers with a stern recess. Floor area per animal - 440 cm ² (minimum allowable area 250 cm ² ).

Feed for keeping laboratory animals PK-120-1, prepared according to GOST R 50258-92 in accordance with the norms approved by order M3 of the USSR No. 755 of 12.08.77, was given ad libitum into the aft recess of the steel lattice cover of the cage (when measuring the level of glucose, animals were deprived of food for 18 hours). Veterinary certificate No. 247 No. 0294922 (LLC Aller-Petfood, Russia) certificate of conformity No. POCRU.PR98.N00093/0051289 valid from 05/17/2011 to 05/16/2014.

Animals received water purified in accordance with SOP OZH-OS-4 and normalized for organoleptic properties, in terms of pH, dry residue, reducing substances, carbon dioxide, nitrates and nitrites, ammonia, chlorides, sulfates, calcium and heavy metals in accordance with SOP AB -38, based on GOST 51232-98 Drinking water. General requirements for the organization and methods of quality control. Water in standard drinking bowls with steel spouts was given ad libitum.

Wood pellets 6 mm (OOO ZooSPb, Russia) were used as bedding. The animals were kept under controlled environmental conditions (19.5-21°C and relative air humidity 61-75%). The light regime was 12 hours of light and 12 hours of darkness. The ventilation mode was set to provide about 15 room volumes per hour, CO ₂ concentration not more than 0.15% by volume, ammonia not more than 0.001 mg/l. Air temperature and humidity were recorded daily. There were no significant deviations of these parameters during the period of acclimatization and during the experiment.

Study design.

Characteristics of the studied groups, the route and duration of the introduction of the studied substances

Group No. Gender, number of animals Substance injected Dose Route and duration of administration 1 m 5 — — Subcutaneously 2 M 5 Distilled water 3 M 10 Basta* 4.5 µg/kg daily once a day for 80 days 4 m 10 Exenatide 45 mcg/kg 5 m 10 Peptide D 6.0 µg/kg 6 m 10 Metformin (Siofor* 500) 85.7 mg/kg Intragastrically, daily once a day for 80 days

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In accordance with the experimental schedule in animals on day 0, experimental diabetes mellitus was induced using a single intraperitoneal injection of streptozotocin (STZ) at a dose of 65 mg/kg, previously diluted in citrate buffer (pH=6.5). Nicotinamide was administered intraperitoneally to all animals 2 hours before at a dose of 230 mg/kg (5% solution) [35, 37].

The level of glucose in the peripheral blood was measured in rats on an empty stomach (the animals were deprived of food for the night) before the induction of the pathology, then 1, 3, and 9 days after the induction of the pathology. After the start of the administration of the studied substances, the glucose level was measured every 10 days and on the day of euthanasia. Registration of body weight was carried out on the same days as the measurement of glucose levels. During the experiment, the test for allodynia was performed on the 60th, 70th and 80th days of drug administration. In alcoholic neuropathy, a tactile reactivity test was performed on days 1, 50, and 100 of drug administration.

On the day of euthanasia, a venous blood sample was taken from the animals to determine the level of glycosylated hemoglobin. The following organs were also taken for pathomorphological examination: sciatic nerve, part of the mesentery artery, part of the retinal artery, part of the gastrocnemius muscle, pancreas.

Graph of experiment and manipulation

Manipulation Induction of pathology Registration of body weight Measurement of glucose levels in peripheral blood* Conducting an allodynia test __________ Administration of drugs Measurement of glycosylated hemoglobin _________ Euthanasia ___________

Note: *Measurement of glucose level was also performed on the 1st, 2nd, 3rd, 9th, 10th and 14th day after the introduction of STZ additionally.

Example 6. Determination of glucose in the blood of experimental animals.

Peripheral blood glucose was measured in rats using the One Touch® glucometer and One Touch® test strips using the glucose oxidase method. The rats were deprived of food but not water the night before the glucose level was measured. The measurement process was as follows: an animal with the desired label was removed from the cage, the tail was treated with an antiseptic, a vein was punctured with a needle from a syringe, when a drop of blood was released, a glucometer with a test strip was brought up. The obtained data were entered into the primary map.

In FIG. Table 1 shows that against the background of the use of the studied drugs, there was a general trend towards a decrease in the concentration of glucose. Repeated measures two-way analysis of variance (ANOVA) revealed a statistically significant effect of study drugs on peripheral blood glucose concentration (F4, ₂₅ =3.49, p=0.02). The subsequent intergroup comparison significantly showed the difference between the group receiving metformin, starting from the 20th day of drug administration (p<0.05, Bonferroni test), as well as the groups receiving the claimed peptide (peptide D), starting from the 40th day of drug administration (p <0.05, Bonferroni test) and exenatide, starting from the 50th day of drug administration (p<0.05, Bonferroni test), compared with the control group.

Pairwise comparison between pre-treatment baseline glucose (0-point) and the last day of therapy (day 80) showed a statistically significant difference between metformin (p=0.017, t-test) and peptide D (p=0.011, t-test) groups. -test) (Fig. 1).

As a result, it can be concluded that the claimed peptide D had a hypoglycemic effect during therapy lasting 80 days, starting from the 40th day of administration. The incretin-like drug exenatide also showed a hypoglycemic effect, starting from the 50th day of administration, although its severity was lower than that of peptide D. The administration of Byetta® had no effect on the level of glucose in the peripheral blood, but the reference drug metformin showed, as expected , hypoglycemic properties, starting from the 20th day of therapy.

Example 7. Determination of glycosylated hemoglobin in peripheral blood.

Determination of glycosylated hemoglobin in venous blood was carried out using the Diabetes-Test, HbA _1C reagent kit (000 Phosphosorb, Russia), by affinity chromatography of glycosylated and non-glycosylated hemoglobin fractions in rat blood hemolysate.

On the 81st day of daily administration of the study drugs, blood samples were taken from the tail vein of experimental animals to determine glycosylated hemoglobin in the hemolysate of erythrocytes of the blood of animals. The data obtained are given in table. 1.

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Table 1. The content of glycosylated hemoglobin in the blood of animals, %, M±m

Group η Glycosylated gsmoglobin.% Intact group 5 4.2±0.59 Control group 4 15.6± 1.63* Basta* 4 8.1 ± 1.16' Exenatide 4 7.9 ±2.03' Peptide D 4 53 ±033' Metformin 6 5.7±005'

Note:

# - differences are statistically significant compared to the intact group, t-test for independent variables at p < 0.05, * - differences are statistically significant compared to the control group. Boferroni test at p < 0.05.

From the data in Table. 1 shows that in animals of the control group there was a statistically significant increase in the concentration of HbA _1C by 4 times compared with the intact group (p=0.0002, t-test). These changes characterize the pronounced development of experimental pathology.

Against the background of the use of the study drugs, a statistically significant decrease in the concentration of HbA _1C was found (F ₄ , ₁₇ =11.83, p<0.0001). The subsequent intergroup comparison revealed the maximum effect of therapy in the groups treated with the claimed peptide D (p<0.0001) and metformin (p<0.0001), in the groups treated with Byetta® and exenatide, the effect was not so pronounced (p=0 .0018 and p=0.0015, respectively).

Example 8 Assessment of tactile allodynia in rats with diabetic neuropathy.

The rat was placed in a plastic cage with a slatted floor made of metal wire and left in this cage for 5 min for the extinction of orienting reactions. The average effective threshold of tactile reactivity was determined according to the method of Chaplan et al. [38] using a set of 8 standard von Frey hairs (Stoelting, USA). The stiffness of the hairs, expressed as the minimum force required to bend the hair, increased logarithmically with absolute values from 0.692 g to 28.840 g.

In each rat, the threshold was first determined on the left paw and then on the right paw. The tip of the hair was touched to the middle of the plantar surface of the paw with the force necessary to bend the hair, and the hair was held in this position for 6–8 s. A positive response was recorded if the animal abruptly withdrew its paw during touching or if the removal of the hair was followed by a sharp flexion of the paw.

Testing was started with a hair corresponding to a force of 3.630 g. Stimuli (hairs) were then presented in ascending or descending sequence. In the case of a positive response, a hair with a lower stiffness was presented, and in the case of a negative response, a hair with a higher stiffness was presented. After the initial determination of the sensitivity threshold, 4 more hairs were presented according to the same principle.

The psychophysiological average effective threshold of tactile reactivity was calculated according to the method described by Dixon [39].

In table. 2 presents the results of experiments to study the effect of 80-day administration of the study drugs on the degree of development of tactile allodynia.

In the performed experiments, by the 60th day of drug administration, the manifestation of tactile allodynia in the control group was maximum (F1, ₁₄ = 669.8, p<0.0001), which indicates a formed model of diabetic neuropathy, with a pronounced pain syndrome.

For further evaluation of the obtained results, statistical processing was carried out using a two-way analysis of variance with repeated measurements, which made it possible to determine the significance of the drug administration factor on the expression of tactile allodynia (F ₄ , ₄₃ =10.93; p<0.0001). Subsequent intergroup comparison (Bonferroni test) showed a decrease in the severity of tactile allodynia in the group treated with peptide D, starting from the 60th day of administration. Table 3 presents the values of the criterion F for all substances used. Subsequent intergroup comparisons (Bonferroni test) confirmed a significant effect only in the group that received the claimed peptide D.

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Table 2. Effect of study drugs on tactile allodipy in rats. Tactile allodypia was assessed prior to administration of study drugs. Data are presented as mean paw withdrawal threshold (r) (M±w).

Group Baseline 60-day administration of drugs 70 days of drug administration 80-dsp drug administration Intact group 28.8210.00 27.7410.80 26.9411.88 27.7410.80 Control group 28.8210.00 2.7710.71* 3.1010.78* 3.46Yu.61 Byeta* 28.8210.00 5.7211.61 3.411035 63911.46 Exenatide 28.1410.69 5.3311.17 5.7211.18 8.2412.73 Peptide D 28.8210.00 16.1514.18* 13.911339* 15.0513.67* Metformin 28.8210.00 4.9410.90 4.9810.87 4.2810.45

Note:

# - statistically significant differences compared to the intact group, 1-test for independent variables at p<0.05, * - statistically significant differences compared to the control group, Bonferroni - test at p<0.05.

Table 3. Results of statistical processing of data on the expression of tactile allodynia in the background

Thus, when studying the effects of the studied drugs, the following was shown: Byeta®, exenatide and metformin, when compared individually, slightly reduced the severity of the manifestation of allodynia, however, subsequent intergroup comparison did not confirm a statistically significant effect. The introduction of the claimed peptide D, starting from the 60th day of administration, significantly reduced the manifestations of tactile allodynia in rats with diabetic neuropathy (Tables 2-3). This observation allows us to conclude that long-term treatment (starting from the 60th day of administration) with the claimed peptide D has a therapeutic effect on one of the most common complications of type 2 diabetes, peripheral diabetic neuropathy, which is probably due to the neuroprotective effect of the claimed peptide D.

Example 9 Assessment of tactile allodynia in rats with alcoholic neuropathy.

Tactile allodynia was assessed in the same way as in example 7. Symptoms of alcoholic neuropathy (tactile allodynia) were observed starting from week 8 (56-60 days) of 30% alcohol consumption by rats using the forced drinking method [40] (Fi 12= 26.25; p=0.0003; Fig. 2), which indicates an established model of alcoholic neuropathy with severe pain.

For further evaluation of the obtained results, statistical processing was carried out using a two-way analysis of variance with repeated measurements, which made it possible to determine the significance of the drug administration factor on the degree of development of tactile allodynia (Fi ₁₀ =8.79, p=0.014). Subsequent intergroup comparison (post hoc) showed a decrease in the severity of tactile allodynia in the group treated with the claimed peptide D on the 100th day of administration compared with the control group at the corresponding measurement point (p<0.05; Bonferroni test; Fig. 2).

Summarizing the above, we can conclude that the claimed peptide D has neuroprotective properties against neurodegenerative processes that develop in alcoholic and diabetic peripheral neuropathy.

Pathological study.

All experimental animals were subject to pathomorphological examination at the end of the study.

Pathological examination included necropsy, macroscopic examination, histological examination of the following internal organs: middle part of the sciatic nerve, retinal artery, part of the mesenteric artery, part of the gastrocnemius muscle, pancreas. Necropsy was performed under the direct supervision of a pathologist. Samples of these tissues were excised and fixed in a 10% neutral formalin solution, and the eyeball in Davidson's fixative for 24 h, then the material underwent standard processing for the manufacture of histological and histochemical preparations with a thickness of serial paraffin sections of 5-7 μm. For microscopic examination, sections of tissues and organs were stained with hematoxylin and eosin. Samples of nervous tissue were stained with hematoxylin and eosin, toluidine blue according to Nissl, pikrofuksin according to Van Gieson. To identify myelin fibers and myelin degradation products, transverse and longitudinal sections of nerves obtained on a freezing microtome were stained with Sudan black according to Lyson [41, 42,

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43]. Nuclei were counterstained with Mayer's hematoxylin. Gastrocnemius muscle samples were stained with hematoxylin and eosin and picrofuchsin according to Van Gieson.

Morphological examination of histological preparations was carried out using a light-optical microscope Carl Zeiss (Germany) at a magnification of 100, 200 and 400. Microphotography was performed using an Axio Scope A1 digital camera (Germany). Morphometry was performed semi-automatically and manually using the Axio Vision Rel software. 4.8. and the PhotoM 1.21 image editor.

To assess the effect of the studied substances, a comparative histological assessment of their influence was carried out:

1. On the islet apparatus of the pancreas, where the area of the islets was measured.

2. On the arteries of the muscular type and the microvasculature (arterioles and capillaries) of the retina, the arteries of the mesentery, where a morphometric measurement of the diameter of the capillaries and the thickness of the intima of the arteries was carried out.

3. On the condition of the sciatic nerve.

4. On the state of the striated muscles.

Example 10. Assessment of the state of the intima of the mesenteric artery of rats.

In FIG. 3 shows data on the effect of the studied substances on the state of the intima of the mesenteric artery of rats. Pairwise comparison of the intact and control groups showed that there was a significant increase in the thickness of the intima of the vessels in the control group (p<0.0001, t-test). This increase in the thickness and swelling of the intima of the vessels testifies in favor of the development of endotheliopathy processes. Subsequent one-way analysis of variance (ANOVA) showed that long-term administration of the test substances prevented an increase in the thickness of the mesenteric artery intima in rats (F ₄ , ₂₅ =12.87; p<0.0001). Intergroup comparison showed a significant decrease in the thickness of the vascular intima in all groups compared with the control group (p<0.05, Bonferroni test). The maximum effect was in the group receiving the claimed peptide D (p<0.0001, Bonferroni test).

Example 11. Assessment of the state of the nervous tissue of the sciatic nerve.

Based on the established pathomorphological changes in the nervous tissue, it can be concluded that the modeling of STZ-induced type 2 diabetes mellitus was accompanied by a pronounced picture of diabetic neuropathy, which was characterized by a decrease in the thickness and diameter of the nerve trunks, deformation and swelling of the myelin sheaths, damaged nerve fibers, swelling of the interstitial tissue, excessive accumulation of lipids in the epi- and perineurium, proliferation of endoneural connective tissue.

Individual fibers were in a state of hydropic dystrophy. Also, in the foci of demyelination, there were accumulations of small sudanophilic granules, indicating the ongoing breakdown of myelin.

The introduction of metformin had no therapeutic effect on the nervous tissue. In tissue samples of this group, gross damage to the nerve trunks and fibers was found, manifested by severe polymorphonuclear infiltration of the perineurium and epineurium with focal degeneration of the myelin sheaths, as well as swelling and deformation of the fibers, the appearance of myelin decay products in the form of sudanophilic granules adjacent to the basement membrane of intact myelin fibers.

With the introduction of Byetta® and exenatide, only a slight accumulation of lipids in the perineurium and endoneurium was observed. Obviously, degenerative phenomena in this group were significantly lower or did not appear at all. On the contrary, the introduction of the claimed peptide D had a positive therapeutic effect, while there were no pronounced morphological differences in the structure of the sciatic nerves from the group of intact animals.

Pathological analysis of the nervous tissue indicators showed that the thickness of the nerve trunk significantly decreased in the control group compared to intact animals (p<0.0001; t-test), which may indicate degenerative changes in the tissue and is confirmed by the pathomorphological picture of the pathology development ( Table 4).

Table 4. Morphometric parameters of the nervous tissue under the influence of the test substances

Groups Indicators Nerve trunk thickness, µm The area of the connective tissue of zndonsvria, micron ² Intact 3403*8.7 25963*74.1 Control 2033*6.9* 39623*314.6' Byetta 2133*37.4 2111.7*239.9* Exenatide 256.8*113 2284.4*2113* PeptideD 303.6*20.3* 1956.1*273.4** Metformin 382*23.4 28263*298.9*

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Note:

# - difference from the intact group (p<0.05);

* - difference from the control group (p<0.05);

* * - difference from the control group (p<0.01).

As previously noted, the pathomorphological picture of the nervous tissue samples in the group treated with metformin indicates swelling of the nerve trunk and an excessive increase in its thickness, which indicates an increase in the pathology, and not a therapeutic effect. In this regard, a statistical analysis was carried out without including the above group. One-way analysis of variance (ANOVA) revealed a significant effect of the administration of the test substances on the thickness of the nerve trunk (F3.34=4.83; p=0.007). Subsequent intergroup comparison (post hoc) confirmed a significant increase in the thickness of the nerve trunk in the group treated with the claimed peptide D (p<0.01; Bonferroni test), compared with the control group. When analyzing such a pathomorphometric indicator as the area of the endoneurial connective tissue, it was found that when modeling the pathology, there was a significant proliferation of the endoneural connective tissue (p<0.0018; t-test), which may indicate the predominance of degenerative manifestations over reparative ones [Karpova and Krishtop, 2013]. One-way analysis of variance (ANOVA) showed a significant effect of the introduction of the test substances on the area of endoneural connective tissue (F ₄ , ₂₅ =8.58; p=0.0002). Subsequent intergroup comparison (post hoc) confirmed a significant decrease in the area of endoneural, connective tissue in all groups (p<0.01; Bonferroni test). The maximum effect was recorded in groups taking the claimed peptide D (p=0.0002; Bonferroni test) (Table 4), which can be interpreted as an increase in reparative processes in the nervous tissue.

Example 12. Assessment of the state of muscle tissue (calf muscle).

Based on the established pathomorphological changes in the striated muscle tissue of the gastrocnemius muscles, it can be concluded that the development of experimental DM 2 was accompanied by a pronounced picture of muscular dystrophy, which was characterized by thinning and deformation of muscle fibers, moderate growth of the surrounding connective tissue, in some cases with the replacement of atrophied collagen fibers. connective tissue, as well as an increase in the number of muscle cell nuclei (myonuclei). In the connective tissue of the endo- and perimysium in animals with fiber atrophy, a moderate inflammatory infiltration was found.

In the group treated with Byetta® and exenatide, there was a focal proliferation of connective tissue with replacement of damaged muscle fibers and moderate lymphocytic infiltration. In the group where the animals received metformin, against the background of a sharp increase in the myonuclear ratio, there was a minimal focal inflammatory infiltration of the fibers and interstitial tissue.

Against the background of the formed pathology, the introduction of the claimed peptide D had a pronounced therapeutic effect on muscle tissue. Pathological changes occurred in these groups in isolated cases and were minimal compared to the control group.

When analyzing the cross-sectional area of muscle fibers, it was found that the development of experimental type 2 DM is accompanied by a significant decrease in the cross-sectional area of muscle fibers (p<0.0001; t-test) with a parallel decrease in the number of myonuclei in the fibers (about 70%), which can be interpreted as signs of muscular dystrophy. One-way analysis of variance (ANOVA) showed a significant effect of the introduction of the test substances on the cross-sectional area of muscle fibers (F ₄ , ₂₂ =13.67; p<0.0001). Subsequent intergroup comparison (post hoc) confirmed a significant increase in the cross-sectional area of muscle fibers in all groups (p<0.01; Bonferroni test), with the exception of the Byetta® group (p=0.051; Bonferroni test). The maximum effect was recorded in the groups taking exenatide (p<0.0001; Bonferroni test) and the claimed peptide D (p=0.0004; Bonferroni test) (Table 5).

Table 5. Cross-sectional area of muscle fibers of the gastrocnemius muscle under the influence of the studied substances (M±m)

Groups Cross-sectional area of fibers, µm ² Intact 1450*120 Control 551.1*45* Basta* 9683*115 Exenatide 1763*142 Peptide D 1472*282 Metformin 1286*157

Note:

# - difference from the intact group (p<0.05);

* - difference from the control group (p<0.05);

** - difference from the control group (p<0.0001).

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Example 13 Pancreatic islet apparatus.

In table. 6 presents generalized data on the effect of the studied substances on the islet apparatus of the pancreas of experimental animals. When comparing the intact and control groups in pairs, it was shown that there was a significant decrease in the islets of Langerhans in the control group (p<0.0001, t-test). This decrease in the area of the islet apparatus indicates the development of pathology, which also correlates with an increase in the level of glucose in the peripheral blood. Pairwise comparison of the control group with the experimental group revealed a statistically significant effect of the introduction of all the studied substances on the area of the pancreatic islets (Table 7). This observation indicates that prolonged administration of the studied substances leads to an increase in the area of the islets and thereby promotes the processes of repair of the islet apparatus of the pancreas.

Table 6. Morphometric parameters of the state of the pancreatic islet apparatus after 80 days of administration of the study drugs

Note:

# - difference from the regular group (p<0.05);

* - difference from the control group (p<0.05, t-test).

Table 7. Results of statistical processing of data on the area of pancreatic islets after repeated administration of the study drugs, (Student's t-test)

Index R value Control rO.OOOG Byeta p-0.019* Exenatide p-0.04* PeptideD p-0.011* Metformin p-0.001 *

Note:

# - compared with the group of intact rats (p<0.001);

* - compared with the control group of rats (p<0.05).

Thus, the pathomorphological analysis of organs and tissues revealed that the modeling of streptozatocin-induced type 2 diabetes mellitus was accompanied by endotheliopathy, a decrease in the area of the pancreatic islet apparatus, as well as a pronounced picture of the development of peripheral diabetic neuropathy and degenerative changes in the striated muscles of the leg.

The maximum and pronounced therapeutic effect was observed after 80 days of administration of the claimed peptide D. The administration of this compound had a positive therapeutic effect on complications of type 2 diabetes mellitus, such as diabetic neuropathy, muscular dystrophy, endotheliopathy, and had a positive effect on reparative processes in the islet apparatus. pancreas. Analysis of histological samples correlate with clinical findings. In particular, a pronounced therapeutic effect was noted in the manifestation of signs of allodynia and a decrease in the content of glycosylated hemoglobin in the erythrocytes of experimental animals. A pronounced hypoglycemic effect of this compound was also noted, which was comparable to that of metformin.

Considering the foregoing, it can be concluded that the claimed peptide D is a highly effective agent for the treatment and prevention of complications of type 2 diabetes mellitus, in particular diabetic neuropathy, and also has a neuroprotective effect against alcoholic neuropathy.

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Claims (2)

CLAIM 1. Polypeptide of formula: H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Gly-Glu-Glu-GluAla-Val-Arg-Leu-Phe-Ile -Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-D-Arg-D-ArgD-Arg-D-Arg-D-Arg -D-Arg-D-Arg-D-Arg-Gly-OH, effective in the treatment and prevention of type 2 diabetes mellitus and its complications, selected from diabetic neuropathy, muscular dystrophy and endotheliopathy. 2. The use of a polypeptide according to claim 1 for the treatment and prevention of type 2 diabetes mellitus and its complications, selected from diabetic neuropathy, muscular dystrophy and endotheliopathy. Fig. 1 - concentration of glucose in peripheral blood under the influence of the studied preparations, M±m. mmol/l. The first column is the point before the start of drug administration, the second column is the last day of therapy (80th day). * - statistically significant differences in the groups that received the study drugs and the control group, Bonferroni test, at p < 0.05: # - statistically significant differences in the groups, between the initial level and the last day of therapy (80th day of drug administration), t- test, at p<0.05 Time (days) Fig. 2 - Effect of peptide D at a dose of 10 μg/kg on tactile allodymia in rats with alcoholic neuropathy. For 100 days, the animals were injected with peptide D according to the study plan. Tactile allodynia was assessed before modeling alcoholic neuropathy, on the 1st day of drug administration, on the 50th and 100th day of daily administration. Data are presented as mean paw withdrawal threshold (g) (M±m) - 19 040166 Fig. 3 - Thickness of the intima of the mesenteric artery after 80 days of administration of the studied preparations, μm, # - difference from the intact group (p<0.05); * - difference from the control group (p<0.05, Bonferroni test); ** - difference from the control group (p<0.001. Bonferroni test)