RU2563234C2 - Medication for prevention and correction of diabetes manifestations - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to field of pharmacology and deals with preparation for prevention and treatment of diabetes. Claimed is application of N-phenylacetyl-L-prolylglycine ethyl ether and created on its base nootropic and neuroprotective medication Noopept as medication for prevention and treatment of diabetes. N-phenylacetyl-L-prolylglycine ethyl ether and Noopept in dose 0,5 mg/kg in combined preventive-therapeutic, preventive and therapeutic, including regimen of delayed therapeutic administration attenuates expression of all metabolic manifestations of diabetes, namely demonstrates antihyperglycaemic effect, recovers normal glucose tolerance under stress conditions, prevents loss of body weight, reduces development of diabetic neuropathy. Noopept surpasses other perorally active standard antidiabetic medication Janow (Sitaglipin) by indices of tolerance to glucose load.

EFFECT: totality of obtained results in combination with numerous clinical data about good tolerance of -phenylacetyl-L-prolylglycine ethyl ether and Noopept makes it possible to consider expedient to apply it by novel indication - as medication of pathogenetic therapy of diabetes, activity of which is aimed at prevention of its progress.

2 cl, 3 dwg, 6 tbl, 5 ex

Description

The use of the original nootropic and neuroprotective drug Noopept (N-phenylacetyl-L-prolylglycine ethyl ester) as an agent for the prevention and treatment of diabetes is proposed. It was shown that the administration of streptozotocin toxin to rats in diabetic doses of 45-50 μg / kg (once intraperitoneally) leads to the development of such signs of diabetes as increased blood glucose, accelerated weight loss, increased pain sensitivity, polyuria. Noopept, administered at a dose of 0.5 mg / kg repeatedly (in the combined prophylactic-therapeutic, prophylactic and therapeutic, including the regimen of delayed therapeutic administration) weakens the severity of all these metabolic manifestations of diabetes. In experiments on a model of developing diabetes caused by repeated administration of streptozotocin in sub-diabetic doses (30 mg / kg), the high antidiabetic activity of Noopept, administered orally at a dose of 5 mg / kg, was demonstrated. Noopept is superior to another orally active standard antidiabetic drug Januvius (Sitagliptin) in terms of glucose tolerance. The combination of the results obtained in combination with numerous clinical data on the good tolerability of this nootropic drug (www.noopept.ru) allows us to consider it appropriate to use it according to a new indication - as a means of pathogenetic therapy of diabetes, the action of which is aimed at preventing its progression.

Field of Invention

The invention relates to medicine, in particular to pharmacology, and relates to a 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 suffer from diabetes, and according to statistics cited by Shaw J.E., 220 million people worldwide suffer from this pathology [A.A Spasov et al. // Ext. and wedge. Famacol. 2011. 74 (11), 14-6]. It is predicted that by 2030 this figure will double [J.E.Shaw, R.A.Sicree and P.Z. Zimmet // Diabetes Research and Clinical Practice, 2011, V.87. No. 1, P.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 8 million in 1994 will increase to 18 million by 2020.

Currently, preference is given in the treatment of diabetes to drugs from the sulfonylurea derivatives groups [P.M. Ashcroft, F. Reimann // Problems of Endocrinology. 2001. - No. 6. - from. 43-47], biguanides [S.R. Salpeter, N. S. Buckley, J. A. Kahn and al. // Am J Med. 2008. 121: 149-157], a derivative of thiazolidinedione [A.S. Ametov, E.V. Sokareva // Breast Cancer. 2008. No. 28, 1858]. Most recently, drugs from the group of dipeptidyl peptidase-4 inhibitors (DPP-4), whose effect is based on increasing the concentration of incretins that promote insulin synthesis, are beginning to occupy an important place [R.E. Pratley, A. Salsali // Curr. Med. Res.Opin. 2007. 23, 4, 919-931; S.H. Havale, // Bioorg. Med. Chem. 2009. 17, 5, 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 [A.A. Aleksandrov // Diabetes mellitus: Consilium medicum. 2001. Vol. 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 [L. M. Forman, D. A. Simmons, R. H. Diamond // Ann Intern Med. 2000. 132, 2: 118-21] and an increased risk of developing cardiovascular pathology [A. Rohatgi, D.K. McGuire // Cardiovasc Drugs Ther. 2008. Jun; 22 (3): 233-4]. 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 [M. Elashoff, A. V. Matveyenko, B. Gier et al. // Gastroenterology. 2011. 141, 1: 150-6].

All these facts make it clear 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.

It is known that one of the pathogenetic factors in the development of diabetes is the excessive activation of free radical oxidation processes [M.I. Balabolkin // Diabetes mellitus. 2002.4: 5-16; N.I. Fadeeva // Dis. for the degree of Cand. honey. sciences. 2000. P.217]. This process is considered as a universal mechanism that combines the main biochemical pathways of the toxic effect of hyperglycemia on the body. The consequences of diabetes, such as cardiovascular diseases, diabetic neuropathy, nephropathy, damage to the heart and peripheral vessels, the retina and lens, a set of cognitive impairments are considered mainly as a result of damage to the corresponding organs by free radicals - fragments of molecules that have an unpaired electron due to oxygen atoms [P.Ready, X. Zhu, G. Perry, el al. // J Alzheimers Dis. 2009. v.16, n. 4, 763-774; K. Maiese // Expert Rev Cardiovasc Ther. 2008. v.6, n.3, 281-284]. One of the most important consequences of the enhancement of free radical oxidation processes is the ability of the free radicals formed to react with phospholipids of cell membranes. The accumulation of peroxide (RO2 *), alkoxyl (RO *), alkyl radicals (R *), superoxide anion, singlet oxygen and cytotoxic peroxynitrites and their reaction with phospholipids of cell membranes leads to structural changes in membranes, damage to proteins, lipids and nucleic acids and DNA fragmentation. As a result, structural changes in the membranes occur, impaired permeability and loss of elastic properties [P. Packer, I.G. Obrosova, J. G. Mobable et al. // Emerging New Therapeutical Strategies Curr Med Chem. 2005. 12 (3): 267-275].

In diabetes mellitus, not only the formation of free radicals is activated, but also the activity of natural factors of endogenous antioxidant defense is reduced: superoxide dismutase, catalase, glutathione peroxidase, i.e. the balance between the prooxidants and the components of the antioxidant defense system is violated towards the predominance of activity of the former [A.P. Rolo and S.M. Palmeira. // Toxicology and Applied Pharmacology. 2006. vol. 212, n.2, pp. 167-178]. Excessive accumulation of free radicals leads to the activation of protein kinases, increased protein glycation, and the accumulation of NAD (P) H, which activates proinflammatory processes (S. Golbidi, M. Badran, and I. Laher. // Experimental Diabetes Research. 2012. Review Article, ID 941868, 1-16]. It is known that the onset of diabetes is characterized by only a slight decrease in the activity of antioxidant enzymes, and with a long course of the disease, the antioxidant properties of the blood sharply worsen and lipid peroxidation (LP) processes are significantly activated. Let us note that deficiency of antioxidant systems and accumulation of free radicals in β-cells of the pancreas are more pronounced than in other organs [M T. Lortz, Th. Nachtwey, AEKarlsen et al. // Diabetes. 2000. Vol. 49. P. 1123-1130].

Data on the effectiveness of antioxidants for the prevention and comprehensive treatment of diabetes is mixed. There is evidence of a positive effect of Mexidol [O.V. Zanozina, NN Borovkov, M.I. Balabolkin et al. // Bulletin of experimental biology and medicine. 2006. Appendix 1; I.A. Volchegorsky, L.M. Rassokhina, I.Yu. Miroshnichenko // Problems of endocrinology. 2008.V. 54, No. 5, p. 43-49]. It was established that the most clear therapeutic effect was obtained with the use of Mexidol at a dose of 200 mg (intravenous drip). For a famous antioxidant. α-lipoic acid, it was shown that 10-day perfusion at a dose of 500 mg enhances insulin sensitivity in patients with diabetes mellitus (N.A. Chernikova, B. Abaeva, M.A. Prudnikova et al. // Endocrinology. 2010 , Volume 12, No. 12, 11-17]. However, the data on the effectiveness of lipoic acid in diabetes, given in foreign publications, are heterogeneous. Along with reports of the presence of positive effects [S. Golbidi, M. Badran and I. Laher. // Frontiers in Pharmacology 2011. V.2, Art 69, 1-15], the negative results of the use of lipoic acid are also given [Huang and Gitelman // Pediatr. Di abetes. 2008. v.9, 69-73]. It is emphasized that even with the course use of lipoic acid in doses up to 1800 mg per day, improvement is noted mainly in relation to the manifestations of diabetic polyneuropathy [D.Ziegler, A.Ametov, A.Barinov et al. // DiabetesCare. 2006, 29, 2365-2370], but not the main metabolic manifestations of diabetes [AM de Oliveira, PHRondó, Luzia el al. // Diabetes Res. Clin. Pract. 2011, 92, 253-260]. Other researchers also caution against overstating the effectiveness of existing antioxidants in diabetes [J.S. Johansen, A.K. Harris, D.J Rychly el al. // Cardiovascular Diabefology. 2005, v.4. n.5, 1-11]. It should be noted that the use of lipoic acid is accompanied by the development of adverse side reactions; the most common adverse reactions to lipoic acid include its gastrotoxicity when taken orally, skin irritation and itching, as well as headache with parenteral administration [M. Reljanovic. // Free Radio Res. 1999.v.31. N 3, 171-179].

Despite the relatively large range of drugs for suppressing free radical oxidation reactions, problems arise associated with the use of existing agents, such as the toxicity of some phenolic antioxidants, displacement of endogenous antioxidants using synthetic compounds [S.V. Okovity. // Clinical pharmacology of antioxidants // FARMindeks prakt. 2003. Issue 5. - P.85-111; M.V. Lucc, I.V. Zarubina, P.D. Shabanov // Psychopharmacology and biological narcology. 2008.V.8. - Issue 1-2, part 1. S.2255-2263].

Thus, at present, the problem of treating diabetes with drugs with an antioxidant component of action is not resolved, and a search for new drugs of this type for the prevention and treatment of diabetes is required.

The original diopeptide preparation Noopept (GVS-111; ethyl ester of N-phenylacetyl-L-prolylglycine) developed at the Institute of Pharmacology showed a set of nootropic and neuroprotective effects [S.V. Seredenin, T.A. Voronina, T. Gudasheva el al. // US Patent. 1995. No. 5,439,930; T.A. Gudasheva, T.A. Voronina, RU Oslrovskaya el al. // Eur. J. Med. Chem. 1996. 31, 151-157; RU Ostrovskaya, TAGudacheva, SBTrofimov et al. In: Biological basis of individual sensitivity to Psychotropic drugs. Graffhan Press Ltd. Eds Gaviraghi G., Longo V., Seredenin S., Edinburgh, UK, 1994, 79-91]. The drug demonstrated in various experimental models in vitro and in vivo a pronounced normalizing effect on the ratio of the activity of pro- and antioxidant systems. It has been shown that even with a single administration in small doses (0.1 mg / kg), it prevents the accumulation of molecular products of POL-diene conjugates, malondialdehyde and Schiff bases in the brain tissue and blood plasma [A.V. Lysenko, R.U. .Ostrovskaya, E.I.Uskova et al. // Expert. the clinic. pharmacology. 1997, 60, 15-18]. Noopept increases the activity of enzymes of the endogenous antioxidant system: superoxide dismutase, catalase, ceruloplasmin in the subcellular fractions of the brain and blood plasma of rats [AM Menzheritsky, AV Lysenko, SV Demyanenko et al. // Neurochemistry. 2003. Vol. 20, 4, 281-286]. The ability of the drug to activate the endogenous antioxidant system is confirmed in terms of its effect on the blood serum of healthy volunteers [T.N. Fedorova, K.S. Us, R.U. Ostrovskaya // Neurochemistry. 2007. Vol. 24, No. 1, 69-73]. In experiments on cultures of human cortical neurons (aborted fruits), Noopept's ability to eliminate the enhancement of oxidative processes caused by the addition of hydrogen peroxide to healthy fruit neurons or the genetically predetermined hyperactivation of peroxidation, characteristic of fruits with prenatally diagnosed Down syndrome, was established. It is shown that Noopept in this indicator is significantly more effective than vitamin E, traps of free radicals PBN and Piracetam [A. Pelsman, C. Hoyo-Vadillo, SBSeredenin el al. // Int J Dev Neurosci. 2003, 21, n. 3, 117-124]. Higher neuroprotective activity than Piracetam was also described on the model of Fe ²⁺ induced death of rat cerebellum neurons (N.A. Andreeva, E.V. Stimalshuk, N.K. Isaev et al. // Bull. Biol. and honey 2000. 130, No. 10, 418-421]. The listed data indicating Noopept’s ability to cause a shift in the ratio of the activity of pro- and antioxidant systems to the prevalence of the latter, were the basis for studying the effect of Noopept on the severity of diabetes manifestations. Noopept as an object of this study wa Other aspects of Noopept's multicomponent mechanism of action also played a role: its ability to enhance the expression of neurotrophic factors [Ostrovskaya R.U., Gudasheva T.A., Tsaplina A.P. et al. // Bulletin of the exp. biol. honey. 2008. No. 9. From 309-312], weaken the effects of pro-inflammatory cytokines [Kovalenko L. P., Shtpaeva E. V., Alekseeva S. V. et al. Bull. Exp. Biol. Med. 2007, 144, 7, 54-57 ] and have a multicomponent antithrombotic effect [Ostrovskaya R.U., Lyapina L.A., Pastorova V.E. Experiment and clinic. pharmacology. 2002, 65, 2, 34-37].

It is known that in the experiment the development of hyperglycemia can be reproduced by the introduction of diabetogenic toxins alloxan and streptozotocin, which have a direct cytotoxic effect on pancreatic β-cells. The advantage of streptozotocin is a relatively higher metabolic stability, a longer duration of hyperglycemia, less pronounced acidosis and mortality in experimental animals [T. Szkudelski. // Minireview Physiol. Res. 50: 536-546, 2001]. Streptozotocin is an alkylating compound derived from nitrosourea, which:

1) promotes the generation of free radicals;

2) alkylates DNA, activates poly-ADP-ribose synthetase, inhibits DNA synthesis and proliferation of β-cells;

3) inhibits the active transport of calcium and calmodulin-activated protein kinase [D.A. Rees // Diabet Med. 2005. Apr; 22 (4): 359-70].

SUMMARY OF THE INVENTION

Our goal was to study the effect of Noopept on the severity of the effect of the diabetic toxin streptozotocin (STZ). This goal was achieved by studying the effect of Noopept in an experiment with different methods of administration (different modes and routes of administration) on the model of developed (examples 1-4) and developing (example 5) CT3 diabetes. The technical result of the invention is the sugar-lowering and glucose tolerance-improving effect of N-phenylacetyl-L-prolylglycine ethyl ester and Noopept drug on a model of streptozotocin diabetes. The experiments were conducted on male Wistar white rats weighing 210-240 g (from the beginning of the experiment) obtained from the Pillar nursery of the Russian Academy of Medical Sciences. 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 (with the exception of 12 hours preceding the introduction of STZ in a model of developed diabetes or determination of glucose tolerance in a model of developing diabetes when the food was removed). 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.

The results obtained in these experiments are illustrated by the following examples.

Example 1. The effect of combined (prophylactic and therapeutic) administration of Noopept on a model of advanced diabetes.

The experiment was performed on 30 rats randomly divided into 3 groups of 10 animals each. 1. Animals of the passive control group (GPK1). They were daily injected with saline (RF) for 29 days. 2. Rats of the active control group (HAC1). They were administered RF for 14 days, then once Streptozotocine (Sigma) at a dose of 45 mg / kg, dissolved in RF, then the administration of RF continued for the next 14 days. 3. Rats of the experimental group (OG1). They were prophylactically injected with Noopept daily at a dose of 0.5 mg / kg, dissolved ex tempore in RF, then CTZ was once administered. and further for 14 days continued the introduction of Noopept. In these experiments, as in the experiments described in examples 2-4, both STZ and Noopept or FR were administered intraperitoneally.

Determination of the glucose level in blood taken from the tail vein (One Touch Ultra glucose meter, USA) was carried out after 3, 14 and 28 days (i.e., 14 days after the termination of Noopept administration) after the administration of STZ. To determine the pain sensitivity, thermal effect was used in the tail-flick test (TSE Analgesia System, USA), and the latent period of tail withdrawal was noted.

In the statistical processing of data, the significance of differences between groups for all indicators was determined using the non-parametric Mann-Whitney test (after a preliminary assessment by Kruskall-Wallis).

To clarify the comparative effectiveness of Noopept with different modes of administration and its comparison with other antidiabetic drugs, the coefficient of antihyperglycemic activity (Ag) was calculated by the formula:

$$\mathrm{BUT}g=\frac{gl.\mathrm{FROM}T3-gl.(\mathrm{FROM}T3+N\mathrm{about}\mathrm{about}PePt)}{gl.\mathrm{FROM}T3-gl.FR}\times \mathrm{one\; hundred}\%,$$

where hl STZ - glucose level in the blood of animals GAK1,

ch. STZ + Noopept - glucose level in the blood of animals GO1,

ch. FR - the level of glucose in the blood of animals GIC1.

Δ body weight was also calculated - the average value of the difference in body weight between the current and previous measurements.

According to published data, a glucose content of more than 13-15 mmol / L is considered as an indicator of diabetes [A. Kuhada, M. Bishnoia, V. Tiwuria el al. // Pharmacology Biochemistry and Behavior. 2009. V.92, Iss. 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 an average of 16.96 mmol / L. When testing after 14 and 28 days, hyperglycemia in the active control group persisted (testing at a later date in these experiments was not conducted). Noopept in the conditions of its long-term administration (prophylactic and therapeutic, for a total of 28 days) eliminates the hyperglycemic effect of the diabetic toxin (Fig. 1), reducing the level to values close to those of the passive control group. The coefficient of antihyperglycemic activity of Ag after 14 days was 95.2%. It is important to emphasize that 14 days after the cancellation of Noopept, its antihyperglycemic effect persisted - the coefficient remained at the same high level - 96.79%.

An important indicator of the diabetic effect of STZ is weight loss. Noopept prevents this effect (Fig. 2): if the animals of the active control group lost an average of 34 ± 16.3 g in weight (the difference between the initial mass and the mass 14 days after the introduction of STZ), then against the background of the Noopept action, they not only lost weight, but also gained weight (an average of 29.3 ± 3.67 g). This effect persisted after 28 days, i.e. 14 days after discontinuation of Noopept therapy. Thus, in these experiments, Noopept was shown to be highly effective for combined therapeutic and prophylactic administration, and it was established that this effect persists after discontinuation of therapy.

Example 2. The effect of the prophylactic administration of Noopept on a model of advanced diabetes.

The experiments were performed on 30 rats randomly divided into 3 groups of 10 animals each. 1. Animals of the passive control group (GPC2) They were injected daily intraperitoneally (iv) with physiological saline (RF) for 15 days. 2. Rats of the active control group (HAC2), which were injected with RF for 2 weeks, then CTZ was administered once in a dose of 45 mg / kg, dispersed in physiological saline. 3. Animals of the experimental group (OG2), which were prophylactically daily for 14 days injected with freshly prepared Noopept solution (0.5 mg / kg), and then STZ 45 mg / kg. Determination of glucose level in blood taken from the tail vein (One Touch Ultra glucose meter, USA) in these experiments, as well as in the experiments described in examples 3 and 4, was carried out 3, 14 and 28 days after STZ. Animals were weighed every 3 days.

Under the conditions of prophylactic administration, Noopept also exhibits an antihyperglycemic effect (table 1). After 3 days, Ar was 41%, while after 14 days there was a tendency to a further increase - 67.6%. Against the background of the preventive administration of Noopept, the loss in body weight was 2 times lower than in the active control group (3 days after the introduction of STZ), and after 14 days the animals even began to gain weight (table 2).

Table 1 The effect of Noopept, administered prophylactically for 14 days, on hyperglycemia caused by the introduction of STZ 45 mg / kg iv groups 3 days after STZ or FR 2 weeks after STZ GPK2 6.22 ± 0.18 5.9 ± 0.13 HAK2 20.1 ± 1.87 p≤0.05 (comparison with CCP2) 20.27 ± 3.2 p≤0.05 OG2 (PREVENTION) 14.4-1: 1.81 p≤0.05 (comparison with HAK2) 10.55 ± 1.66 p≤0.05 Ag 41% 67.6%

table 2 The change in body weight of rats in terms of body weight D (the average value of the difference in body weight between the current and previous measurements in grams) with the prophylactic administration of Noopept (0.5 mg / kg iv for 2 weeks) to STZ (45 mg / kg) groups 2 weeks before the introduction of STZ 3 days after STZ or FR 2 weeks after STZ GPK2 19.2 ± 4.97 5.2 ± 3.62 32.1 ± 6.22 HAK2 10.8 ± 1.97 -22.2 ± 3.68 p≤0.05 (comparison with CCP2) -10 ± 6.47 p≤0.05 OG2 (PREVENTION) 12.25 ± 3.4 -13.25 ± 3.13 p≤0.05 (comparison with HAC2) 15.25 ± 8.53 p≤0.05

In experiments with determining the threshold of pain sensitivity, it was shown that the latent time of tail withdrawal in the passive control group was 3.01 ± 0.34 s. In diabetic rats, it was reduced to 2.16 ± 0.35 s, which corresponds to the literature on the ability of STZ to cause diabetic neuropathy [A.I. Vinik. R.E. Maser, B.D. Mitchell el al. // Diabetes Care. 2003.26 (5): 1553-79; P.S.van Dam // Diabetes Metab Res Rev. 2002. 18 (3): 17 6-84].

It was found that in rats that were treated with Noopept prophylactically, the threshold of pain sensitivity was significantly increased (4.63 ± 1.22 s) compared with the group of rats with active control.

Example 3. The effect of therapeutic administration of Noopept on a model of advanced diabetes.

The experiments were performed on 3 groups of rats (N = 10 in each). 1. The passive control group (GPKZ) consisted of rats, which were administered daily RF for 15 days. 2. Animals of the active control group (GACZ) were once administered STZ, and then RF was administered daily for 14 days. 3. The experimental group (OGZ) consisted of rats, which were first administered with STZ, and then immediately after STZ Noopept 0.5 mg / kg was administered intraperitoneally; this treatment lasted for 14 days.

It was shown that 3 days after the first administration of Noopept, the glucose level of OG did not differ significantly from that of HAC (Ar was only 8.4%), however, after 14-day therapy, Ar was 28.9%, and in the next 14 days, i.e. . after the cessation of therapy, it continued to grow and amounted to 42.35% (table 3). There was a decrease in body weight in the active control group; in rats administered Noopept, the loss in body weight was less pronounced, and after 28 days it stopped (table 4).

Table 3 The glucose content (mmol / l) in the blood of rats under the conditions of a therapeutic (started immediately after STZ) two-week administration of Noopept v / br at a dose of 0.5 mg / kg / day groups 3 days after STZ or FR 2 weeks after STZ 4 weeks after STZ GPPZ 5.6 ± 0.06 5.8 ± 0.06 5.5 ± 0.04 GAKZ 14.7 ± 2.38 p≤0.05 20.36 ± 2.6 p≤0.05 (comparison with GPKZ) 14.0 ± 2.11 P≤0.05 OGP (treatment) 13.93 ± 2.64 p≤0.05 16.15 ± 3.12 p≤0.05 (comparison with GAKZ) 10.4 ± 1.87 P≤0.05 Ag 8.40% 28.90% 42.35%

Table 4 Change in rat body weight in terms of Δ body weight (the average value of the difference in body weight between the current and previous measurements in grams) under the conditions of treatment (started immediately after STZ) two-week administration of Noopept at a dose of 0.5 mg / kg / day / br groups 3 days after STZ or FR 2 weeks after STZ 4 weeks after STZ GPPZ 1.3 ± 2.19 -2.5 ± 4.63 38.1 ± 10.91 GAKZ -25.5 ± 3.3 p≤0.05 (comparison with GPKZ) -19.2 ± 5.18 p≤0.05 -4.1 ± 4.18 p≤0.05 OGP (treatment) -30.4 ± 3.77 p≤0.05 (comparison with GAKZ) -7.5 ± 15.15 p≤0.05 0.78 ± 8.78 p≤0.05

Example 4. The effect of delayed administration of Noopept on a model of advanced diabetes.

The experiments were performed on 3 groups of rats (N = 10 in each). 1. Group (GPK4) passive control, which daily FR in / b was administered for 4 weeks. 2. The active control group (HAC4), which was first given once an intravenous injection of STZ, and then, like the passive control group, were administered intraperitoneally administered FR every day for 4 weeks. 3. The experimental group (OG4), which investigated the possible "delayed" therapeutic effect of Noopept. First, STZ was once administered in / br, then RF was administered in / br within 2 weeks, and then Noopept was administered daily at a dose of 0.5 mg / kg / br for 2 weeks.

In this experiment, in which the beginning of the therapeutic effect was delayed for 2 weeks, the glucose level on the third day after the introduction of STZ in groups 2 and 3 was equally increased. The Noopept therapy, begun 14 days after STZ, led to the weakening of hyperglycemia (Ar was 37.3%), and after 28 days (against the background of already stopped therapy), the coefficient continued to increase and amounted to 52% (table 5). The decrease in body weight in rats from the active control group took place during the entire observation period - 28 days. In rats of the group with delayed administration of H after 14 and 28 days, weight loss was replaced by its increase.

Table 5 The glucose content (mmol / l) in the blood of rats under conditions of delayed therapeutic administration of Noopept (0.5 mg / kg / day for 2 weeks). groups 15 days after STZ (3 days of noopept therapy) 17 days of noopept therapy GPK4 5.8 ± 0.12 6.1 ± 0.2 HAK4 21.9 ± 2.45 p <0.05 (comparison with GPK4) 24.4 ± 4.28 p≤0.05 OG4 (treatment) 15.95 ± 3.52 p <0.05 (comparison with HAC4) 14.88 ± 3.76 p≤0.05 Ag 37.30% 52.00%

Example 5. The effects of Noopept and Januvia, administered orally, on the dynamics of experimental diabetes (model of fractional administration of toxin).

Metabolic disorders in the pancreatic insular apparatus precede the clinical manifestation of diabetes, which occurs only after the death of 80-90% of insulin-producing cells. This fact dictates the feasibility of searching for pathogenetic therapies aimed at preventing the progression of the disease in its early stages. Obviously, for the implementation of long-term prophylactic use of the drug, the most appropriate is the use of the oral route of administration. For Noopept, this path is completely acceptable, since it was previously established that dipeptides, unlike peptides of a more complex chemical structure, remain active when administered orally - an effect due to the presence of a specific ATP-dependent dipeptide transport system in intestinal enterocytes [M.Sala-Rahanal, DDFLoo B.A. Hirayama et al. // J. Physiol. 2006. V.574. P.149-166]. Pharmacokinetic studies confirmed the fact of the specific bioavailability of Noopept for the brain under the conditions of oral administration of the drug and it was shown that to achieve the same concentration of Noopept in the blood plasma under conditions of parenteral and oral administration, a 10-fold increase in the dose is required [S. S. Boyko, S. A. Korotkov , R.U. Ostrovskaya et al. // Bull. Exp. Biol. Honey. 2000. vol. 129, No. 4, 426-429]. This fact is fully consistent with the results of pharmacodynamic studies of Noopept with parenteral and oral administration of Noopept [T.Kh. Mirzoev, G.A. Romanova, E.V. Kravchenko et al. // Bull. Exp. Biol. Honey. 2001. vol. 132, No. 10, 404-408].

There is convincing evidence of the participation of the so-called incretins - hormones of the gastrointestinal tract: glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) in enhancing insulin secretion by β-cells. Since both of these peptides have low biological stability and cannot be used as drugs, it was proposed to use peptidomimetics of the indicated peptides or inhibitors of the enzyme carrying out their hydrolysis of dipeptidyl peptidase 4 (DPP-4) for the treatment of diabetes [M. Lotfy,. /. Singh, N. Kalasz el al. // Open Medicinal Chemistry Journal 201 J. Vol. 5. P. 82-92]. The first orally active inhibitor of DPP-4, introduced into medical practice in the USA in 2007-2008, was the drug sitagliptin (Januvia). Currently, it is also widely used in the Russian Federation.

It is known that a single administration of streptozotocin at doses of 45-60 mg / kg in the standard single administration regimen causes severe degenerative changes in the pancreatic β-cells, which are manifested, in particular, in prolonged severe hyperglycemia and a decrease in glucose tolerance. Models of repeated administration of small doses of streptozotocin mimic the dynamics of type 2 diabetes to a greater extent [V.I. Novikov. O.V. Molotov, A.P. Subcheck // Diabetes mellitus. 1999. No. 2. S. 37-39; K. Srinivasan. P. Ramarao // Indian J Med Res 2007. 125, 451-472].

This example illustrates the results of a comparative study of the effects of Noopept and the gold standard for treating diabetes, Januvia, on a model of developing diabetes with oral administration of both drugs.

The experiments were carried out on sexually mature (210-240 g weight) male Wistar rats. received from the nursery "Pillar" RAMS. The animals were kept in vivarium of the FSBI “V.V. Zakusova ”RAMS, 10 animals per cage with free access to standardized food and water, with the exception of 12 hours prior to determining the level of background (fasting) glycemia and glucose tolerance. Studies were carried out from 10 ⁰⁰ to 14 ⁰⁰ . The experiments were performed on 4 groups of rats (N = 12 in each).

1. For rats from the passive control group, physiological saline (RF) was administered i.v. for three consecutive days, in the same period and for the next 16 days, the RF was injected into the rats through a gastric tube. 2. Streptozotocin (Sigma), a diabetic toxin causing selective oxidative stress, DNA alkylation, and apoptosis in pancreatic β-cells, was injected into rats from the active control group. Cytotoxin was administered i.v. at a dose of 30 mg / kg for 3 consecutive days. 5 minutes before STZ, FZ was administered inside; the introduction of Federal Law continued further for 16 days. 3. The first experimental group in which streptozotocin (STZ) was administered i.v. at a dose of 30 mg / kg for 3 consecutive days. 5 minutes before STZ, Noopept was administered to rats orally at a dose of 5 mg / kg; administration of the drug was continued after a triple administration of STZ for another 16 days. The choice of this dose of Noopept is due to the fact that Noopept under the conditions of intraperitoneal administration showed antidiabetic activity at a dose of 0.5 mg / kg, and, according to our previous experience, when switching from the parenteral route of administration of dipeptides to the oral one, a 10-fold increase in the dose is required to exhibit a similar effect . 4. Animals of the second experimental group of STZ were injected / br at a dose of 30 mg / kg for 3 consecutive days. 5 minutes before STZ, Januvia was administered orally at a dose of 5 mg / kg; administration of the drug was continued for a further 16 days. The choice of this dose of Januvia was made on the basis of numerous literature data [Ferreira L. Teixeira-de-Lemos E., Filipa Pinto F. et al. // Mediators Inflamm. 2010. Art. AD 592760; Lotfy M, Singh J., Kalasz H el al. // Open Medicinal Chemistry Journal 2011. Vol. 5. P.82-92].

Determination of glucose level in blood taken from the tail vein (One Touch Ultra glucose meter, USA) was carried out on the 4th (counting from the day of the first injection of STZ), then on the 12th and 19th day. Along with the determination of the basal (fasting) blood glucose level in rats of all four groups, a glucose tolerance test was performed, for which it was administered intraperitoneally at a dose of 1000 mg / kg; blood glucose was determined 1 and 2 hours after exercise. Animals were weighed every 3 days.

If in the group of passive control the initial glucose level was 4.9 mmol / L, and after loading after 1 hour it did not significantly differ from the control (5.28 mmol / L), then in animals with HAC, which was administered STZ, the day after the third injection of STZ, there was not only an increase in spontaneous glucose level (up to 10.84 mmol / L), but also a decrease in glucose tolerance - 1 hour after exercise the glucose level in the blood was 17.15 mmol / L, and after 2 hours - 17.93 ± 2.05 mmol / L. On the 12th day of the experiment, the background glucose level began to decrease, but glucose tolerance remained impaired: on the 19th day of the experiment, this group fully normalized the background level while the glucose load tolerance was still reduced (table 6). The body weight of rats at all periods of observation in this and experimental groups remained unchanged.

Analyzing the diabetes models used, it should be emphasized that in experiments in which STZ was administered once at doses of 45-50 mg / kg, the level of basic hyperglycemia, in full accordance with published data, even after 28 days after the administration of the toxin was not less than 16 mmol / l, then on the model of fractional administration, it did not exceed 10.8 mmol / l during the period of maximum severity (4th day of the experiment), and already 9 days after the introduction of the toxin it reached 6 mmol / l. It is important to emphasize that on the used model of fractional administration of STZ in the presence of a basic glucose level close to normal, tolerance to glucose load remained impaired. Since it is known that reduced glucose resorption with a normal background level of glucose is characteristic of the initial phase of diabetes [Dedov II, Shestakova MV // Diabetes. Algorithms for specialized medical care for patients with diabetes mellitus. 2011, No. 3], it can be concluded that the applied regimen for the administration of STZ (30 mg / kg for 3 consecutive days) simulates the development of diabetes. This assumption is also supported by the absence of weight loss - an effect characteristic of the action of STZ administered once at a higher dose [Skudelski T. // Physiol. Res. 2001. Vol. 50, No. 6. P. 536-546].

In the group of rats to which Noopept was administered, the very next day after the end of STZ administration, firstly, a pronounced decrease in the background glucose level (up to 5.54 mmol / L) was observed, which corresponds to the calculated indicator of the relative antihyperglycemic activity of Ag 90.4 % In addition, which is very important, Noopept already at the beginning of the experiment improved glucose load tolerance - the glucose tolerance indicator did not significantly differ from that in the passive control group. 9 days after the end of the exposure to the diabetic toxin (day 12 of the experiment), in the rat group treated with Noopept, all the indices did not differ from the passive control group, i.e. Noopept restored basal glycemia and normal glucose tolerance under stress. This corresponded to an increase in Ar indices for both background glycemia and exercise glycemia. Finally, when testing 16 days after the end of the introduction of STZ in the active control group, there was a restoration of the spontaneous glycemia level with some improvement in glucose load tolerance, and the indices in the experimental group treated with Noopept did not differ from those in the passive control group.

As follows from the data shown in table 6, in rats that were introduced Januvia, on the 4th day of the experiment, there was a restoration of the basic level of glycemia, which corresponded to a high rate of Ar (97.9%). Although this indicator was slightly higher than that for Noopept, the effect of Januvia on glucose tolerance, especially in the first hour, was less pronounced than that of Noopept (Fig. 3). On the 12th day of the experiment (9 days after the end of the toxin administration), when the background glycemia level started to decrease in the active control group, but glucose load tolerance was reduced (15.67 and 9.86 mmol / L 1 and 2 hours after load, respectively), in the group of rats treated with Yanuvia, the background glucose content was lower and glucose tolerance was better than in the active control group. On the 19th day of the experiment (16 days after the end of the toxin administration) in the active control group, there was a restoration of the background level of glycemia, but glucose load tolerance after 1 hour remained reduced, which was reflected in a high glucose level (12 mmol / L) through 1 hour after exercise. In the group of rats treated with Yanuvia, the blood glucose level against the background of the load decreased only to 8.8 mmol / L, and the value of Ar was only 43.6%, i.e. was almost two times less than that in the group treated with Noopept (84.2%).

Table 6 The effect of Noopept and Januvia on the background level of rat blood glucose (mmol / l) and hyperglycemia 1 hour and 2 hours after glucose administration (1000 mg / kg iv) on a model of developing diabetes (30 mg / kg iv administration of STZ) br for three consecutive days) (M ± SM). * On the 19th day of the experiment in the 2-hour interval after exercise, no determination was made 4th day 12th day 19th day * background 1 hour 2 hours Background 1 hour 2 hours Background 1 hour FR + FR 4.98 ± 0.24 5.28 ± 0.13 5.1 3.82 ± 0.17 5.5 ± 0.18 4.46 ± 0.1 3.98 ± 0.16 4.7 ± 0.4 STZ + FR 10.84 ± 1.31 17.15 ± 1.74 17.93 ± 2.05 6.74 ± 1.4 15.67 ± 1.75 9.86 ± 1.9 5.27 ± 0.44 12 ± 1.9 STZ + Noopept 5.54 ± 0.14 6.07 ± 0.39 6.15 ± 0.25 3.91 ± 0.12 5.18 ± 0.26 4.34 ± 0.08 4.32 ± 0.19 5.85 ± 0.4 Ag% Noopept 90.4% 93.35 91.7% 96.9% 103% 102.2% 73:% 84.2% STZ + Januvia 5.11 ± 0.28 10.04 ± 1.3 7.25 ± 1.25 4.8 ± 0.13 6.04 ± 0.8 5.9 ± 0.43 4.78 ± 0.29 8.81 ± 0.8

Thus, the Noopept drug in the context of the used model of developing diabetes is not inferior to the "gold standard" - the orally active antidiabetic drug, Januvia. If in the first days after the end of the toxin administration the indicator of antihyperglycemic activity with respect to the background glucose level for both drugs was close, then later on in the group treated with Yanuviya, it decreases more sharply than in the group of rats administered Noopept. By the end of the experiment, Noopept’s glucose tolerance was 2 times higher than Januvia.

The totality of the data indicates the ability of the drug Noopept to weaken the severity of the metabolic effects of diabetic toxin. This effect was revealed both in conditions of a pronounced form of diabetes, caused by a high dose of streptozocin, and in mild diabetes, due to the repeated administration of diabetic toxin in small doses. It is very important to emphasize that the effectiveness of noopept identified in this study is maintained under conditions of oral administration.

Previous experimental studies have shown that Noopept, even under conditions of prolonged administration, does not show signs of toxic effects [L.P. Kovalenko, N.M. Smolnikova, S.V. Alekseeva et al. Experimental pharmacology and toxicology. 2002, 65, No. 1b, 62-64]. The clinical use of the drug as a nootropic also did not reveal side effects [www. Noopept.ru].

N-phenylacetyl-L-prolylglycine ethyl ester and Noopept drug, which has a nootropic and neuroprotective effect, demonstrate their effectiveness both for the prevention and treatment of diabetes: they act as a means of multicomponent pathogenetic therapy, the action of which is aimed at preventing the progression of diabetes.

A brief description of the drawings.

In FIG. Figure 1 shows the effect of noopept with the combined (prophylactic and curative, for 28 days) action of a drug administered at a dose of 0.5 mg / kg bp on blood glucose under conditions of a developed diabetes model (administration of STZ at a dose of 45 mg / kg one-time). Data are presented as M ± SEM; abscissa - time, days after the introduction of STZ; along the ordinate axis is the blood glucose content, mmol / l. Significant differences (p <0.05) according to the non-parametric Mann-Whitney criterion are presented as: * - significance of differences between passive control (RF) and active control (STZ); # - significance of differences between active control (STZ) and the Noopept group.

In FIG. Figure 2 shows the effect of noopept with combined (prophylactic and therapeutic) administration on rat body weight dynamics under the conditions of a developed diabetes model (administration of a single dose of STZ at a dose of 45 mg / kg). Data are presented as M ± SEM; abscissa - time, days after the introduction of STZ; along the ordinate axis, the difference in rat body weight (indicator Δ) for different observation periods, grams: for the 14th day, Δ between the 14th and 3rd day after the introduction of STZ; for the 28th day - Δ between between the 28th and 14th day after the introduction of STZ.

Significant differences (p <0.05) according to the non-parametric Mann-Whitney criterion are presented as: * - significance of differences between passive control (RF) and active control (STZ); # - significance of differences between active control (STZ) and Noopept.

In FIG. Figure 3 presents data comparing the efficacy of Noopept (5 mg / kg) and Januvia (5 mg / kg), administered orally on a model of developing diabetes caused by repeated administration of STZ (30 mg / kg / b for 3 consecutive days). Indicators of the 4th day (1st day after the end of the last introduction of STZ). The abscissa shows the time points: on an empty stomach, 1 and 2 hours after glucose loading (1000 mg / kg iv); along the ordinate axis is the blood glucose content, mmol / l. Significant differences (p <0.05) according to the non-parametric Mann-Whitney criterion are presented as: * - p <0.05 for the difference between passive control (FR) and active control (FR and STZ); # - p <0.05 for the difference between the active control group (RF and STZ) and the group treated with Yanuviya (Januvia and STZ), ** - p <0.05 for the difference between the active control (STZ) and the group treated with Noopept (Noopept and STZ).

Claims (2)

1. The use of ethyl ester of N-phenylacetyl-L-prolylglycine as a means for the prevention and treatment of diabetes. 2. The use of the drug Noopept as a means to prevent and treat diabetes.

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