EA021236B1 - N-acyl derivatives of aminoacyl-2-cyanopyrrolidine - inhibitors of prolyl endopeptidase and dipeptidyl peptidase-iv, having hypoglycemic, antihypoxic, neuroprotective action and action of cognitive function improvement - Google Patents

Abstract

The subject of the present invention is Nα-acyl derivative of aminoacyl-2-cyanopyrrolidine of formula- inhibitor of prolyl endopeptidase and dipeptidyl peptidase-IV, having hypoglycemic, antihypoxic, neuroprotective action and action of cognitive function improvement.

Description

The invention relates to the field of biologically active compounds for the treatment of type 2 diabetes mellitus, as well as diseases associated with neurodegeneration and cognitive impairment.

BACKGROUND OF THE INVENTION

Diabetes is one of the most pressing problems of medicine. According to official figures, 3 million people in Russia suffer from diabetes, of which more than 2.7 million are patients with type 2 diabetes, however, according to epidemiological studies, the real number of patients with type 2 diabetes in Russia is 3 -4 times higher; according to the Ministry of Health and Social Development, mortality from diabetes is 6.7 cases per 100,000 population (Ametov A.S. Type 2 diabetes mellitus. Problems and solutions, published by GEOTAR - Media, 2011, p. 704; Dedov I.I., Shestakova M.V. Diabetes mellitus, M., 2003; Shestakova M.V., Dedov I.I. Diabetes mellitus and chronic kidney disease, ed. Medical Information Agency, 2009).

Type 2 diabetes mellitus (insulin-independent diabetes) is a metabolic disease characterized by chronic hyperglycemia that develops as a result of impaired insulin secretion or the mechanisms of its interaction with tissue cells (A. Ametov, Type 2 diabetes mellitus. Fundamentals of pathogenesis and therapy; M. , 2003). This type of disease is due to a decrease in the sensitivity of tissues to the action of insulin (insulin resistance), which is synthesized in normal or even increased amounts in the initial stages of the disease. The diet and weight loss of the patient in some cases help to normalize carbohydrate metabolism and reduce glucose synthesis in the liver from non-carbohydrate (non-carbohydrate) raw materials. Over time, excessive secretion of insulin depletes the β-cells of the pancreas, which makes it necessary to inject insulin (Balabolkin M.I., Klebanova E.M., Kreminskaya V.M. Treatment of diabetes mellitus and its complications. Manual for doctors. - M .: Medicine, 2005, p. 18-21).

Type 2 diabetes accounts for 85-90% of all types of diabetes mellitus and most often develops in people over 40 years old and is usually associated with obesity. The disease progresses slowly. Secondary symptoms are characteristic of it, ketoacidosis rarely develops. Over time, complications develop: micro- and macroangiopathy, nephro- and neuropathy, retinopathy, etc. (Ostapova

V.V. Diabetes. M., 1994; Dedov I.I., Suntsov Yu.I., Kudryakova S.V. The epidemiology of diabetes. Diabetes. A guide for doctors. Universum Publishing. M., 2003, p. 75-93).

Available methods for treating type 2 diabetes have not changed significantly over the years, but have revealed a number of limitations. Although physical exercise and lowering calories in your diet can effectively improve your diabetic condition, adherence to this treatment is very difficult due to your well-established sedentary lifestyle and excess food intake. Increasing plasma insulin levels by administering insulin secretion stimulants - sulfonylureas (tolbutamide, glipizide) or meglitinide, which stimulate pancreatic cells to secrete more insulin, and / or by injecting insulin, can lead to higher insulin concentrations, especially insulin, to stimulate, especially insulin -resistant tissues. Biguanides increase insulin sensitivity, leading to some correction of hyperglycemia, but phenformin and metformin (biguanides) can induce lactic acidosis and nausea / diarrhea. Glitazones (derivatives of 5-benzylthiazolidine-2,4-dione) are a recently described class of compounds with the potential to improve many symptoms of type 2 diabetes. Glitazones increase insulin sensitivity in muscles, liver and adipose tissue and lead to partial or complete correction of elevated plasma glucose levels without hypoglycemia. However, some glitazones (troglitazone) cause serious side effects (e.g. hepatotoxicity).

In recent years, the role of gastrointestinal hormones in the regulation of insulin secretion, and therefore in the regulation of glucose homeostasis in the human body, has been shown. Of these, the most significant role is played by the glucagon-like peptide 1 (OLP-1, HIP-1) and the gastrointestinal intestinal peptide (glucose-dependent insulinotropic polypeptide, HIP, O1P), which are called incretins - gastrointestinal hormones produced in response to food and causing stimulation of insulin secretion (Ogkkou S., 1crp5sp 1., Mabkab 8., Knock! 1.1. Rhod1isadop rhobiscus ίη p1a§a £ пош п ки Н п-бере бере 61 61 аг 8 8 аг 8 8 81 81 81 81 81 81 81. 1 C1T 1pus51: 1991, 87, 415-423; Ametov A.S. Regu The secretion of insulin secretion in normal and type 2 diabetes mellitus: the role of incretins. Russian Medical Journal. 12/14/2006, v. 14, No. 26).

Since the half-life of GLP-1 is very short, it cannot be used in clinical practice as a natural preparation. In this regard, inhibition of the enzyme dipeptidyl peptidase-4 (DPP-ΐν), which destroys incretins in the body, seems to be a promising direction. The main function of the DPP-ΐν enzyme is to quickly inactivate oligopeptides in different tissues and organs by cleaving two amino acids from the Ν-terminal part. The target peptides for DLPIV are located in the intestine - HIP-1 and HIP and it has been shown that the enzyme DPP-ΐν quickly inactivates HIP-1 and HIP immediately after their secretion in the intestine. However, with inhibition of DPP-ΐν, the level and activity of incretins increases, and their effect is prolonged. Inhibition of DPP-ΐν significantly increases the level of active incretins in the blood as well. As a result, the imbalance in the ratio of insulin / glucagon is normalized: glucose-dependent insulin secretion by β-cells increases, and the secretion of glucagon by α-cells is suppressed. This allows you to effectively control glucose levels against the background of a significant reduction in the risk of hypoglycemia, especially severe episodes, due to glucose-sensitive action. Improving islet cell function leads to improved glycemic control. Since incretins are only produced by eating, inhibition of DNI-GU does not increase insulin levels between meals, which could lead to excessively low blood sugar (hypoglycemia). Therefore, inhibition of DPP-1U increases insulin without increasing the risk of hypoglycemia, which is a dangerous side effect associated with the use of insulin secretion stimulants (Antsiferov MB, Dorofeeva LG, New approaches in the treatment of type 2 diabetes mellitus: glucagon-like peptide-1 and exenatide (Bayeta. Pharmateka. No. 11 (145) 2007, pp. 14-19).

Thus, DPP-1U inhibitors are a new class of sugar-lowering drugs, the main effect of which is aimed at increasing the activity of the body's own incretins, which provides a better advantage / risk ratio compared to other classes of drugs for the treatment of type 2 diabetes mellitus. The data of numerous international studies have confirmed that drugs based on DPP-ΐν inhibitors increase the level of endogenous insulin, reduce the level of glucagon in the blood plasma and suppress gluconeogenesis in the liver, reduce insulin resistance, providing complete glycemic control. The most famous drugs of this class are sitagliptin (Januvia) and vildagliptin (G alvus) - drugs that have achieved tremendous commercial success in the pharmaceutical market (Aksbet R., Ktriek M.8., BipssGog! TK., E! A1. EGGes! OG b1rerebyb1 ReBa8e-4 ίηΗϋίΐοτ kyadiri ak topoShetaru op Dusetu soi1to1 ίη raeSchk \ νί11ι! ur 2 b1ae1e8. H1ae1e8 Sage: 2006, 29, 2632-2637; ίίббададад оп оп оп оп Д исокисок соп соп соп соп соп1111111 о 24 24 24 24 24 24 \екеккί рай рай рай рай рай рай рай рай рай рай 2 2 2 2 2 2 2 Ф Ф Ф Ф Ф Ф 2 Ф 2 2 2 2 2 2 2 2 Готототототототототототототототот Г Готототототот Г Г Гототототототототот Гототототототототтот. 2007ототототт.. 2007... 2007 2007 200790 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007ов 2007 2007 2007ов 2007ов 2007 2007 2007 2007 2007 2007 2007 200790 2007 2007 2007ов 2007 2007ов 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007овов 2007ов 2007 2007 2007 2007ов 2007 2007 2007ов 2007 2007ов 2007ов 2007 200790 2007 2007 2007овов 2007 2007 2007ов 2007 2007 2007 2007овов 2007 2007 200790ов 2007 2007 2007 2007 2007 2007 2007 2007 2007 2007ов ., Shestakova MV Algorithms specialized medical care to patients with diabetes mellitus. M., 2007).

The search for new drugs for the treatment of type 2 diabetes among DPP-1U inhibitors is still ongoing. In particular, mention may be made of hexahydrodiazepinone derivatives according to KI 2301803, Y-substituted-2-cyanopyrrolidines according to KI 2180901, ΌΡΡ-ΐν inhibitors for the treatment of neurodegeneration and cognitive impairment according to KI 2394570, as well as the treatment of type 2 diabetes 2328283.

The problem solved by this group of inventions is to obtain a new approach to the creation of drugs for the treatment of type 2 diabetes mellitus, specifically to compounds from the Ν '' group - acyl derivatives of aminoacyl-2-cyanopyrrolidine, in which mono and dicarboxylic acids can be used as substituents - preferably glutaral, maleic and amber. The result obtained by solving this problem is to obtain a substance that increases the activity of inhibiting dipeptidyl peptidase-ΐν, as well as having a more pronounced antiglycemic effect.

The claimed group of substances combines the properties of PEP inhibitors and prodrugs - an inhibitor of DDPΐν (one of the key enzymes of the pathogenesis of type 2 diabetes), formed during metabolic transformations, and has a hypoglycemic, antihypoxic, neuroprotective and cognitive improving effect.

To achieve the set result, предлагается '' is proposed - an acyl derivative of aminoacyl-2-

The compound may have, in particular, an inhibitor of prolyl endopeptidase and dipeptidyl peptidase-ΐν action, or a hypoglycemic, neuroprotective, antihypoxic effect and effects that improve cognitive functions.

To achieve the result, it is also proposed to use the compounds of formula (1) as agents for the treatment of type 2 diabetes mellitus, or as agents for the treatment of diseases accompanied by hypoxia, neurodegeneration and cognitive impairment.

In addition, a dosage form comprising a compound of formula (1) as an active substance is provided.

Ν '' - acyl derivatives of aminoacyl-2-cyanopyrrolidine, their properties and the method of preparation are not described in the literature.

Ν '' - acyl derivatives of aminoacyl-2-cyanopyrrolidine can be introduced into the body by any

- 2 021236 in a suitable manner, including oral, buccal, sublingual, rectal, intravaginal and transdermal administration, as well as intravenous, subcutaneous and intramuscular injection. Oral administration in the form of tablets or capsules is preferred. The dose can be set by the attending physician, taking into account all significant factors. Typically, a single dose may include from 1 to 500 mg, preferably from 5 to 150 mg.

The invention is illustrated in the drawing, which shows the effect of various concentrations of the compound ΑΙοΡίάΝ and sitagliptin on the activity of purified dipeptidyl peptidase IV.

Synthesis of Compounds

In the synthesis, optically active L-series amino acids were used. The composition of the reaction mixtures and the individuality of the obtained compounds were controlled by TLC on glass plates with silica gel (1 <1C5s1ds1 60P254, Megsk). Substances were detected by staining with ninhydrin (5% solution in n-butyl alcohol, 110 ° C) and benzidine reagent after chlorination.

Optical rotation [a] _{s was} determined on a Retkt-E1tet 241 polarimeter in 10 cm cuvettes at room temperature (18 ° C). IR spectra were recorded on a Regksh E1teg spectrophotometer (England). Melting points were determined on a Boethius instrument.

Accepted abbreviations (in addition to standard ones): Boc ₂ O - di-tert-butylpyrocarbonate; RTB - 8pyrrolidin-2; IMR - dimethylformamide; RGr - pentafluorophenyl.

Steps for the synthesis of alanyl-2-cyano-pyrrolidine hydrochloride (A1a-Rgb-C'K compound I) and Ν-3 carboxypropionyl-alanyl-2-cyano-pyrrolidine (HOCOS ₂ CH ₂ CO-A1a-Rtb-C ^ compound II)

1) Vos-A1a-Rgo-OH.

2) Βο ^ Α ^^ το-Ν ^.

3) Vos-A1a-RTb-SK

4) NS1 A1a-RTb-SK

5) NOSOSN2SN2SO-A1a-RTb-SK

The preparation method is based on the interaction of the activated ester of Ν-protected alanine with proline salts, followed by isolation of the Ν-protected dipeptide and its conversion into an amide, which was then converted into Ν-protected nitrile, the protective group was removed and the target product was isolated in the form of hydrochloride.

experimental part

1. Vos-A1a-Rgo-OH

A suspension of 1.3 g (11 mmol) of proline, 1.6 g (11 mmol) of K ₂ CO ₃ and 0.1 g of tetrabutylammonium hydroxide in 10 ml of dioxane was stirred for 15 minutes, 2.9 g (10 mmol) of Ν-oxysuccinimide tert-butyloxycarbonyl-L-alanine ester and stirred for 10 hours. Diluted with water (25 ml), stirred for 1 hour, extracted with a mixture of hexane and ether (1: 1, 15 ml), the aqueous solution was acidified with 1 M hydrochloric acid to pH 2-3, extracted ethyl acetate (50 + 20 ml), the extract is washed with brine, dried with magnesium sulfate and evaporated. Obtain 2.3 g (80%) of chromatographically pure tert-butyloxycarbonylalanylproline with so pl. 155-156 ° C, Kg 0.47 (toluene-acetone-acetic acid 100: 50: 1), which, without further purification, is used to obtain the amide in the next stage.

2. Vos-A1a-Rto-MN ₂

A suspension of 2.2 g (7.7 mmol) of Ν-tert-butyloxycarbonylalanylproline, 1 g (13 mmol) of ammonium bicarbonate, 0.3 ml of pyridine and 2 ml (9 mmol) of di-tert-butylpyrocarbonate in 10 ml of acetonitrile was stirred for 16 hours , diluted with water (30 ml), extracted with a mixture of ether with hexane (1: 1, 15 ml) and then with a mixture of chloroform with n-propanol (9: 1; 50 + 20 ml). The extracts are combined, washed with brine and dried. The solvent was evaporated, the residue was triturated with ether and hexane to give 2.0 g (73%) of tert-butyloxycarbonylalanylproline amide as a white crystalline powder, mp. 112113 ° C, [α] _ο - 10.5 (s 1, ethanol). K _g 0.35 (B). Found%: C 54.3; H 7.6; N, 14.5. C ₁₃ H ₂₃ No. O ₄ . Calculated%: C 54.73; H, 8.13; N, 14.72.

3. Vos-A1a-Rgb-S '%

To a solution of Boc-A1a-Rto-MH ₂ (2.0 g, 7.0 mmol) in 10 ml of pyridine cooled to 5 ° C, benzenesulfonyl chloride (2.5 ml, 19 mmol) was added portionwise, stirred at room temperature for 16 hours, and evaporated in vacuo. The residue was dissolved in ethyl acetate, washed with water, 5% sulfuric acid solution, water, brine and dried with MgSO ₄ . The solvent was evaporated, the residue was crystallized from ether to give 1.5 g (80%) of the expected product as a white crystalline powder, mp. 78-79 ° C, | α | [; - 163 (c 0.5, methanol), K _g 0.25 (heptane-acetone 1: 1); IR (KC1): 2226 cm ^-1 C ^ group. Found%: C 58.2; H 8.1; N, 15.4. C ₁₃ H ₂₁ No. O ₃ . Calculated%: C 58.41; H 7.93; N, 15.71.

4. H-A1a-Rgb-C '^ HC1 (I)

5 ml of trifluoroacetic acid are added to a solution of Boc-Ala-Prb-C '% (1.3 g, 5 mmol) in 5 ml of dichloromethane and kept at room temperature until the stain of the starting product disappears on TLC (about 1 h). The solution was evaporated in vacuo until the trifluoroacetic acid was distilled off, 10 ml of a 1 M solution of HC1 in an organic solvent (in dioxane, in alcohol or in acetic acid) were added to the residue and evaporated to dryness again. The residue is crystallized by trituration in ether. The precipitate is filtered off, washed with ether and dried in vacuo. Obtain 0.7 g of chromatographically homogeneous, white crystalline substance with so pl. 162-164 ° C, K _£ 0.42 (chloroform-methanol, 4: 1, [a] _s - 142.1 (s 1, ethanol). Found%: C 47.3; H 7.1; N 20 5. C ₈ H ₁₃ MsOH 1. Calculated%: C 47.18; H 6.93; N 20.63.

5. Nososchschschsso-AP-Rgb ^

A solution of 0.22 g (2.2 mmol) of succinic anhydride in 3 ml of dichloromethane is added to a solution of H-A1a-Rgb-C.'X HC1 (0.41 g, 2 mmol) in 3 ml of dichloromethane, 0.25 ml is added triethylamine and the mixture was incubated at room temperature for 3-5 hours (TLC control). The reaction mixture was washed with 5% citric acid solution, water, brine, dried with magnesium sulfate and evaporated. In the residue receive the target product in the form of a resin. The residue was triturated with ether, the ether was drained, and the remaining resin was crystallized from a mixture of ethyl acetate and hexane. Obtain 0.4 g of 75% of the target product in the form of a white crystalline powder with so pl. 90-92 ° С, К _£ 0.27 (chloroform-methanol, 9: 1), [а] _с - (103) (с 1, ethanol). Found%: C 53.67; H, 6.61; N, 15.55. C1 ₂ H ₁₇ ^ O ₄ . Calculated%: C 53.92; H, 6.41; N, 15.72.

Prolyl endopeptidase inhibitory activity of ^ 3-carboxypropionyl-alanyl-2-cyanopyrrolidine and dipeptidyl peptidase-ΐν inhibitory activity of A1a-Rgb-CH hydrochloride (compound I), its antidiabetic, antiglycemic, neuroprotective, cognitive and cognitive-enhancing effects are new in the following examples.

Example 1. The effect of the compounds §is-A1a-Rgb-C ^ A1a-Rgb-CX and sitagliptin on the activity of dipeptidyl peptidase-ΐν (DPP-ΐν) and related DPP-ΐν enzymes from untreated blood plasma of patients with type 2 diabetes, healthy volunteers and experimental animals.

Materials and research methods

The substances of the compounds were hydrochloride A1a-Rgb-C.'X and ^ 3-carboxypropionyl-alanyl-2cyanopyrrolidine. The commercial preparation sitagliptin (YANUVIA® from Megsk) was selected as a reference sample. The active principle of sitagliptin from the dosage form was obtained by extraction with water in an ultrasonic disintegrator UZDGN-01, followed by purification by liquid chromatography with reversed phase on a LIP-2 column (C18, Lyeoteich). The resulting preparation was homogeneous by thin-layer chromatography on silica gel plates (K1ke1de1 υν254, Megsk) when manifested in iodine vapor. The solution was dried in a stream of nitrogen gas and used to study the effect on the activity of dipeptidyl peptidases ΐν and II, prolyl endopeptidase and prolidase.

Blood was collected from the ulnar vein of healthy volunteers and patients with type 2 diabetes, as well as from white outbred adult male rats, using trisubstituted sodium citrate as an anticoagulant 0.109 M. Plasma was obtained by centrifuging whole blood in the cold for 30 min at 2500 / d in a 16-B centrifuge (Vesktai-Soibeg, USA). Plasma was carefully separated from the settled cells and used to purify DPP-ΐν. Prior to isolation, plasma was stored at -80 ° C.

Getting enzyme preparations

All work on obtaining enzyme preparations was carried out at 4 ° C.

Obtaining dipeptidyl peptidase-ΐν. Blood plasma was diluted 5 times with 0.02 M Tris-HC1 buffer with a pH of 7.5, containing 0.1 mm Dithiothreitol and 1.0 mm EDTA. To the diluted plasma, crystalline (XN ₄ ) ₂ §O _{4 was} added with stirring to 70% saturation (560 g / L). The precipitate was collected by centrifugation, the precipitate was dissolved in 0.05 M Ш-phosphate buffer with a pH of 7.0, containing 1.5 M (ХН ₄ ) ₂ §О ₄ and applied to a column (2.6 / 15 cm) of phenylsepharose (Ategkcat- Ragtaaa). Proteins were eluted with a decreasing gradient (XN ₄ ) ₂ §O ₄ in phosphate buffer. Fractions containing DPP-4 activity were collected, concentrated by the addition of (ХН ₄ ) ₂ §О ₄ , and the resulting precipitate was dissolved in 0.02 M Tris-НС1 buffer with pH 7.5 containing 0.1 mM dithiothreitol and 1.0 mM EDTA and applied to a column (2.6 / 90 cm) with Sephadex 0-100 (ATEGKAT-PAGTAAA). Proteins were eluted with the same buffer at a rate of 30 ml / h. Active fractions were collected, concentrated by ultrafiltration on an i-30 membrane (ATUOP) and chromatographed on a concanavalin-A-Sepharose (ATEGKAT-Pbagtash) column. The final purification was carried out by ion-exchange chromatography on a MopoO column (Integkkat-Pbagtash). The resulting preparation DPPΐν was a dimer with a molecular weight during gel chromatography on AsA-34 220 K gel. The enzyme was homogeneous upon polyacrylamide gel electrophoresis in the presence of doctyl sulfate No. v and had a molecular weight of about 110 Kb for the monomer. The purified DPP-ΐν was concentrated by ultrafiltration and stored at -80 ° C.

Obtaining dipeptidyl peptidase-ΐΐ

The enzyme was obtained from the placenta of cattle. The separation of the activities of DPP-ΐΐ and DPP-ΐν in the process of isolation was controlled by hydrolysis of the Li5-A1a-AMS substrate at pH 5.5 (specific for DPP-ΐΐ), in contrast to the hydrolysis of 01y-Progo-AMS, which is cleaved with an optimum pH of 7, 5. To isolate DPP-ΐΐ, a modified procedure was used compared to that described for human placenta. Partial purification of DPP-2 from the homogenate was carried out by fractionation with ammonium sulfate, chromatography on β-cellulose (WaStai) and phenylsepharose (Ateccat-Pbagtash). The resulting enzyme preparation was stored at -80 ° C.

- 4 021236

Obtaining Prolyl Endopeptidase (PEP)

Prolyl endopeptidase was purified from human blood erythrocytes by ion exchange chromatography on S-Sephadex A-50 (Atet8yat-Ryatas1a), gel chromatography on Sephadex 0-75 (Ateg8yat-Ryatas1a) and high-performance chromatography on Colon MopoPyatPyatPyatPyatPyatPyat system . The obtained PEP preparation was a monomer and was homogeneous upon polyacrylamide gel electrophoresis in the presence of Na dodecyl sulfate and had a molecular weight of 75 Kb. The purified probe was concentrated by ultrafiltration and stored at -80 ° C.

Determination of proteinase activity

The unit of enzyme activity was taken to be its amount that cleaves 1 μmol of substrate in 1 min.

The activity of dipeptidyl peptidase-ΐν (EC 3.4.14.5, SE26, DPP-ΐν) was determined by hydrolysis of the O1u-Prgo-7-amino-4-methylcoumarin (Vasyet) substrate fluorometrically.

Measurement protocol

20 μl of the enzyme solution (0.5 to 5 μg of the enzyme) was sequentially mixed with 760 μl of 0.02 M Tris-HC1 pH 8.0 buffer and 20 μl of the O1y-Progo-AMS solution (1 mg / ml in ΌΜδΟ) (total ν = 800 μl), vigorously mixed and incubated at 37 ° C for 20 min. The reaction was stopped with 200 μl of 20% acetic acid. The fluorescence of 4-methyl-coumaryl-7-amide was measured at an excitation wavelength of 380 nm and a fluorescence of 460 nm.

The specific activity was determined by the formula

where E is the fluorescence of the sample (380/460 nm); C — fluorescence of a mixture containing 20 μl of substrate and enzyme, 760 μl of buffer (pH 6.0), and 200 μl of 20% acetic acid; B — fluorescence of a mixture containing 20 μl of substrate, 780 μl of buffer and 200 μl of acetic acid; δ is the fluorescence of a mixture containing 20 μl of substrate, 780 and 200 μl of acetic acid and 1 μl of a solution of 7-amino-4 methylcoumarin (1 nmol).

The activity of dipeptidyl peptidase-ΐΐ (EC 3.4.14.2, DPP-Ιΐ) was determined by hydrolysis of the Li8A1a-7-amino-4-methylcoumarin (Vasiet) substrate fluorometrically at pH 5.5 (for the hydrolysis of the O1y-RgoAMS substrate, the optimum was observed at pH 8).

Measurement protocol

20 μl of the enzyme solution (0.5 to 5 μg of the enzyme) was sequentially mixed with 760 μl of 0.1 M Na phosphate buffer pH 5.5 and 20 μl of the Li8-A1a-AMS solution (1 mg / ml in δδ) (total ν = 800 μl), vigorously mixed and incubated at 1 = 37 ° C for 20 min. The reaction was stopped with 200 μl of 20% acetic acid. The fluorescence of 4-methyl-coumaryl-7-amide was measured at an excitation wavelength of 380 nm and a fluorescence of 460 nm.

The specific activity was determined by the formula

where E is the fluorescence of the sample (380/460 nm); C - fluorescence of a mixture containing 20 μl of substrate and enzyme, 760 μl of buffer (pH 5.5) and 200 μl of 20% acetic acid; B — fluorescence of a mixture containing 20 μl of substrate, 780 μl of buffer and 200 μl of acetic acid; δ is the fluorescence of a mixture containing 20 μl of substrate, 780 μl and 200 μl of acetic acid and 1 μl of a solution of 7-amino-4 methylcoumarin (1 nmol).

Determination of dipeptidyl peptidase-ΐ (EC 3.4.14.1; cathepsin C, DPP-ΐ) was carried out in an experiment using the preparation of cathepsin C from bovine spleen (C-8511, δφιηη-ΛΗπΟι) with specific activity> 5 units / mg protein. The activity of DPP-ΐ was measured in two ways - spectrophotometrically with a substrate Shk ^ et-p-nitroanilide (Shk ^ et-pYA, Vasyet, Ne1be1betd, Oegtapu) and fluorometrically with a substrate Oyy-Agd-7-amino-4-methylcoumarin (01y-Agd -AMS, Vasyet).

The spectrophotometric method for determining the activity of DPP-ΐ consisted in the fact that from 0.25 to 10 μg of the enzyme preparation DPP-ΐ were incubated for 5 min at 37 ° C in 500 μl of activation buffer (100 mM Na ₂ NRA ₄ with pH 6.0 containing 20 mM BaCl, 1 mM EETA and 4 mM cysteine), 400 μl of substrate buffer (100 mM Ba-phosphate buffer with pH 6 containing 20 mM BaC1, 1 mM EETA, 125 mM Bq ^ et-pbA and 1% was added ΌΜδΟ). The release of p-nitroaniline was recorded for 10 min on a 50-50 spectrophotometer (Veskstap, ^ A) at 410 nm. The amount of p-nitroaniline was determined based on a molar extinction coefficient of 8800 M ^-1 l ^-1 cm ^-1 . In the fluorometric method for determining the activity of DPP-О, O1y-Agd-7-amino-4-methylcoumarin (01yAgd-AMS, Vasiet) was used as a substrate. The release of 4-methyl-coumaryl-7-amine was recorded on a Όδ-5-B spectrofluorometer (Retksh-E1tet).

Measurement protocol

20 μl of the enzyme solution (0.5 to 5 μg of the enzyme) was sequentially mixed with 760 μl of 0.1 M Na phosphate buffer pH 6.0 and 20 μl of the O ^ y-Agd-AMS solution (1 mg / ml in ^ ΜδΟ ) (total ν = 800 μl), vigorously mixed and incubated at 1 = 37 ° C for 20 min. The reaction was stopped with 200 μl of 20% acetic acid. The fluorescence of 4-methyl coumaril-7-amide was measured at an excitation wavelength of 380 nm and a fluorescence of 460 nm.

The specific activity was determined by the formula

where E is the fluorescence of the sample (380/460 nm); C — fluorescence of a mixture containing 20 μl of substrate and enzyme, 760 μl of buffer (pH 6.0) and 200 μl of 20% acetic acid; B — fluorescence of a mixture containing 20 μl of substrate, 780 μl of buffer and 200 μl of acetic acid; δ is the fluorescence of a mixture containing 20 μl of substrate, 780 and 200 μl of acetic acid and 1 μl of a solution of 7-amino-4 methylcoumarin (1 nmol).

To determine the activity of dipeptidyl peptidases 8/9, the so-called gelatinase method was used, which distinguishes it from other dipeptidyl peptidases that do not hydrolyze gelatin. An enzyme preparation was obtained from human peripheral blood leukocytes by various chromatographic procedures.

The activity of prolyl endopeptidase (EC 3.4.21.26, PEP) was determined similarly to the determination of DPPIV using a fluorogenic substrate 2-A1a-Progo-AMS, where 2 = benzyloxycarbonyl). An enzyme purified from human red blood cells was used in the experiment.

When determining prolidase (KF3.4.13.9), we used purified prolidase from pig kidneys (Bshta-AIPSk P6675). The enzyme selectively hydrolyzes dipeptides of the Haa-Rgo type (partially Haa-Nur) and does not hydrolyze the dipeptide Rgo-Rgo. The principle of enzyme determination is based on monitoring the enzyme-catalyzed cleavage of the C1u-L-Prgo substrate at 40 ° C and pH 8.0 in a spectrophotometer cuvette at a wavelength of 242 nm in a 1-cm quartz cuvette. For 1 unit of activity, the amount of the enzyme hydrolyzing 1 μmol C1u-Rgo in 1 min was taken.

The following reagents from Bshta-AIPSK were used in the determination:

A) 50 mM Tris-HCl buffer, pH 8.0 (100 ml is prepared from Tn / tbase (T-1503), 1N HCl is adjusted to pH 8.0 at 40 ° C); B) 30 mm solution of glutathione (RLS) (prepared immediately before use 5 ml in deionized water from reduced glutathione (C-4251)); C) 20 mm solution of MpC1 ₂ (10 ml of solution is prepared in deionized water (MpC1 ₂ -4H ₂ O, M-3634)); D) 25 mM C1u-L-Prgo (30 ml is prepared in solution A using C1u-L-Prgo (C-3002). The pH is adjusted to 8.0 at 40 ° C, using 1N HCl or NaOH if necessary); D) a prolidase solution (a solution is prepared immediately before use with an enzyme content of about 5 mg / ml in cold solution A).

Measurement protocol

The activated enzyme was prepared by mixing (in ml) the following solutions exactly in the order listed: solution A (buffer) - 2.40; Solution B (MpC1 ₂ ) - 0.40; solution B (RLS) - 0.10; solution D (prolidase) - 0.20. Then mix and incubate at 40 ° C for 20, 25 and 30 minutes. Immediately after the end of activation, put in a quartz cuvette of a ектаυ-50 spectrophotometer (Vektap-SoiYeg): solution G (C1uRgo, ml) in experiment = 2.70, in control = 2.70; solution B (MnCl ₂ , ml) in experiment = 0.20, in control = 0.20.

Stirred and heated to 40 ° C. Optical density is recorded at 242 nm to a stable value. Then add the activation mixture with the enzyme - 0.50 and the activation mixture without the enzyme - 0.50. Quickly mix and record a decrease in optical density of about 5 minutes

Calculations (DAg42nm / MIN Experience - (DAg42nm / MIN Control) (3.1) (3.4)

Units farm / ml = --------------------------------------------- ----- (0.0254) (0.5) (0.2) where 3.1 is the volume (in ml) of the activation mixture; 3.4 - volume (in ml) of the reaction mixture; 0.0254 is the millimolar extinction coefficient for C1u-Rgo at 242 nm; 0.5 is the volume (in ml) of the enzyme used in the determination; 0.2 - volume (in ml) of enzyme used during activation.

Determination of the degree of inhibition of (1C ₅₀ ) peptidases

In the experiment, inhibitor preparations were used at a concentration of 0.1, 1, 10, and 100 μM. For each concentration, measurements were performed in the presence of different concentrations of C1u-Progo-AMS (0.006, 0.024, 0.10, 0.39, 1.56, 1.25, 6.25, 25, and 100 pM. Experiments were performed in three parallel The parameters of inhibition were determined using the Pn5t-4 program (CarbPab BoP \\ ae 1ps., Bap O1edo, CA, IBA).

Measurement protocol

To 20 ml of an enzyme solution (about 5 mg / ml protein) or untreated blood plasma from type 2 diabetes patients and healthy volunteers, 740 μl of 0.02 M Tris-HCl with pH 8.0, 20 μl of A1aPrbA or sitagliptin solution were added and incubated with 37 ° C for 30 minutes Then, 20 μl of a solution of the substrate in ΌΜδΟ was added to the mixture and continued to incubate under the same conditions for 20 min. The reaction was stopped by adding 200 μl of a 20% solution of acetic acid. The fluorescence of 4-methylcoumaryl-7-amide released during the enzymatic reaction was measured at an excitation wavelength of 380 nm and a fluorescence of 460 nm.

Statistical analysis and a graphical representation of the results were carried out using the software package Pn / m 4 (SarbPab Boyzge, 1ps.). Significance of differences with control

6 021236 was evaluated using the Mann-Whitney test.

Research results

1. The effect of the compound ΛΙα-ΡιΠ-ΕΝ and sitagliptin on the activity of dipeptidyl peptidase-ΐν (Д11ПIV) and related DPP-ΐν enzymes from crude blood plasma of patients with type 2 diabetes and healthy volunteers.

It was found that the value of the activity of dipeptidyl peptidase-ΐν (DPP-ΐν) in the blood plasma of healthy volunteers (n = 17) was 2.17 ± 0.07 nmol / min / ml. In blood plasma from patients with type 2 diabetes (n = 9), this indicator was 3.74 ± 0.21 nmol / min / ml. These figures were taken as 100% and indicators for assessing the effect of drugs were calculated as a percentage in relation to the control, i.e. determined the residual activity of dipeptidyl peptidase-ΐν in% (table. 1).

The results of studying the effect of substances on the activity of DPP-ΐν in blood plasma samples are presented in table. one.

Concentrations of ΛΙα-ΡΓά-С HC1 and sitagliptin for plasma inhibition of dipeptidyl peptidase-ΐν (DPP-4) were 1, 10, and 100 nmol.

It was shown that the activity of DPP-ΐν in blood plasma from healthy volunteers and patients with type 2 diabetes was inhibited by Λ1α-ΡΓά-ί''Ν HC1 and sitagliptin in the concentration range from 1 to 100 nmol. The inhibitory activity of the compound Λ1αάΓά-ί''Ν HC1 significantly (P <0.05) exceeded that for the sitagliptin used as a comparison drug.

Table 1. The effect of A1a-Rgb-CH §is-L1a-Rgb-SM and sitagliptin on the activity of dipeptidyl peptidase-ΐν in the blood plasma of patients with type 2 diabetes (n = 9) and healthy volunteers (n = 17)

A drug The residual activity of dipeptidyl peptidase IV (%) The concentration of the compound nmol 0 one 10 one hundred Plasma 1 (control, healthy volunteers) 100.019.2 Plasma 1 + Sitagliptin 100.019.2 43.815.7 * 12.313.5 * 1.510.4 * Plasma 1 + A! A-Rgd-С№ 100.019.2 31.5ΐ3.9 * # S.412.5 * # 0.810.2 * # Plasma 2 (control, type 2 diabetes) 100.0 ± 5.9 Plasma 2 + Sitagliptin 100.015.9 34.616.8 * 23.816.7 * 3.111.8 * Plasma2 + A! A-Rgs! -CM 100.015.9 29.818.2 * 17.515.2 * 1.9YU.8 * # Plasma1 + Eis-A1a-RgFSM 100.019.2 99.017.3 102.0111.3 96.718.9

* - significance of differences with control at P <0.05.

# - significance of differences L1a-Prgy-HCl HC1 in comparison with sitagliptin at P <0.05.

Study of the influence of Α1; · ι-ΡΓά-ί '.' Ν and sitagliptin on the activity of related DPP-ΐν enzymes: dipeptidyl peptidase-ΐ (DPP-ΐ), dipeptidyl peptidase-ΐΐ (DPP-ΐΐ), dipeptidyl peptidase-8/9 ( 8/9), prolidase and prolyl endopeptidase (PEP) - was performed using the plasma of healthy volunteers.

It was found that A1a-RTb-CM HC1 and sitagliptin did not have inhibitory activity against dipeptidyl peptidase-ΐ (DPP-ΐ), dipeptidyl peptidase-ΐΐ (DPP-ΐΐ). dipeptidyl peptidase-8/9 (DPP-8/9), prolidase and prolyl endopeptidase (PEP) (Table 2).

Table 2. Comparative inhibitory efficacy of 5> is-A1a-Rgb-C.'K A1a-RTb-SM and sitagliptin on the activity of related DPP-ΐν enzymes (dipeptidyl peptidase-ΐ (DPP-ΐ), dipeptidyl peptidase-ΐΐ (DPP-ΐΐ) , dipeptidyl peptidases-8/9 (CI! 1-8 / 9), prolidases and prolyl endopeptidases (PEP)

Compound 1C50 (μmol / L) DPP-4 DPP8 / nine DPP-2 DPP-1 PEP Prolidase 5is-A1a-Rgs1-SY > 10,000 > 1000 0 > 10,000 > 10,000 o.ozyu.o 00 > 1,000,000 Αΐ3-ΡΓά-0Ν 0.00210.00 0 511 14001320 142891384 25001870 > 1,000,000 Sitagliptin 0.02510.00 nine 912 32001160 91501490 18001230 > 1,000,000 Compound Relative efficiency DPP-4 DPP 8/9 DPP-2 DPP-1 PEP Prolidase ΑΙθ-Ρίά-ΟΝ HC1 one 6500 1,200,000 160,000 3250000 40,000,000 5isA1a-Rgs1-SM 0 0 0 0 one 0

Thus, the obtained data showed that the compound A1a-Rgb-CM HC1 has a high inhibitory activity against dipeptidyl peptidase-ΐν purified from human plasma, both in healthy volunteers and in patients with type 2 diabetes. The well-known comparison drug for the treatment of type 2 diabetes - sitagliptin (YANUVIA®) also showed high inhibitory activity. The values of 1С ₅₀ for the inhibition of DPP-ΐν in human studies, calculated from the Cheng-Prussoff equation, were for the AI-PM-СЫ НС1 compound in the range 2.0 ± 0.3 nmol / L, and for sitag-7 021236 liptin - 25.0 ± 9.0 nmol / L, which corresponds to the data on sitagliptin, known from the literature (Όίδ ует ует о ίΑ Ь Ь Ь 1)))), and 8c1cc1n'e ορορΙίόνΙ Ι ер Р Ь Ь Ь Ь от от 8 8 8 8 8 8 2 2 Ту Ту Ту Ту Ту Ту ре Α. От Ь гу гу ап б б б η η Е. Е. Е. Е. Ь Ь ег.. Г Sitgep! Bidding Фπ MeFsta Ь ет 18 18 18 18 18, 2007, 7. S. 557-568, 557).

ΑΗ-Ρτά-ΟΝ and sitagliptin did not show inhibitory properties with respect to other proteinases - dipeptidyl peptidases I, II, 8/9, prolyl endopeptidase and prolidase.

2. The effect of δ ^ -Α ^ Ρτά-ΟΝ and ΑΗ-Ρτά-ΟΝ on the activity of dipeptidyl peptidase-Γν (DPP-Щ) and prolyl endopeptidase in the blood plasma of experimental animals.

The studies were performed on white mongrel sexually mature male rats weighing 250-280 g (n = 6) according to the methods described above. 8is-L1a-Rgb-C% was administered once, intraperitoneally at a dose of 5 mg / kg. ΑΗ-Ρτά-ΟΝ HC1 was administered once, intraperitoneally at doses of 1 and 2 mg / kg (n = 6). To the control animals (n = 6), distilled water was injected intraperitoneally in a similar volume. The activity of PEP and DPP-SH was recorded at various time intervals after the introduction of substances.

It was found that δ ^ -Α ^ Ρτά-ΟΝ (5 mg / kg) with a single intraperitoneal administration statistically significantly reduces the activity of prolyl endopeptidase (PEP) several times within 5 hours. The maximum inhibition of enzyme activity is observed 1-2 hours after the introduction of δ ^ -Α ^ Ρτά-ΟΝ and then the PEP inhibitory effect gradually decreases, although it remains at a reliable level for 5 hours.

δ ^ -Α ^ Ρτά-ΟΝ (5 mg / kg) with a single intraperitoneal administration statistically significantly reduces the activity of DPP-SH. A decrease in the activity of DPP-U under the influence of δ ^ -Α ^ Ρτά-ΟΝ is observed gradually, reaching a statistically significant decrease 3 hours after the substance is introduced, and then the effect gradually increases and reaches a maximum after 5-6 hours.

As can be seen from the table. 3, the activity of DPP-Γν in rat blood plasma under the influence of ΑΠ-Ρτά ^ Ν HC1 (2 mg / kg) is significantly and statistically significantly reduced. The effect of the substance is detected 1 hour after the introduction of the substance and persists for 6 hours of observation. ΑΠ-Ρτά ^ Ν HC1 does not affect the activity of the probe during 6 hours of observation.

Table 3. The effect of δ ^ -Α ^ Ρτά-ΟΝ and ΑΠ-Ρτά ^ Ν HC1 on the activity of dipeptidyl peptidase-Sch (DPP-Sch) and prolyl endopeptidase (PEP) in rat plasma

Time after introductions compounds Zis-A1a-Rgs! -CM (5 mg / kg) A1a-Rga-SY NS! (2 mg / kg) PEP dpp-ιν PEP dpp-ιν Before introduction 2.53 ± 0.17 10.33 ± 0.37 2.53 ± 0.17 10.33 ± 0.37 1 hour 0.67 ± 0.07 * 9.87 ± 0.53 2.04 ± 0.26 4.77 ± 0.31 *

2 hours 0.75 ± 0.05 * 8.35 ± 0.43 2.68 ± 0.31 3.51 ± 0.78 * 3 hours 0.91 ± 0.04 * b, 95 ± 0.53 * 2.71 ± 0.41 4,5b ± 0,26 * 4 hours 1.19 ± 0.11 * 5.37 ± 0.72 * 2.45 ± 0.19 4.87 ± 0.42 * 5:00 1.79 ± 0.12 * 4.97 ± 0.71 * 2.94 ± 0.17 5.09 ± 0.48 * 6 hours 2.18 ± 0.15 4.28 ± 0.42 * 2.63 ± 0.51 4.94 ± 0.53 *

* - significance of differences with control at P <0.05.

Thus, δ ^ -ΑΠ-Ρ ^^ Ν (5 mg / kg) with a single intraperitoneal administration reduces the activity of prolyl endopeptidase (PEP) 1 hour after administration and this effect persists for 5 hours, whereas the activity of DPP- Щ under the influence of δ ^ -Α ^ Ρτά-ΟΝ decreases gradually and a reliable result is observed only 3 hours after administration and then the effect intensifies, reaching a maximum after 5-6 hours. In contrast, δΗαΑΠ-Ρτά ^ Ν compound ΑΗ-Ρτά-ΟΝ НС1 does not affect the activity of PEP, but has a reliable and long-lasting (6 h) inhibitory effect on ac DPP-Shch activity.

Additionally, it is known that acylated peptides are deacylated during the metabolism in mammals by the enzymatic method (ΤιιΗίβ ^ ί ίη Eu / Уuoteps1a1ige 1992 [ΑΑάά ^ ^ΡΡΡ888888 δ δап,,, δ δ δ88888888888888888888888888888888888888888888888888, -12-12-12-12-12 £ ог ог,,,,, ΚΒΝ 0-12-227164-5 ( Lamask), 0-12-227165-3 (raregbask)] mb, δ and ρ1step 1 (1993), δ and ρ1step 2 (1994), δ and ρ1tep 3 (1995), δ and ρ1tep 4 (1997), a ρ δ and ρ1tep 5 (ίη Eb. , 223, 1-5; Eig. I. Hsbt. 1995, 232, 1-6; Eg. I. Hsbeth. 1996, 237, 1-5; Eig. I. Hsbeth. 1997, 250; 16, I. Wuset. 1999, 264, 610-650; he8resyue1u) [Soruld! SEVM]). This indicates that in the process of metabolism δ ^ -Α ^ Ρτά-ΟΝ is deacylated to ΑΗ-Ρτά-ΟΝ.

Example 2. Antidiabetic, hypoglycemic activity of δΗαΑΠ-Ρτά ^ Ν in comparison with ΑΠbr! -SU and sitagliptin in the experiment of streptozotocin model of diabetes mellitus.

The study was conducted on male Wistar rats weighing 290-320 g. We used an experimental model of type 2 diabetes mellitus induced by repeated administration of pre-diabetic doses of streptozotocin toxin to rats (Novikov V.I., Molotkov O.V., Podcheko A.P. The protective effect of T-activin in experimental diabetes mellitus. Diabetes mellitus No. 2, 1999, pp. 37-40. Kolesnik Yu.M., Vasilenko GV, Abramov AV The condition of the islet apparatus of the pancreas in experimental diabetes mellitus rats of varying severity. toology

- 8 021236

1992, v. 54, No. 12, p. 24-27). Streptozotocin (STZ) was administered intraperitoneally at a dose of 30 mg / kg 3 times with an interval of 24 hours (in total, the animal received 90 mg / kg STZ). Glucometry was performed 24 hours later and 7 days after the last (3rd) administration of STZ. Blood glucose was measured with a glucometer: first after 12 hours of fasting, and then 1 hour after glucose loading, which was carried out by intraperitoneal injection of a glucose solution at a dose of 1 g / kg, intraperitoneally. Along with this, the weight gain of animals was determined on the first day of the introduction of STZ and on the 4th day after the introduction of STZ.

5th A1a-Prg-C'.U was administered intraperitoneally at a dose of 3 mg / kg three times 4 hours before the introduction of STZ for 3 days with an interval of 24 hours.

Ala-Pr4-C’U was used as a comparison substance, which was administered intraperitoneally at a dose of 3 mg / kg three times 10 minutes before the administration of STZ for 3 days with an interval of 24 hours.

As a comparison drug used sitagliptin (Januvia firm Megsk), which is the most widely used drug in the treatment of type 2 diabetes. Sitagliptin was administered intraperitoneally at a dose of 5 mg / kg three times 10 minutes before the introduction of STZ for 3 days with an interval of 24 hours.

It was found that in the control animals 24 hours after the last (3rd) administration of streptozotocin (STZ, 30 mg / kg, (WB)), there was a statistically significant increase in the level of glucose in the blood of rats compared with the values of the control animals, as measured after 12 hours of fasting, and 1 and 2 hours after glucose loading (Table 4).

A high level of glucose in the blood, significantly exceeding the indices of the control animals, remains even 7 days after the last (3rd) administration of STZ, both when measured after 12 hours of fasting, and 1 and 2 hours after glucose loading.

Table 4. The effect of 8is-A1a-Prg-C'U in comparison with A1a-Prg-CU and sitagliptin on blood glucose in the rat streptozotocin (STZ) model of diabetes mellitus

Group Blood glucose level 24 hours after the last (3rd) administration of STZ Blood glucose level 7 days after the last (3rd) administration of STZ 12 hour Glucose load (1 g / kg vnb) 12 hour fast Glucose load (1 g / kg vnb) starvation 1 hour 2 hours 1 hour 2 hours Control, diets. Water 4.2 ± 0.2 6.0 ± 0.2 - - - -

STZ 30x3 Vnb 7.3 ± 0.5 # 14.6 ± 0.6 # ^L 9.4 ± 0.4 ^L 8.5 ± 1.5 14.7 ± 1.5 b ^l 10.5 ± 1.5 Sitagliptin 5x3 mg / kg, vnb for Yumin to STZ 30x3 5.8 ± 0.3 13.4 ± 1.0 # ^L 1O, 3 ± 1.3 ^L 5.6 ± 0.4 * 12.3 ± 1.4 ^L 11.5 ± 2.3 A1a-Rgb-SY 3x3 mg / kg, vnb for Yumin to STZ 30x3 5.4 ± 0.1 6.9 ± 0.2 * ^L 6 ± 0.2 * 4.8 ± 0.5 * 7.1 ± 0.6 * ^L 6 ± 0.4 * ^l A1a-Rgb-SM 3x3 mg / kg, vnb 4 hours before STZ 30x3 6.9 ± 1.9 12.6 ± 3.2 8.1 ± 3.1 8.1 ± 2.1 9.2 ± 3.7 11.8 ± 3.1 5isA! Argdci 3x3 mg / kg, vnb 10 minutes before STZ 30x3 7.4 ± 1.2 12.3 ± 2.3 8.1 ± 1.3 6.2 ± 1.2 9.8 ± 2.5 9.2 ± 1.8 ZISA1ARGYSY 3x3 mg / kg, vnb 4 hours before STZ 30x3 5.9 ± 0.2 7.3 ± 0.3 * ^L 6.9 ± 0.3 * 5.1 ± 0.2 * 6.6 ± 0.5 * ^L 7.4 ± 0.6 * ^L

# - P <0.05 significance of differences according to the Mann-Whitney test in comparison with the control group (distilled water).

* - P <0.05 significance of differences according to the Mann-Whitney criterion in comparison with the STZ group.

l - P <0.05, the significance of differences according to the paired student criterion compared with values against 12 hours of fasting.

It was found that 8is-A1a-Prg-S'U (3 mg / kg three times 4 hours before the introduction of STZ for 3 days with an interval of 24 hours) also statistically significantly reduces the elevated blood sugar level of animals. 24 hours after the last administration of STZ and after glucose load 8is-A1a-Prg-S'U statistically significantly reduces the sugar level by 2 times. 7 days after the last administration of STZ 5isA1a-Prg4-SU, the glucose level also statistically significantly decreases both against the background of 12-hour fasting (2.2 times) and after glucose loading (1.4 times) compared with the group of animals receiving STZ (table. 4).

A1a-Prg-C'U (3 mg / kg three times 10 minutes before the introduction of STZ for 3 days with an interval of 24 hours) has a pronounced ability to reduce elevated blood sugar levels in animals. 24 hours after the last administration of STZ and after glucose loading A1a-Prg4-C '. HC1 statistically significantly reduces (by 2.1 times) the sugar level. 7 days after the last administration of STZ A1a-Prg-S'U HC1 also statistically significantly reduces the level of glucose both against the background of 12-hour fasting (1.8 times) and after glucose loading (1.7 times) compared with a group of animals treated with STZ

- 9 021236 (tab. 4).

Sitagliptin (5 mg / kg three times 10 minutes before the introduction of STZ for 3 days with an interval of 24 hours) statistically significantly reduces (1.5 times) the glucose level only against the background of 12 hours of starvation 7 days after the last administration of STZ, but not reduces the elevated STZ level of glucose after 24 hours and 7 days after the last administration of STZ and glucose load (Table 4).

The data obtained indicate that 8is-A1a-Prgy-SY (administration 4 hours before STZ), as well as AP-PTE-SY (administration 10 minutes before STZ), at a dose of 3 mg / kg have a pronounced, hypoglycemic activity in a model of type 2 diabetes induced by streptozotocin in rats, which is expressed in their ability to statistically significantly reduce the level of glucose in the blood of animals 24 hours and 7 days after the last administration of STZ both after starvation and against the background of glucose loading.

5> is-A1a-Rgb-CU and A1a-Rgb-CU significantly outperform the well-known drug sitagliptin in hypoglycemic activity, which, with a similar regimen of administration at a higher dose (5 mg / kg), reduces glucose levels only with one measurement - after 12 hours starvation 7 days after the last administration of STZ, and does not affect the elevated STZ glucose level after 24 hours and 7 days against the background of glucose loading.

Thus, 5> is-A1a-Prg-CU and A1a-Prg-CU at a dose of 3 mg / kg have a pronounced, hypoglycemic activity in the model of type 2 diabetes induced by streptozotocin in rats and significantly exceed the known drug sitagliptin in activity. used to treat type 2 diabetes.

Unlike A1a-Rgb-CU and sitagliptin, which have a hypoglycemic effect after a short latent period (administration 10 minutes before STZ), 5> is-A1a-Rgb-CU has a delayed hypoglycemic effect, which manifests itself 4 hours after administration of the substance . The delayed hypoglycemic effect of 5> is-A1a-Rgb-CU is associated with its metabolism and mechanism of action, namely, its ability to turn into A1a-Rgb-CU and acquire the properties of a dipeptidyl peptidase-ΐν inhibitor.

Example 3. The effect of 5> is-A1a-Rgb-CU on impaired cognitive function.

A study of the effect of 5> is-A1a-Rhb-CY on impaired cognitive functions was carried out in experiments on white outbred adult rats males weighing 250-280 g. The method of scopolamine amnesia of the conditioned passive avoidance reflex (passive avoidance reaction) was used according to the Guidelines for the study of the activity of substances with nootropic activity (Voronina T.A., Ostrovskaya R.U., 2005) set forth in the Guide to the experimental (preclinical) study of new pharmacological substances (Federal Service for the Supervision of Health and Social Regional Development, Federal State Institution Scientific Center for Expertise of Medical Devices, ed. Medicine, ed. Khabriev R.U., M., 2005, pp. 308-320).

The study was carried out in a certified LaRaueie PMgitsh Co installation (USA). The installation is a dark chamber 400x400x400 mm in size with an electrode floor. The dark chamber is connected through a square guillotine door measuring 60x60 mm with a hinged platform measuring 250x70 mm. The mounted platform is illuminated by a 60 W lamp located at a height of 400 mm. A dark chamber is located on the laboratory bench, and the platform hangs above the floor at a height of 800 mm. Rats were placed on the illuminated platform before entering the dark chamber of the installation with their tail to the inlet and the latent time of the reflex was recorded. Then, in a dark chamber, the rat received a single pain irritation with an electric current (0.3 mA) lasting 10 s (training).

To impair cognitive function (amnesia of a trained passive avoidance reaction), rats were injected with a scopolamine M-cholinergic receptor blocker at a dose of 1.15 mg / kg intraperitoneally 30 minutes before learning the conditioned passive avoidance reflex (passive avoidance reaction), then the passive avoidance reaction study was performed and the reflex was reproduced after 24 hours Passive avoidance reaction. The test for the reproduction of the trained and the presence of amnesia was carried out when the animal was re-placed in the installation 24 hours after training with registration for 3 minutes of the latent period of the first entry of the rat into the dark experimental chamber, where it had previously received pain irritation, and the number of animals that did not enter the dark camera compartment, in%.

8is-A1a-Prgy-CU was administered at a dose of 2 mg / kg once intraperitoneally 40 minutes before training. As the reference drug, the well-known nootropic drug Piracetam was used at a dose of 500 mg / kg (once, intraperitoneally, 40 minutes before training).

Statistical processing of the results was carried out using statistical packages Vu81a1 for \ νίηύο \ Υ5. Used Mann-Whitney test and criterion x ²

Research results

Control without amnesia. When placed on an illuminated platform, rats with a short latent period enter the dark chamber and receive pain irritation through the electrode floor (training). When the reflex is reproduced 24 hours after training, the rats remember the trained one and when they are placed on the illuminated platform, the animals remain there for the main time of the experiment and enter the dark dangerous chamber a little, where they received pain irritation (training) the day before (Table 5).

Amnesia control. The introduction of scopolamine before training causes amnesia, which is expressed in a statistically significant (P <0.05) significant shortening (4.6 times) of the duration of the latent 10 021236 period of entry into the dark dangerous chamber when reproducing passive avoidance reaction and reliable (P <0, 05) reduction (complete absence) of animals that did not enter the dark dangerous compartment of the camera at all during reproduction, i.e. remembering the pain irritation caused there the day before (Table 5). The data obtained indicate a pronounced amnesic effect of scopolamine.

8is-L1a-Rgb-CX at a dose of 2 mg / kg (once, intraperitoneally) statistically significantly (P <0.05) increases (2.54 times) the duration of the latent period of entry into the dark dangerous chamber when reproducing passive avoidance reaction and the number of animals without amnesia, which did not go into the dark compartment of the camera at all during playback, i.e. remembering the pain irritation caused there the day before (in the control with amnesia - 0%, and after the administration of 8isL1a-Prg-CX in 33.3%) (Table 5).

Piracetam at a dose of 500 mg / kg (once, intraperitoneally) has a similar antiamnestic effect (Table 5).

Table 5. The effect of 8is-L1a-Rgb-SK at a dose of 2 mg / kg (once, intraperitoneally) and piracetam at a dose

500 mg / kg (once, intraperitoneally) for amnesia of the conditioned passive avoidance reflex (passive avoidance reaction) caused by scopolamine in the certified 'LaGauue 1pz1gishep1 So installation

Groups Training Playback after 24 hours: Latent reflex time, s Latent time reflex with Count rats not% logged in dark camera Control without amnesia 24.3 ± 9.3 135.7 ± 31.0 66.7% Control - Amnesia (scopolamine) 16.0 ± 1.3 29.5 ± 19.8 * 0% # bisA1a-Rgs1-SM 2 mg / kg + Scopolamine 17.7 ± 7.5 75.2 ± 7.4 & 33% ^l Piracetam 500 mg / kg + Scopolamine 16.0 ± 2.10 68.4 ± 8.3 & 20% ^Λ

* - the significance of differences from control without amnesia at P <0.05 according to the Mann-Whitney criterion;

# - significance of differences from control without amnesia at P <0.05 according to the criterion by criterion x.

& - significance of differences from control with amnesia (scopolamine) at P <0.05 according to the Mann-Whitney criterion;

^L - significance of differences from control with amnesia (scopolamine) at P <0.05 according to criterion x.

Thus, 8is-L1a-Prgb-SU at a dose of 2 mg / kg has the ability to restore impaired cognitive functions, providing a clear anti-anamnestic effect in the test of amnesia of the conditioned passive avoidance reflex caused by the scopolamine blocker of M-cholinergic receptors in a certified setting . According to the severity of the antiamnestic effect, 8is-L1a-Rgb-SK at a dose of 2 mg / kg is not inferior to the reference nootropic drug piracetam (500 mg / kg).

Example 4. The study of the neuroprotective effect of 8is-L1a-Rgb-SK.

The neuroprotective effect of 8is-L1a-Rgb-SK was evaluated by the ability of a substance to eliminate neurodegenerative, neurotoxic changes caused by maximum electric shock (Voronina T.A., Nerobkova T.N. Guidelines for the experimental (preclinical) study of new pharmacological substances. Federal Service for Surveillance in healthcare and social development, Federal State Institution Scientific Center for Expertise of Medical Utilities, ed. Medicine, ed. Khabriev R.U., M., 2005).

The experiment was performed on white outbred male rats weighing 250-280 g. In order to obtain neurodegenerative brain lesions, the maximum electroconvulsive shock (MES) was used. An electric stimulus (50 Hz, 150 mA) of 0.2 s duration was applied to the animals through corneal electrodes. The neuroprotective effect of 8is-L1-Pr0-CX was assessed by its effect on the following indicators: coma duration (onset of which was judged by the loss of the overturning reflex with the development of complete adynamia, total muscle hypotension, the absence of corneal reflexes and pain sensitivity), the latent period of active movements (time from the moment the animal comes out of the coma and the restoration of the overturning reflex to the appearance of spontaneous motor activity), the total duration of seizures, the duration of tonic and clonic phase of convulsive manifestations.

Neurological deficits in violation of locomotor and orientational research activity after maximum electric shock were evaluated in the Open Field Test. An open field test was performed twice: the first time — 48 hours before electroshock and 30 minutes after the administration of 8is-L1a-Pr0-CX and the second time — 3 hours after electroshock. This scheme made it possible to evaluate both the effect of the substance on the spontaneous behavior of animals (administration before electroshock) and the protective effect of 8is-L1a-Rgb-CX in conditions of neurodegenerative brain damage (administration of the substance after electroshock). In addition, neurological deficits in impaired coordination of movements were evaluated in

- 11 021236 test of the Rotating rod in the installation of Co1a Κοά (firm ϋ§ο Vazier. Italy). Zis-A1a-Prgy-SI was administered at a dose of 3 mg / kg once intraperitoneally 40 minutes before the start of the experiments.

The presence of a neuroprotective effect in the studied substance was also judged by its ability to reduce the severity of post-convulsive neuropsychiatric disorders. namely, amnesia of the conditioned reflex of passive avoidance (passive avoidance reaction). The study was carried out in a certified IaRaueie 1pz1gitep1 Co installation (USA). The installation is a dark chamber 400x400x400 mm in size with an electrode floor. The dark chamber is connected through a square guillotine door measuring 60x60 mm with a hinged platform measuring 250x70 mm. The mounted platform is illuminated by a 60 W lamp. located at a height of 400 mm. A dark chamber is located on the laboratory bench. and the platform hangs above the floor at a height of 800 mm. First, rats were trained. The animal was placed on the illuminated platform before entering the dark chamber of the installation with its tail to the inlet and the latent time of the reflex was recorded. Then, in a dark chamber, the rat received a single pain stimulation with an electric current (0.3 mA) for 10 s (training). A test for the reproduction of trained passive avoidance reaction was carried out 48 hours after training. registering for 3 min the latent period of the first entry of the rat into the dark dangerous chamber. where she previously received pain irritation. and the number of animals. didn’t enter the dark compartment of the camera at all. in %. Then, after the test for the safety of the trained. for amnesia. which is accompanied by neurodegenerative brain disorders. rats received the maximum electroshock and after 3 hours amnesia was recorded in them according to the latent period of the first rat entry into the dark dangerous chamber. where she previously received pain irritation. and the number of animals. did not enter the dark compartment of the camera at all. in %. Zis-L1a-Prgy-SI was administered intraperitoneally at a dose of 3 mg / kg twice: 30 minutes before training and 30 minutes before reproduction.

Statistical processing of the results was carried out using statistical packages Vy31a1 for \ ¥ ίηάο \ Υ3. Mann-Whitney test and x ² criterion were used.

Research results

1. The effect of ZIS-L1a-Prgy-SI (3 mg / kg. Once. Intraperitoneally) on neurological indicators (duration of coma and latent period of the beginning of active motor movements after coma). the severity of convulsive syndrome and motor behavior in the open field after conducting maximum electroshock in rats.

Installed. that Zis-L1a-Prgy-SI has a pronounced ability to reduce neurological disorders. caused by maximum electric shock (MES). The neuroprotective effect of Zis-A1a-PrgyCI was expressed in the ability of the substance to statistically significantly decrease (by 1.8 times) the duration of the coma compared to the control. and also reduce (by 6 times) the time from the moment of restoration of the flipping reflex (coma) to the appearance of spontaneous motor activity (Table 6).

Table 6. The effect of Zis-A1a-Prgy-SI (3 mg / kg. Once. Intraperitoneally) on neurological indicators (duration of coma and latent period of the onset of active motor movements after coma) and the severity of convulsive syndrome after maximum shock (MES) in rats

Group, parameters The control 8isA1a-Rgi-SM Seizure intensity, points 4.0 ± 0.37 3.83 ± 0.31 Duration of the tonic phase convulsive manifestations, with 12.17 ± 0.83 11.0 ± 0.97 Clonic phase duration convulsive manifestations, with 57.5 ± 6.68 53.0 ± 1.92 Total duration convulsions with 68.0 ± 6.98 64.0 ± 1.57 Coma duration, s 174.5 ± 7.25 96.33 ± 15.95 * Latency onset active motor movements after a coma, with 561.5 ± 88.39 92.5 ± 40.47 * The death of animals in% twenty 0

* - P <0.05 significance of differences relative to control according to the Mann-Whitney criterion.

Zis-L1a-Prgy-S’I at a dose of 3 mg / kg slightly reduced the percentage of death of animals after maximum electric shock: in the control, death in 20% of rats; after Zis-A1a-Prg6-SI - the death of 0% rats. Zis-A1a-Pr6-SI did not affect the intensity and duration of seizures. caused by maximum electric shock. like tonic. and clonic phases (table. 6).

When testing rats after administration of Zis-L1a-Prgy-SI on a rotating rod (Ko1a Κοά) with a constant rotation speed of 15 rpm, no change in behavior compared with the control was detected: in the control group, the retention time on the drum was 83.0 + 29.12 s. and in the experimental group 106.5 + 26.32 s.

Approximate research and motor activity in the open field in rats. which were once injected with Zis-A1a-Prz-SI at a dose of 3 mg / kg (vnb). did not differ from the parameters. registered in the control group (table. 7).

- 12 021236

Table 7. The effect of 5> is-A1a-Rgb-C. ^ (3 mg / kg, once, intraperitoneally) on the motor activity of rats in an open field before and after maximum electric shock (MES)

Group To MES 3 hours after MES Horizon Active Vertical. active Mink reflex Horizon active Vertical. active Mink reflex The control 18.0 ± 2.38 9.5 ± 1.23 7.7 ± 1.15 17.8 ± 3.57 5,511,23 6,712.85 Zis-A1- rgd-ΟΝ 15.2 + 2.36 10.5 ± 2.86 5.5 ± 1.77 12.3 ± 3.84 3.3 + 1.12 * 3.7 + 1.09

* - P <0.05, the significance of differences according to the paired student criterion relative to the parameters obtained before the presentation of MES.

3 hours after the maximum electroshock (MES), the animals treated with A1a-Rgb-C '. ^ HC1 showed a decrease in the number of intersections of squares, uprights and peeps in holes (mink reflex) compared to the background values obtained during the first test in the Open field. Considering the positive effect of A1a-Prgb-C '. ^ NS1 on learning and amnesia of passive avoidance reaction caused by MES, a decrease in the tentative research behavior during repeated testing under the conditions of the Open Field technique can be interpreted as habituation (addiction), which is observed for substances with nootropic action .

2. The effect of 5> is-A1a-Prg-C. ^ (3 mg / kg, once, intraperitoneally) on the study of passive avoidance reaction and amnesia of passive avoidance reaction caused by the maximum electroconvulsive seizure.

Control and experimental (after the introduction of §is-A1a-Rgb-S0 animals, when placed on an illuminated platform with a short latent period, enter a dark chamber and receive pain irritation through the electrode floor (training). When the reflex is reproduced 48 hours after training, the control and experienced rats remember the trained and, when they are placed on the illuminated platform, the animals remain there for the main time of the experiment and enter the dark dangerous chamber a little, where they received pain irritation (training) the day before.

It was found that rats that received 5> is-A1a-Rgb-CH are better trained and remember the pain irritation received than control animals, which is expressed in a higher latent time of passive avoidance reaction during reproduction (differences with control are statistically unreliable and statistically significant (P <0.05) increase compared with the control of the number of animals that did not enter the dark dangerous compartment at all (Table 8).

After conducting the maximum electric shock in control animals, severe amnesia is observed, which is expressed in a statistically significant increase (P <0.05) in the duration of the latent period of entry into the dark dangerous chamber when reproducing passive avoidance reaction and a significant (P <0.05) decrease in the number of animals, completely not entering the dark dangerous compartment of the camera during playback, i.e. remembering the pain irritation caused there the day before (Table 8). 5> is-A1a-Rgb-S. ^ At a dose of 3 mg / kg statistically significantly (P <0.05) increases (1.4 times) the duration of the latent period of entry into the dark dangerous chamber when playing passive avoidance reaction and decreases (by 1 , 8 times) the number of animals without amnesia that did not go into the dark compartment of the chamber at all during reproduction (Table 8).

Table 8. The effect of 5> is-A1a-Rgb-S. ^ (3 mg / kg, ip) on the training of the conditioned passive avoidance reflex (passive avoidance reaction) and passive avoidance amnesia caused by the maximum electroconvulsive seizure (MES)

Group Passive avoidance reaction training for 48 hours to MES Passive passive avoidance reaction LV1, s To MES, LV2, s The number of rats not entering the dark compartment to the MES,% After spare part after MES, distillery The number of rats who did not enter the dark compartment after MES,% The control 24,417,47 135.2 ± 29.24 60 89.3 ± 15.86 # 33, Z ^l ZisA1a-Rgs! ΟΝ 28.8 ± 5.39 152.3 ± 27.67 83.3 * 123,0134.91 $ 60 *

# - P <0.05 significance of differences according to the Mann-Whitney criterion relative to the indicator before MES.

$ - P <0.05 significance of differences according to the Mann-Whitney test with respect to control.

^L - P <0.05 significance of differences by the criterion x ² relative to the indicator before MES.

* - P <0.05 significance of differences according to the criterion x ² relative to the control.

LV1 - latent time during training. LV2 - latent time when playing passive avoidance reaction. LP3 - latent time with amnesia of passive avoidance reaction.

The data obtained indicate that 5> is-A1a-Rgb-C. ^ Has a pronounced neuroprotective activity, which is expressed in its ability to reduce neurological deficits caused by maximum electroshock, namely, a significant decrease (1.8 times) in the duration of coma and time (6 times) from the moment of exit from a coma to the appearance of spontaneous motor activity, as well as in the ability of a substance to improve animal learning and reduce reflex amnesia

- 13 021236 with neurodegeneration of the brain caused by maximum electroshock.

Example 5. The study of the antihypoxic action of δis-Λ1a-P ^ b-CN, A1a-Rgb-CU.

The study of antihypoxic activity was carried out in accordance with the Guidelines for the experimental study of drugs proposed for clinical study as antihypoxic agents (compiled by Lukyanova L.D., Gatsura V.V., Pastushenkov L.V., Uryupov O.Yu., Lesnovskaya E. V., M., Ministry of Health of the USSR, Pharmacological Committee, 1990) according to two methods: normobaric hypoxia with hypercapnia in a hermetic volume and hypobaric hypoxia in a continuous exhaust chamber.

The study was carried out on male outbred adult mice weighing 23-28 g. According to the method of normobaric hypoxia with hypercapnia (canned hypoxia) of mice of the same weight (spread of no more than 2 g per group), one animal was placed in hermetically sealed cans of 230 cm ³ . The survival time (standby time) of animals under hypoxia was recorded.

Creating hypobaric hypoxia in the exhaust flow-pressure chamber with an absorber of CO _2. Mice were raised to a height of 11,000 m (198.7-185 mm Hg) at a speed of 25-50 m / s. Two mice were simultaneously placed in the pressure chamber — one of which was a control, and the other was experimental with the injected substance. The survival time (standby time) of animals under hypoxia was recorded.

The substances were administered once intraperitoneally 10 minutes before placing the animal in a jar. The animals in the control group were injected with distilled water.

Control animals placed under conditions of normobaric hypoxia with hypercapnia die on average within 33 minutes (Table 9).

Table 9. Antihypoxic effect of substances under conditions of normobaric hypoxia

Compound Dose (mg / kg) Time (min) The control - 33.05 ± 1.44 ΑΙβ-Ρτά-ΟΝ one 50.20 ± 1.77 * 5is-A1a-Prgy-SM 2 47.10 ± 1.71 *

* - P <0.05 significance of differences according to the Mann-Whitney criterion compared with the control group.

It was established that A1a-Rgb-CH at a dose of 1 mg / kg and δis-Λ1a-P ^ b-CN at a dose of 2 mg / kg have a pronounced antihypoxic effect under conditions of normobaric hypoxia with hypercapnia in a hermetic volume, which was expressed in a statistically significant increase in time survival during hypoxia (table. 9).

AN-PM-SY at a dose of 1 mg / kg and δis-A1a-P ^ b-CN at a dose of 2 mg / kg at a dose of 1 mg / kg have a pronounced antihypoxic effect under hypobaric hypoxia, which was expressed in a statistically significant increase in survival time with hypobaric hypoxia (table. 10).

Table 10. Antihypoxic effect of substances under hypobaric hypoxia

Compound Dose (mg / kg) Time (min) The control - 28.42 ± 1.92 A1a-Rgs) -CM one 45.20 ± 3.77 * 8is-A1a-Pr0-SM 2 43.10 ± 4.32 *

* - P <0.05 significance of differences according to the Mann-Whitney criterion compared with the control group.

Thus, the compounds A1a-Rgb-CU, δis-A1a-P ^ b-CN and O1n-Rgb-CU have an antihypoxic effect under conditions of normobaric hypoxia with hypercapnia in a hermetic volume and hypobaric hypoxia.

Example 6. The study of side effects and acute toxicity of the compound δis-A1a-P ^ b-CN.

The study of side effects and acute toxicity δis-A1a-P ^ b-CN was carried out in accordance with the Guidelines for the study of acute toxicity set forth in the Guide to the experimental (preclinical) study of new pharmacological substances (Federal Service for the Supervision of Health and Social Development, Federal State the establishment of a scientific center for the examination of means of medical use, published by Medicine, as amended by Khabriev R.U., M., 2005, 832 pp.).

To study possible side effects and acute toxicity, δis-A1a-P ^ b-CN was administered to mice once intraperitoneally at doses of 30 and 60 mg / kg, i.e. in doses exceeding the therapeutic dose (2 and 3 mg / kg), respectively, 10-15 times and 20-30 times. Observation of animals was carried out for 14 days. The control animals were injected once, intraperitoneally, in the same volume with distilled water.

It was found that in animals after the administration of δis-Λ1a-P ^ b-CN, there was no change in the mucous membranes and the condition of the coat; the number of urination and bowel movements remained normal; not detected ptosis, salivation, piloerection, abnormal shaking, sneezing, sipping, contraction of the masticatory muscles; emotional status, grooming, locomotion remained at the normal level; there was no aggression, no passive behavior, activation, and stereotype were observed; no changes were noted

- 14 021236 reactions to sound or touch, tentative-research behavior, pain, pineal and corneal reflexes have not changed, muscle tone has been preserved; there were no paresis, convulsions, twitching (tab. 11).

Under the influence of compound 8is-A1a-Rgb-C '%, there is no violation of coordination of movements in the test of a rotating rod (Ko1a Kob Ido Wazbe, Italy), the ability of animals to stay on an inverted mesh platform and on the climbing reflex to the top of the mesh platform is not impaired. The death of animals was not observed 24 hours after the administration of 8is-A1a-Rgb-CH nor during the entire 14-day observation period (Table 11).

Table 11. The study of side effects and acute toxicity of 8is-A1a-Rgb-C '% in mice when observed for 14 days | Indicators ί Groups, 6cc-A1a-Rgb-SY |

Comte R· 30 mg / kg 60 mg / kg h 24 h 4 day 14 day one h 24 h 4 day 14 day Changing the nature of the coat 0 0 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 0 0 The presence of pipo-erection 0 0 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 0 0 Violation of the rhythm and depth of breath. movement 0 0 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 0 0 Change in auditory response 0 0 0 0 0 0 0 0 0 Increased shyness 0 0 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 0 0 Change in pain response thresholds 0 0 0 0 0 0 0 0 0 Change in posture, catalepsy 0 0 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 0 0 0 0 0 0 0 0 5 s holding on an inverted mesh platform one hundred 10 0 10 0 one hundred one hundred 10 0 10 0 one hundred one hundred Relocating with an inverted set. platform up one hundred 10 0 10 0 one hundred one hundred 10 0 10 0 one hundred one hundred The presence of pineal. reflex one hundred 10 0 10 0 one hundred one hundred 10 0 10 0 one hundred one hundred The presence of corneal. reflex one hundred 10 0 10 0 one hundred one hundred 10 0 10 0 one hundred one hundred Sedation 0 0 0 0 0 0 0 0 0 The presence of a stereotype. 0 0 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 0 0 G ebel animal 0 0 0 0 0 0 0 0 0

Thus, when studying the possible side effects and acute toxicity of 8is-A1a-RgbCN, it was found that after a single intraperitoneal administration of 8is-A1a-Rgb-C '^ in doses of 30 and 60 mg / kg (i.e., in doses exceeding the therapeutic dose by 10-30 times) there were no deviations from the norm in the general condition of animals, in their behavior, in reactions to stimuli, in performing elementary reflexes, locomotor reactions, in emotional status, in neurological status and there are no signs of intoxication and death of animals 24 hours after the introduction of 8is- A1a-Rgb-CH nor during the 14-day registration period.

Claims (5)

CLAIM 1. The compound of the formula - 15 021236 2. The use of a compound according to claim 1 as an inhibitor of prolyl endopeptidase and dipeptidyl peptidase-ΐν. 3. The use of compounds of claim 1 as an agent for the treatment of type 2 diabetes mellitus. 4. The use of the compounds according to claim 1 as a means for the treatment of diseases accompanied by hypoxia, neurodegeneration and cognitive impairment. 5. A dosage form containing, as an active substance, a compound according to claim 1 for the treatment of type 2 diabetes mellitus or the treatment of diseases accompanied by hypoxia, neurodegeneration and cognitive impairment.