RU2613314C2 - Small molecules with ngf-like activity and antidiabetic properties - Google Patents

Abstract

FIELD: pharmacy.

SUBSTANCE: group of inventions refers to application of the original group of dimeric dipeptides, created based on the structure β-bending of the 4th nerve growth factor loop of a rat and a human, respectively, GC-2 Rat (r) (hexamethylenediamide bis-(N-monosuccinyl-glutamyl-lysine)) and GC-2 Human (h) (hexamethylenediamide bis- (N-monosuccinyl-glycyl-L-lysine)), as antidiabetics.

EFFECT: attenuation of hyperglycaemia during treatment of first and the second type of diabetes.

2 cl, 2 tbl, 6 dwg, 3 ex

Description

The use of original dimeric dipeptides created on the basis of the β-bend structure of the 4th loop of rat and human nerve growth factors, respectively, of GK-2 Rat (r) (bis- (N-monosuccinyl-glutamyl-lysine hexamethylene diamide)) and GK-2 is proposed Human (h) (bis- (N-monosuccinyl-glycyl-L-lysine hexamethylene diamide)), as antidiabetic agents. The proposed NGF-like mechanism of action of GK-2 (r) and GK-2 (h) involves the protection of pancreatic beta cells and stimulation of insulin secretion in response to hyperglycemia. There are currently no antidiabetic drugs with an NGF-like mechanism of action. However, such drugs could be used to treat type 1 and type 2 diabetes. It was shown that administration to rats of streptozotocin toxin at a dose of 40 mcg / kg (once intraperitoneally) leads to the development of such signs of diabetes as an increase in blood glucose and weight loss. Chronic combined (prophylactic and therapeutic) intraperitoneal (ip) administration of GK-2 (r) at a dose of 0.5 mg / kg or GK-2 (h) at a dose of 0.1 mg / kg weakens the severity of these manifestations of diabetes. In addition, on the model of streptozotocin diabetes, the anticoagulant effect of GK-2 (r) was revealed, which is of great importance, since in diabetes mellitus there is a violation of blood microcirculation and an increased risk of thrombosis. The data obtained allow us to consider it appropriate to use GK-2 (r) and GK-2 (h) as antidiabetic agents, as well as molecular tools for studying the involvement of NGF in the pathogenesis of diabetes.

Field of Invention

The invention relates to medicine, in particular to pharmacology, and relates to means for the prevention and treatment of diabetes.

State of the art

Diabetes mellitus (DM) is the third leading cause of death in most countries of the world. According to WHO estimates, more than 180 million people currently suffer from diabetes, and according to statistics cited by Shaw J.E., 220 million people worldwide suffer from this pathology [A. Spasov. et al. // Expert. and wedge. pharmacol. 2011. V.74. No. 11. S.14-6]. It is predicted that by 2030 this figure may double [Shaw J.E., Sicree R.A. and Zimmet P.Z. // Diabetes Research and Clinical Practice. 2011. V.87. No. 1. R.4-14]. The rate of increase in the prevalence of this disease is threatening. The number of patients with diabetes in Russia according to official statistics from an indicator of 8 million in 1994 may increase by 2020 to 18 million.

Currently, the preference for the treatment of diabetes is given to drugs from the groups of sulfonylurea derivatives [Ashcrof F.M., Reimann F. // Problems of endocrinology. 2001. No.6. P.43-47], biguanides [Salpeter S.R., Buckley N.S., Kahn J.A. and al. // Am J Med. 2008. No. 121. P.149-157], a derivative of thiazolidinedione [Ametov A.C., Sokareva E.V. // Breast cancer. 2008. No. 28. S.1858]. Preparations from the group of dipeptidyl peptidase-4 (DPP-4) inhibitors, the effect of which is based on increasing the concentration of incretins that promote insulin synthesis [Pratley R.E., Salsali A. // Curr. Med. Res. Opin. 2007. V.23. Number 4. P.919-931; Havale S.H. // Bioorg. Med. Chem. 2009. V.17. No. 5. P.1783-802].

A wide range of side effects and absolute contraindications limits the use of these drugs in clinical practice. The use of sulfonylurea derivatives is limited by the development of secondary resistance to them in 5-10% of patients with diabetes mellitus [Aleksandrov A.A. // Diabetes mellitus: Consilium medicum. 2001.V.1. No. 10. C.2-7]. The restriction of the use of biguanides is determined by the possibility of developing lactic acidosis, and thiazolidinedione derivatives by the revealed hepatotoxicity [Forman L.M., Simmons D.A., Diamond R.H. // Ann Intern Med. 2000. V.132. No. 2. P.118-21] and an increased risk of developing cardio-vascular pathology [Rohatgi A., McGuire D.K. // Cardiovasc Drugs Ther. 2008. V.22. Number 3. P233-4]. The use of DPP-4 inhibitors is often accompanied by the development of arthralgia against the background of an increase in the content of uric acid in the blood, but the main risk factor in their use is an increase in the incidence of pancreatitis and pancreatic cancer [Elashoff M., Matveyenko A.V., Gier B. et al. // Gastroenterology. 2011. V.141. No. 1 P.50-6].

All these facts indicate the need to create new highly effective, safe antidiabetic drugs, since only expanding the range of antidiabetic drugs will maximize the compensation of diabetes taking into account the individual characteristics of each patient, improve the quality of life of patients, reduce disability, maintain the working capacity of patients with diabetes, which has a great social and economic importance to society.

High nerve growth factor (NGF) has a high therapeutic potential for treating diabetes and its complications. It is known that NGF plays an important role in the differentiation and maintenance of the functioning of pancreatic beta cells [Polak M., Scharfmann R., Seilheimer B. et al. // Prod. Natl. Acad. Sci. USA 1993. V.90. No. 12. P.5781-85]. Pancreatic beta cells synthesize and secrete NGF, which by autocrine regulation stimulates insulin secretion; beta-cell synthesis and secretion of NGF are enhanced in response to an increase in extracellular glucose concentration [Rosenbaum T., Sanches-Soto M.S., Hiriart M. // Diabetes. 2001. V.50. No. 8. P.1755-62]. Beta cells secreted NGF and insulin are essential for their survival. These properties of NGF - maintaining the viability and functioning of beta cells, as well as the ability to stimulate insulin secretion, determine its potential potential for the treatment of type 1 and type 2 diabetes.

Deficiency of neurotrophic factors is one of the important reasons for the delayed proliferation of P-cells in diabetes [Nielsen J.H., Galsgaard E.D., Moldrup A. et all. // Diabetes. 2001. V.50 No. 1. P.25-9]; this deficiency is associated with the development of complications of diabetes such as peripheral cardiopathy, neuropathy, retinopathy, nephropathy, etc. [Han. H.E., Goss, Goins W.F., Lacomis D.et. all. // Diabetes. 2006. V. 51. Number 7. P.2227-32]. In a model of streptozotocin diabetes, it has been shown that gene therapy of NGF inhibits the development of diabetic neuropathy in mice [Goss J.R., Goins W.F., Lacomis D. et. al. // Diabetes. 2002. V. 51. Number 7. P.2227-32], and when administered in the form of eye drops, NGF inhibits diabetic retinal degeneration in rats [Colafrancesco V., Coassin M., Rossi S., Aloe L. // Ann. 1st Super Sanita. 2011. V.47, No. 3: P.284-9].

Clinical trials of NGF with its systemic administration to patients with diabetic neuropathy were discontinued due to the development of significant side effects (pain at the injection site) and the low severity of the positive effects of such treatment [Pittenger G., Vinik, A. // Experimental Diab. Res. 2003. V.4. Number 4. P.271-85]. The use of the native NGF molecule in the clinic is limited by a wide range of its biological activity, inability to penetrate the blood-brain barrier and pharmacological instability.

At the FSBI Scientific Research Institute of Pharmacology named after V.V. Zakusov’s RAMS based on the β-bend structure of the 4th loop of rat and human NGF, the original dimeric dipeptide mimetics, respectively GK-2 (r) (bis- (N-monosuccinyl-L-glutamyl-L-lysine) hexamethylene) were created [ Seredenin S.B., Gudasheva T.A. // 2010. RF patent No. 2410392] and GK-2 (h) (bis- (N-monosuccinyl-glycyl-L-lysine hexamethylene diamide)).

Synthesis of GK-2 (h)

a) Synthesis of bis- (N-benzyloxycarbonyl-N ^ε -tert-butyloxycarbonyl-lysyl) hexamethylene diamide, (Z-Lys (Boc) NH) 2 (CH2) 6

A solution of 2.575 g (5.4 mmol) of Z-Lys (Boc) -OSu and 0.299 g (2.6 mmol) of hexamethylenediamine in 14 ml of DMF was stirred for 4 hours at room temperature, and a turbid solution formed. The reaction mixture was diluted with 56 ml of water and left for several hours. After solidification, the precipitate was filtered off and washed with water. After drying over P ₂ O ₅ , 2.085 g (96% yield) of a white crystalline product was obtained.

- 9,31 (с 0,29, этанол)Rf 0.90 (A), 0.93 (B), mp. 138-147,

- 9.31 (s 0.29, ethanol)

Spectrum ¹ H-NMR (DMSO-d ₆ ): 1.05-1.69 (12H, m, 2 (C ^β H ₂ , C ^γ H ₂ , C ^δ H ₂ ) Lys; 8H, m, -NH- CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-; 1.35 18H, s, 2 -C (CH ₃ ) ₃ Boc;), 2.87 (4H, m, 2C ^ε H ₂ Lys), 3.01 (4H, m, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-), 3.89 (2H, m, 2C ^α HLys) 5.01 (4H, s, 2OCH ₂ , Z); 6.74 (2H, t, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-), 7, 13-7.39 (10H, m, 2 C ₆ H ₅ Z; 2H, d, 2NNd Lys), 7.81 (2H, t, 2NNt Lys)

b) Synthesis of N-oxysuccinimide ester of N-benzyloxycarbonyl-glycine, Z-Gly-OSu.

To a solution of 5.28 g (28 mmol) of Z-Gly-OH in 80 ml of ethyl acetate was added 3.22 g (28 mmol) of HOSu and 6.36 g (28 mmol) of DCCA. The reaction mass was stirred at 10 ° C for 2 hours. After the disappearance of the starting material (TLC control), the resulting dicyclohexylurea was filtered. The solvent was removed in vacuo. The resulting oil was recrystallized from a mixture of 3 ml of petroleum ether and 8 ml of chloroform. 5.87 g (22.4 mmol, 80%) of the product are obtained in the form of a white crystalline solid. Physico-chemical characteristics coincide with those described in the literature [Anderson G.W., Zimmerman J.E. The use of esters of N-hydroxysuccinimide in peptide synthesis // J. Amer. Chem. Sco. - 1964 .-- 86, N9. - P.1839-1842].

c) Synthesis of bis- (N ^ε -tert-butyloxycarbonyl-lysyl) -hexamethylene diamide, (Lys (Boc) NH) 2 (CH2) 6

1.86 g (2.2 mmol) (Z-Lys (Boc) NH) 2 (CH2) 6 were hydrogenated in methanol (25 ml) over 10% Pd / C (0.37 g) for 1 h 25 min at room temperature. After the disappearance of the starting material (TLC control), the catalyst was filtered off, the solvent was removed in vacuo. The resulting oil was dried over CaCl ₂ and paraffin. Next, planting was carried out with a mixture of petroleum and diethyl ether (3: 1) and filtered. Received 0.99 g of the substance in the form of a white powder (yield 78%).

5,1 (с 1, этанол)Rf 0.48 (A), 0.68 (B), mp. 70-75,

5.1 (s 1, ethanol)

Spectrum ¹ H-NMR (DMSO-d ₆ ): 1.11-1.65 (12H, m, 2 (C ^β H ₂ , C ^γ H ₂ , C ^δ H ₂ ) Lys; 8H, m, -NH- CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-; 18H, s, 2 -C (CH ₃ ) ₃ Boc;), 2.86 (4H, m, 2C ^ε H ₂ Lys ), 3.04 (4H, m, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-), 3.40 (2H, m, 2C ^α H Lys), 6 , 73 (2H, t, 2NHt Lys), 7.78 (2H, t, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-)

d) Synthesis of bis (N-benzyloxycarbonyl-glycyl-N ^ε- tert-butyloxycarbonyl-lysine) hexamethylene diamide), (Z-Gly-Lys (Boc) -NH-) ₂ (CH ₂ ) ₆ .

To a solution of 0.90 g (1.6 mmol) of Lys (Boc) NH) 2 (CH2) 6 in 6 ml of DMF, a solution of 1.103 g (3.8 mmol) of Z-Gly-OSu in 4 ml of DMF was added dropwise with stirring and stirring was continued for 2 h 10 min at room temperature (TLC control). Then 1.5 ml of DEPDA was added and stirred for another 30 minutes, after which the reaction mixture was diluted with 70 ml of water. The product and organic impurities were extracted with chloroform (3: 1) and washed alternately with 25 ml of water, 25 ml of 2% H ₂ SO ₄ and 25 ml of 3% Na ₂ CO ₃ . Then the solution was dried with MgSO ₄ and filtered. After evaporation of chloroform, the product was obtained as a mixture of a yellowish oil with white crystals. 20 ml of diethyl ether was added: when triturated with a spatula, the product crystallized. The ether was filtered off and 1.26 g (84%) of a white powder was obtained.

- 11,0 (с 0,1, этанол)Rf 0.89 (A), 0.95 (B), mp. 120-126,

- 11.0 (s 0.1, ethanol)

Spectrum ¹ H-NMR (DMSO-d ₆ ): 1.05-1.70 (12H, m, 2 (C ^β H ₂ , C ^γ H ₂ , C ^δ H ₂ ) Lys; 8H, m, -NH- CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-; 1.35 18H, s, 2 -C (CH ₃ ) ₃ Boc;), 2.85 (4H, m, 2C ^ε H ₂ Lys), 3.00 (4H, m, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-), 3.65 (4H, d, 2CH ₂ Gly) 4.15 (2H, m, 2C ^α H Lys), 5.03 (4H, s, 2OCH ₂ , Z), 7.25 (2H, t, -NH-CH ₂ -CH ₂ -CH ₂ -CH _2- CH ₂ -CH ₂ -NH-), 7.14-7.37 (10H, m, 2 C ₆ H ₅ Z; 2H, d, 2NHd Lys), 7.81 (2H, t, 2NHt Lys)

d) Synthesis of bis- (glycyl-N ^ε -tert-butyloxycarbonyl-lysyl) -hexamethylene diamide, (Gly-Lys (Boc) NH) 2 (CH2) 6

1.17 g (1.2 mmol) (Z-Gly-Lys (Boc) NH) 2 (CH2) 6 was hydrogenated in methanol (20 ml) over 10% Pd / C (0.23 g) for 20 min at room temperature. After the disappearance of the starting material (TLC control), the catalyst was filtered off, the solvent was removed in vacuo. The resulting oil was precipitated with diethyl ether (20 ml) and filtered. The substance in the form of a white powder was sent to the next stage without further purification.

f) Preparation of bis (N-monosuccinyl-glycyl-N ^ε -tert-butyloxycarbonyl-lysine hexamethylene diamide), (HOOC (CH ₂ ) ₂ CO-Gly-Lys (Boc) -NH) ₂ (CH ₂ ) ₆ .

To a solution of (Gly-Lys (Boc) NH) 2 (CH2) 6 in 5 ml of DMF was added 0.24 g (2.4 mmol) of succinic anhydride. The reaction mixture was stirred at room temperature for 4 hours (TLC control). The reaction mixture was diluted with 20 ml of ethyl acetate and 15 ml of H ₂ O. The product was transferred to the aqueous phase. To it was added 5 ml of 3% Na ₂ CO ₃ . A solution of 1.5 g of Na ₂ SO _{4 was} saturated and 15 ml of butanol and 10% H ₂ SO _{4 were} added to pH = 3. Then washed with 10% Na ₂ SO ₄ . The solvent was completely evaporated. The residue was triturated with ether and filtered. 0.872 g (0.984 mmol) of product are obtained in the form of a white powder (82% yield).

- 10,4(с 1, этанол)Rf 0.80 (A), 0.67 (B), mp. 86-92,

- 10.4 (s 1, ethanol)

Spectrum ¹ H-NMR (DMSO-d ₆ ): 1.11-1.75 (12H, m, 2 (C ^β H ₂ , C ^γ H ₂ , C ^δ H ₂ ) Lys; 8H, m, -NH- CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-; 1.35 18H, s, 2 -C (CH ₃ ) ₃ Boc;), 2.36 (4H, m, 2 CH ₂ Suc), 2.72 (4H, m, 2C ^ε H ₂ Lys), 2.96 (4H, m, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH- ), 3.65 (4H, s, 2 CH ₂ Gly), 4.10 (2H, m, 2C ^α H Lys), 6.71 (2H, t, -NH-CH ₂ -CH ₂ -CH ₂ - CH ₂ —CH ₂ —CH ₂ —NH—), 7.83 (2H, t, 2NHt Lys), 8.13 (2H, d, 2NHt Lys), 8.87 (2H, t, 2NHt Gly), 2 HOOC (CH ₂ ) ₂ CO-exchange with HDO.

g) Preparation of bis- (N-monosuccinyl-glycyl-lysine) hexamethylene diamide, (HOOC (CH ₂ ) ₂ CO-Gly-Lys-NH-) ₂ (CH ₂ ) ₆ ).

- 15.7° (с 1; вода).All obtained (HOOC (CH ₂ ) ₂ CO-GlyLys (Boc) -NH-) ₂ (CH ₂ ) _{6 was} dissolved in acetic acid and treated with 3 ml of 4M HCl / Dioxane. After 40 minutes, the solvent was decanted, the residue was washed with diethyl ether by decantation, dissolved in 5 ml of water and treated with stirring with Amberlit IRA-410 resin until the pH stabilized ~ 5. Purification was performed by reverse phase C8 HPLC in a gradient of 0-13% isopropanol in 0.1M acetic acid. The appropriate fractions were collected (TLC control), evaporated and dried in vacuo. Lyophilized using liquid N2. recrystallized from ethyl acetate-ethanol (3: 1). Received 0.515 g (0.751 mmol) (total yield 29%) of the final product in the form of a white powder, R _f 0.14 (A), R _f 0.38 (B), so pl. 97-100 ° C,

- 15.7 ° (s 1; water).

Spectrum ¹ H-NMR (DMSO-d ₆ ): 1.06-1.85 (12H, m, 2 (C ^β H ₂ , CγH ₂ , C ^δ H ₂ ) Lys; 8H, m, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ - NH-; 1.35 18H, s, 2 -C (CH ₃ ) ₃ Boc;), 2.26 (4H, m, 2CH ₂ Suc) 2.84 (4H, m, 2C ^ε H ₂ Lys), 3.05 (4H, m, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-), 3 69 (4H, s, 2 СН2 Gly), 4.10 (2H, m, 2C ^ε H Lys), 7.65 (2H, t, -NH-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH-), 8.34 (2H, d, 2NHd Lys), 8.64 (2H, t, 2NHt Gly), 2 HOOC (CH ₂ ) ₂ CO- are exchanged with HDO.

The peptide GK-2 (r) showed a high NGF-like neuroprotective activity in nanomolar concentrations in vitro [Gudasheva TA, Antipova TA, Seredenin SB. // DAN. 2010. V.434, No. 4. S.549-52], and also showed effectiveness in an in vivo model of a brain stroke [Seredenin SB, Silachev DN, Gudasheva T.A. and others. Byul. expert. biol. 2011.V.151. No. 5. S.518-521]. In experiments using an in vivo model of focal cortical ischemia, the neuroprotective activity of the compound GK-2 (h) was also detected [Gudasheva TA, Romanova TA, Shikova FM, Kotelnikova СО. et al. // Ex and wedge farm. 2012, No. 10].

It was shown that GK-2 (r), like the native NGF molecule, causes phosphorylation of specific TrkA receptors and phosphorylation of Akt kinase involved in one of the intracellular signaling cascades activated by these receptors [Gudasheva T., Antipova T., Seredenin S. // XXIInd International Symposium on Medicinal Chemistry. 2012. P.299]. From the literature it is known that the influence of NGF on maintaining the viability of beta cells and their secretion of insulin is mediated by TrkA receptors [Rosenbaum T., Sanches-Soto M.S., Hiriart M. // Diabetes. 2001. V.50. No. 8. R.1755-62; Navarro-Tableros, V., Sanches-Soto, M.C., Garcia, S., Hiriart, M. (2004). Autocrine regulation of single pancreatic beta-cell survival. Diabetes. Aug; 53 (8): 2018-23]. In addition, it is known that the Akt-kinase signaling pathway is involved in protecting NGF beta cells from death [Navarro-Tableros, V., Sanches-Soto, M.C., Garcia, S., Hiriart, M. (2004). Autocrine regulation of single pancreatic beta-cell survival. Diabetes. Aug; 53 (8): 2018-23].

Thus, the data obtained suggest that GK-2 (r) and GK-2 (h) act by an NGF-like mechanism and may be effective as antidiabetic agents.

SUMMARY OF THE INVENTION

The invention consists in the use of GK-2 (r) and GK-2 (h) as antidiabetic agents and as tools for studying the molecular mechanisms of NGF involvement in the pathogenesis of diabetes mellitus.

The purpose of the invention is the development of drugs for the treatment of diabetes mellitus based on NGF that can slow the progression of this disease. This goal was achieved by studying the effects of GK-2 (r) and GK-2 (h) in a model of streptozotocin diabetes.

The technical result of the invention is the antidiabetic and hypocoagulant effects of GK-2 (r) and GK-2 (h).

The invention is illustrated by the following examples.

It is known that in the experiment the development of hyperglycemia can be reproduced by the introduction of diabetogenic toxins alloxan and streptozotocin (STZ), which have a direct cytotoxic effect on pancreatic β-cells. The advantage of streptozotocin compared with alloxan is a relatively higher metabolic stability, a longer duration of hyperglycemia, less pronounced acidosis and mortality in experimental animals [Szkudelski T. // Minireview Physiol. Res. 50: 536-546, 2001]. Streptozotocin diabetes, being a generally accepted experimental model of diabetes mellitus, allows simulating both the gradually developing pancreatic beta-cell dysfunction and impaired glucose tolerance and the development of related disorders [Kheifets IA, Spasov AA, Voronkova M. P. et al. // Bull. expert. biol. 2012.V. 153. No. 1. - S. 62-4].

The experiments were carried out on male Wistar white rats weighing 300-380 g (from the beginning of the experiment) obtained from the Stolbovaya RAMS nursery of the RAMS. The animals were kept in vivarium of the FSBI “V.V. Zakusova ”RAMS of 10 animals per cage with free access to standardized food and water. Studies were carried out from 10 ⁰⁰ to 14 ⁰⁰ . The ethical rules for the humane treatment of animals, as set out in Council Directives 86/609 / EEC, were respected.

Example 1. The effect of the combined (prophylactic and therapeutic) administration of GK-2 (r) on a model of streptozotocin diabetes.

The experiment was performed on 38 rats randomly divided into 3 groups: 1st — passive control (n = 12), 2nd — active control (n = 13), 3rd — experimental group (n = 13). 1. The animals of the passive control group (Control) were injected daily with distilled water (DV) for 14 days, then once with physiological saline (RF), then the introduction of water continued for the next 28 days. 2. Animals of the active control group (STZ) were injected with DV for 14 days, then once with STZ (Sigma) at a dose of 40.0 mg / kg, dissolved in RF, then the administration of DV continued for the next 28 days. 3. Animals of the experimental group (STZ + GK-2r) were prophylactically injected daily with GK-2 (r) at a dose of 0.5 mg / kg, dissolved ex tempore in DV, then STZ was once administered, and then the administration was continued for 28 days GK-2 (r) in the same dose. All substances were administered intraperitoneally at a rate of 2 ml / kg.

Determination of glucose level in blood taken from the tail vein (One Touch Ultra glucose meter, USA) was carried out immediately before the introduction of STZ, as well as on the 3rd, 17th and 28th day after the introduction of STZ. Animals were weighed every 3-4 days.

To assess the statistical significance of the intergroup differences, the Kruskal-Wallis test was used, followed by post-hock comparison of the samples using the Mann-Whitney U test and Fisher's exact test. The results were considered reliable at p <0.05. Data were presented as median samples and interquartile intervals.

The therapeutic effect of the dipeptide GK-2 was calculated by the formula:

where hl STZ - blood glucose level in the STZ group, ch. (STZ + GK-2r) - blood glucose level in the group (STZ + GK-2r), Ch. water - blood glucose level in the passive control group.

Within 2 weeks before the introduction of STZ, no intergroup differences in body weight and blood glucose were observed, that is, chronic administration of GK-2 (r) to healthy rats did not affect these parameters.

According to published data, a glucose content of more than 13-15 mmol / L is considered as an indicator of diabetes [Kuhada A., Bishnoia M., Tiwaria V. et al. // Pharmacology Biochemistry and Behavior. 2009. V.92. No. 2. P.251-259]. In our experiments, 3 days after the introduction of STZ, there was an increase in the glucose content in venous blood to 19.3 mmol / L (median of the sample). When testing 14 and 28 days after the introduction of STZ, hyperglycemia remained in the active control group (testing at a later date was not performed in these experiments). On the 3rd day after the administration of STZ, GK-2 (r) significantly reduced blood glucose with a therapeutic effect of 60%. On the 17th day after the administration of STZ, GK-2 (r) reduced the level of glucose in the blood more clearly - the therapeutic effect of GK-2 (r) was 80%.

On the 28th day after the introduction of STZ, the antihyperglycemic effect of GK-2 (r) was also 80% (Figure 1. The antihyperglycemic effect of GK-2r with combined therapeutic and prophylactic administration).

Comparison of GK-2 (r) with currently used antidiabetic drugs showed that GK-2 (r) is superior to some of them, such as glibenclamide, in terms of the effectiveness of the hypoglycemic effect and is comparable to the effect of glyclazide and diabenol [Erejuwa O.O., Sulaiman SA, Wahab MS et. al. // International Journal of Applied Research in Natural Products. 2011. V.4. No. 2. P.1-10; Tahara A., Matsuyama-Yokono A., Nakano R. et. al, II Basic. Clin. Pharmacol Toxicol. 2008. V.103. No. 6. P.560-8; Anisimova B.A., Balabolkin M.I., Vdovina G.P. and others // RF Patent No. 2386634. 2010] (Table 1).

Table 1 Comparison of the hypoglycemic effect of the dipeptide GK-2 (r) and some antidiabetic drugs used in medicine on a model of streptozotocin diabetes in rats. The hypoglycemic effect was evaluated on the basis of measuring blood glucose during chronic therapeutic administration of the drug (A) or on the basis of a glucose tolerance test with a single administration of the drug (B). Drug name Administration mode Therapeutic effect of hypoglycemic action GK-2 (r) 0.5 mg / kg, ip, daily for 4 weeks 80% (A) Glibenclamide 0.6 mg / kg, per os, daily for 4 weeks 32% (A) (Erejuwa O.O., Sulaiman S.A., Wahab M.S. et. Al. // International Journal of Applied Research in Natural Products. 2011. V.4. No. 2. P.1-10) Voglibosis 1.0 mg / kg, per os, 30 minutes before the guest 40% (B)  (Tahara A., Matsuyama-Yokono A., Nakano R. et. Al. // Basic. Clin. Pharmacol. Toxicol. 2008. V. 103. No. 6. P.560-8) Metformin 1000.0 mg / kg, per os, 30 minutes before the test 60% (B) (Tahara A., Matsuyama-Yokono A., Nakano R. et. Al. // Basic. Clin. Pharmacol. Toxicol. 2008. V.103. No. 6. P.560-8) Gliclazide 50.0 mg / kg, per os, 2 hours before the test 70% (B) (Anisimova B.A., Balabolkin M.I., Vdovina G.P. et al. // RF Patent No. 2386634. 2010) Diabenol 25.0 mg / kg, per os, 2 hours before the test 85% (B) (Anisimova V.A., Balabolkin M.I., Vdovina G.P. et al. // RF Patent No. 2386634. 2010)

An important indicator of the diabetic effect of STZ is weight loss. The dipeptide GK-2 (r) completely prevented a decrease in the gain in body weight of rats (Figure 2. The effect of GK-2r on a 4-week increase in body weight of rats under conditions of streptozotocin diabetes).

Thus, in these experiments, the high efficiency of GK-2 (r) with the combined therapeutic and prophylactic administration was shown.

Example 2. The effect of combined (prophylactic and therapeutic) administration of GK-2 (h) on a model of streptozotocin diabetes.

The experiments were performed on 38 rats randomly divided into 3 groups: 1st — passive control (n = 12), 2nd — active control (n = 13), 3rd — experimental group (n = 13).

1. Animals of the passive control group (Water) were daily injected with DV for 14 days, then once with RF, then the introduction of DV continued for the next 28 days.

2. Animals of the active control group (STZ) were injected with DV for 14 days, then once STZ at a dose of 40.0 mg / kg, dissolved in RF, then the introduction of DV continued for the next 28 days.

3. Animals of the experimental group (STZ + GK-2 (h)) were prophylactically injected daily with GK-2 (h) at a dose of 0.1 mg / kg, dissolved ex tempore in distilled water, then STZ was introduced once, and then for 28 days continued the administration of GK-2 (h) in the same dose. All substances were administered intraperitoneally at a rate of 2 ml / kg.

Determination of glucose level in blood taken from the tail vein (One Touch Ultra glucose meter, USA) was carried out immediately before the introduction of STZ, as well as on the 3rd, 17th and 28th day after the introduction of STZ. Animals were weighed every 3-4 days.

To assess the statistical significance of the intergroup differences, the Kruskel-Wallis test was used, followed by post-hock comparison of the samples using the Mann-Whitney U test. The data in the figures are presented as mean values and standard error of the mean (M ± SEM). The results were considered reliable at p <0.05.

To characterize the dynamics of the antihyperglycemic effect of GK-2 (h), the average values of samples of glucose levels in the groups were calculated and the relative indicator of the therapeutic effect (Te) was calculated by the formula:

where hl STZ - blood glucose level in the STZ group, ch. (STZ + GK-2h) - blood glucose level in the group (STZ + GK-2h), Ch. water - blood glucose level in the passive control group.

Before the introduction of STZ, there were no statistically significant differences between groups in terms of glucose level, i.e. GK-2 (h) does not affect blood glucose in healthy animals. The introduction of STZ caused a marked increase in blood glucose. Statistically significant differences between the active and passive control groups were observed at all observation periods: 3 days after the introduction of STZ, these indicators were 17.9 ± 1.6 mmol / L with a glucose level for rats of the passive control group of 5.4 ± 0.1 mmol / l, p <0.05 (Fig. 3A. Antihyperglycemic effect of GK-2b with combined therapeutic and prophylactic administration, 3rd day after administration of STZ); after 17 days - 14.6 ± 1.7 mmol / L compared with 5.3 ± 0.3 mmol / L, p <0.05 (Fig.3B. Antihyperglycemic effect of GK-2h with combined therapeutic and prophylactic administration, 17th day after the introduction of STZ). The glucose content in the blood of rats on the 17th day after the administration of STZ) and after 28 days was 19, ± 2.5 mmol / L compared with 4.8 ± 0.1 mmol / L; p <0.05 (Fig.3B. Antihyperglycemic effect of GK-2h with combined therapeutic and prophylactic administration, on the 28th day after administration of STZ). Since a glucose content of more than 13-15 mmol / l is considered as an indicator of diabetes [Shaw J.E., Sicree R.A. and Zimmet P.Z. // Diabetes Research and Clinical Practice. 2010. V.87. No. 1, P.4-14], it can be concluded that the known hyperglycemic effect of STZ was reproduced in this experiment.

The combined (prophylactic and therapeutic) administration of GK-2 (h) reduced the severity of hyperglycemia already 3 days after the introduction of STZ (14 days of prevention), when the blood glucose level was 14.0 ± 1.9 mmol / l; p <0.05 (Fig. 3A.). It also occurred after 17 days (11.6 ± 1.7 mmol / L) (Fig. 3B.) And 28 days of therapy (12.8 ± 2.3 mmol / L; p <0.05 compared with the group STZ) (Fig.3B). The calculation of the dynamics of the relative indicator of the antihyperglycemic effect of GK-2 indicates a gradual increase in its value during the observation period.

In full accordance with published data [Han N.E., Goss J.R., Goins W.F., Lacomis D. et. all. // Diabetes. 2006. V. 51. Number 7. P.2227-52; T. Skudelski // Physiol. Res. 2001. V.50. No. 6. P.536-46] the introduction of STZ in experiments led to a decrease in the body weight of rats in the active control group, while in the passive control group its gradual increase at all stages of measurement took place. The calculation of the difference in body weight, Δ (g), between that on the day of the introduction of STZ and on the following days showed the following results (Figure 4. The effect of GK-2h on the 4-week increase in body weight of rats in conditions of streptozotocin diabetes): on the 4th day in the passive control group it was + 6.2 ± 1.8 g, in the active control group -16.6 ± 2.7 g (p <0.05); on the 17th day + 23.6 ± 11.9 g and -14.0 ± 8.5 g, respectively (p <0.05); and on the 28th day + 34.5 ± 5.2 g and -6.5 ± 11.3 g, respectively (p <0.05).

Administration of GK-2 (h) attenuated STZ-induced reduction in rat body weight: on day 4, Δ was -11.6 ± 2.6 g (p <0.05 compared with the active control group), on the 17th day-3.2 ± 1.7 g (p <0.05), and on the 28th day the opposite effect was observed - rats began to gain weight, the Δ score was + 7.8 ± 2.3 g (Figure 4) .

Thus, the antidiabetic effect of low molecular weight rat and human NGF mimetics is shown: GK-2 (r) and GK-2 (h). The ability of NGF to maintain the viability of beta cells, protect them from damage and stimulate insulin secretion in response to an increase in glucose concentration is mediated by TrkA receptors [Rosenbaum T., Sanches-Soto M.S., Hiriart M. // Diabetes. 2001. V.50. No. 8. P.1755-62]. In addition, it is known that the Akt-kinase signaling pathway is involved in protecting NGF beta cells from death [Navarro-Tableros, V., Sanches-Soto, M. S., Garcia, S., Hiriart, M. (2004)] . It was shown that GK-2 (r) activates TrkA receptors [Gudasheva TA, Antipova TA, Seredepin SB. // DAN. 2010. V.434, No. 4. S.549-52], and also stimulates the phosphorylation of Akt kinase involved in signaling from these receptors [Gudasheva T., Antipova T., Seredenin S. // XXIInd International Symposium on Medicinal Chemistry. 2012. P.299]. These data suggest that GK-2 (r) has an antidiabetic effect by an NGF-like mechanism. GK-2 (h), similar to rat NGF mimetic, GK-2 (r), reduces hyperglycemia and prevents weight loss caused by the introduction of STZ. Thus, it can be assumed that GK-2 (h) also exerts an antidiabetic effect by an NGF-like mechanism.

Thus, the proposed NGF-like mechanism of action of GK-2 (r) and GK-2 (h) involves the protection of pancreatic beta cells and stimulation of insulin secretion in response to hyperglycemia. The results obtained indicate the promising use of the dipeptides GK-2 (r) and GK-2 (h) as antidiabetic drugs effective in diabetes of both type 1 and type 2.

Example 3. Hypocoagulation effect of GK-2 (r)

The studies were performed on two groups of male Wistar rats weighing 300-350 g, which were once administered intraperitoneally with a dose of 40 mg / kg STZ (active control group n = 8) or physiological saline (passive control group, n = 8). The experiment was carried out under ex vivo conditions: GK-2 (r) was added to the blood plasma taken from animals of both groups.

, I и ci - индексы коагуляции, равные соответственно 160×tgα и ma/r+k. Фибринолитическую активность оценивали по времени лизиса сгустка эуглобулиновой фракции. Статистическая обработка полученных данных проводилась с помощью U теста Манна-Уитни.Blood for the study was taken from the jugular vein of animals of both groups using a syringe containing 3.8% sodium citrate solution (9: 1 ratio) 4, 10, 18, and 45 days after the start of the experiment. Using a One touch Ultra glucose meter (USA), glucose concentration was measured, then the blood was centrifuged for 10 minutes at 2000 rpm. Each sample of the obtained plasma was divided into 2 equal parts (0.7 ml). 0.035 ml of distilled water (series 1) was added to one part of the plasma of rats from the active control group, and 0.350 ml of a freshly prepared solution of peptide GK-2 (r) in distilled water at a concentration of 10 ^-5 M (series 2) was added to the other. The passive control group was treated in a similar manner (series 3 and 4, respectively). All samples were incubated for 10 minutes at 37 ° C. Then, they determined a number of indicators of the state of hemostasis by decoding the data of the thromboelastogram (TEG) recorded on a Hellige thromboelastograph. 0.735 ml of blood plasma incubated with water or a peptide was placed in a cuvette of the device, 0.030 ml of a 1.9% solution of calcium chloride was added and recording was made. The results obtained made it possible to evaluate a number of hemostasis parameters (anticoagulant plasma activity and properties of the resulting clot): r is the reaction time corresponding to thrombin formation, k is the time of onset of clot formation, the appearance of fibrin filaments, ma is the maximum amplitude and angle α (Figure 5. Thromboelastogram) . Based on these parameters, the following indicators were calculated: E is the density of the bunch

, I and ci are coagulation indices equal to 160 × tgα and ma / r + k, respectively. Fibrinolytic activity was evaluated by the time of lysis of the clot of the euglobulin fraction. Statistical processing of the data was carried out using the Mann-Whitney U test.

The blood glucose in rats of the passive control group throughout the experiment was 5.36 ± 0.12 mmol / L. In animals of the active control group, 4 days after the administration of the toxin, the glucose level was 20.0 ± 1.6 mmol / L, after 10 days - 20.1 ± 1.5 mmol / L, after 18 days - 20.8 ± 1 , 3 mmol / L, after 45 days - 14.4 ± 0.9 mmol / L.

Previously, in a model of diabetes caused by the introduction of STZ, it was shown that this toxin causes hypercoagulation [Hansen H.R., Wolfs J.L., Bruggemann L. et all. // Blood Coagul Fibrinolysis. Oct .; 18 (7): 627-36 (2007)]. In our study, it was found that the most pronounced changes in hemostasis (accelerated thrombin generation, a sharp increase in coagulation indices with delayed fibrinolysis) develop 10 days after the introduction of STZ, and by the 45th day there is a gradual normalization of indicators. On the 4th day of the experiment, against the background of hemostasis indices close to normal, the addition of GK-2 (r) to the plasma causes only a slight acceleration in the formation of thrombin, which is neutralized by a slowdown in the formation of fibrin filaments (Table 2). On the 10th day of the experiment, under conditions of accelerated thrombin formation and, accordingly, increased coagulation indices (ci and I) in diabetic rats (series 1), the addition of GK-2 (r) compound (series 2) to the plasma slows down the process thrombin formation, reduction of coagulation indices and acceleration of fibrinolysis. On the 18th day, the hypocoagulation effect of GK-2 (r) is partially preserved and is expressed in a slower formation of thrombin and fibrin, an almost twofold decrease in clot density, and a one and a half fold coagulation index ci. After 45 days, against the background of decreasing hyperglycemia, hemostasis is normalized and the addition of GK-2 (r) to the plasma is not accompanied by additional changes in coagulation indices (Fig. 6A. Effect of GK-2h on rat plasma coagulation index in streptozotocin diabetes and normal, group active control).

table 2 Comparison of TEG after adding distilled water (series 1) or GK-2 (series 2) to plasma taken from diabetic rats. Terms after STZ R mm K mm E unit ci, units I, unit 4 days Episode 1 6.33 ± 0.88 2.67 ± 1.67 157.80 ± 33.22 7.98 ± 2.05 282.70 ± 101.80 Series 2 0.20 ± 0.05 * 4.50 ± 2.29 156.70 ± 36.88 8.42 ± 1.42 307.30 ± 66.75 10 days Episode 1 2.92 ± 0.40 2.58 ± 0.70 90.00 ± 14.30 16.68 ± 5.73 531.40 ± 41.20 Series 2 4.29 ± 0.80 * 2.58 ± 0.92 80.16 ± 14.37 7.11 ± 1.00 * 329.50 ± 34.95 * 18 days Episode 1 2.33 ± 0.75 4.08 ± 0.90 149.10 ± 28.30 9.19 ± 1.18 328.90 ± 21.60 Series 2 3.08 ± 1.02 7.75 ± 2.92 * 90.52 ± 4.93 * 5.19 ± 1.63 * 262.90 ± 66.07 * 45 days Episode 1 6.00 ± 2.04 2.50 ± 1.19 162.80 ± 9.30 p.m. 7.46 ± 0.35 273.00 ± 13.63 Series 2 5.75 ± 2.10 2.50 ± 1.19 151.90 ± 28.10 7.06 ± 0.33 269.10 ± 32.92 * - p <0.05 compared with series 1 (U test Mann-Whitney).

In intact animals, when GK-2 (r) is introduced into plasma, there is a tendency to hypocoagulation (Fig.6B. Effect of GK-2h on rat plasma coagulation index in streptozotocin diabetes and normal, passive control group) and acceleration of fibrinolysis, however this the effect is less pronounced than in diabetic rats.

Thus, the hypocoagulation effect of GK-2 (r) was first detected. Ex vivo experiments do not allow a final conclusion on the mechanisms of hypocoagulation action of GK-2 (r). The literature describes TrkA receptor-mediated ability of NGF to stimulate basophil activity and activate heparin excretion into the blood [Welker P., Grbbe J., Gibbs B. et al. // Immunology. Mar; 99 (3): 418-26 (2000)]. Given the anticoagulant properties of heparin, as well as on the basis of the revealed fact of the interaction of the compound GK-2 (r) with TrkA receptors [Gudasheva TA, Antipova TA, Seredenin SB. // DAN. 2010. V.434, No. 4. S.549-52; Antipova T.A. // Experimental and clinical pharmacology. T.73. No. 12. C.6-8 (2010)], it can be assumed that the ability to enhance the release of heparin is one of the mechanisms of the hypocoagulation action of GK-2 (r) in conditions of diabetes.

The combination of antihyperglycemic and anticoagulant properties of GK-2 (r) is extremely important because with diabetes there is a violation of blood microcirculation and an increased risk of thrombosis.

A brief description of the drawings.

Figure 1 (Antihyperglycemic effect of GK-2r with a combined therapeutic and prophylactic administration) shows the antihyperglycemic effect of GK-2r (r) with a combined (preventive and therapeutic) administration. The abscissa axis is the time after the introduction of STZ (day); the ordinate axis is the blood glucose content (mmol / g). Data are presented as median samples and interquartile intervals. * p <0.001 compared to the Control group (Kruskal-Wallis test followed by a post-hock comparison using the Mann-Whitney U test); ** p <0.0001 compared to the Control group (Kruskal-Wallis test followed by a post-hock comparison using the Mann-Whitney U test); # p <0.05 compared with the STZ group (Kruskal-Wallis test followed by a post-hock comparison using the Mann-Whitney U test), ^ p <0.05 compared with the STZ group (Fisher’s exact test )

Figure 2 (Effect of GK-2r on 4-week weight gain of rats under conditions of streptozotocin diabetes) shows the effect of GK-2 (r) on a 4-week weight gain of rats under conditions of streptozotocin diabetes. The abscissa axis — experimental groups; ordinate axis - weight gain compared with the day of introduction of STZ. Data are presented as median samples and interquartile intervals. * p <0.0001 compared to the Control group; # p <0.01 compared to the STZ group (Kruskal-Wallis test followed by a post-hock comparison using the Mann-Whitney U test).

Figure 3 (Antihyperglycemic effect of GK-2h with combined therapeutic and prophylactic administration) shows the effect of GK-2 (h) with combined (preventive and curative) administration on rat glucose in 3rd (A), 17th (B) and the 28th (C) days after the introduction of STZ. The abscissa axis — experimental groups; on the ordinate axis - blood glucose (mmol / g). The data are presented as average values of the samples. * - the significance of differences in the U Manny-Whitney test between passive control (water) and active control (STZ); # - significance of differences between active control (STZ) and experimental groups (administration of GK-2 (h) 0.1 mg / kg ip, 14 days before and 28 after administration of STZ).

Figure 4 (Effect of GK-2h on a 4-week gain in rat body weight under conditions of streptozotocin diabetes) shows the effect of GK-2 (h) upon combined (prophylactic and therapeutic) administration on the change in body weight (r) from the day STZ was administered ( Δ weight). The abscissa axis is the time after the introduction of STZ (day); the ordinate axis is the change in body weight compared to the day of the introduction of STZ (r). The data are presented as average values of the samples. * - significance of differences in the Mann-Whitney U test between passive control (water) and active control (STZ). # - significance of differences between active control (STZ) and therapeutic administration of GK-2 (H) 0.1 mg / kg.

Figure 5 (Thromboelastogram) shows a standard thromboelastogram and methods for measuring its parameters.

Figure 6 (Effect of GK-2b on rat plasma plasma coagulation index under streptozotocin diabetes and normal) shows the effect of GK-2 (r) on rat plasma plasma coagulation index ci (coagulation index) under normal streptozotocin diabetes. A - active control group (STZ), B - passive control group (control). The abscissa axis is the time after the introduction of STZ (day); the ordinate axis is the value of ci (coagulation index) of rat plasma.

Claims (2)

1. The use of the dimeric dipeptide GK-2 Rat (r) (hexamethylene diamide bis- (N-monosuccinyl-glutamyl-lysine)) as a substance with antidiabetic activity. 2. The use of the dimeric dipeptide GK-2 Human (h) (hexamethylene diamide bis- (N-monosuccinyl-glycyl-L-lysine)) as a substance with antidiabetic activity.

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