RU2480234C1 - Method for prevention and treatment of carbohydrate and lipid metabolism disorder in metabolic syndrome and encephalopathy, use of tetrapeptide and pharmaceutical composition for prevention and treatment of such disorders - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to medicine. There are presented method, use and pharmaceutical composition for prevention and treatment of carbohydrate and lipid metabolism disorders in metabolic syndrome and encephalopathy of atherosclerotic, vascular genesis and diabetic encephalopathy, including the introduction of tetrapeptide Pro-Gly-Pro-Leu of general formula.

EFFECT: higher effectiveness of prevention and treatment of carbohydrate and lipid metabolism disorders in metabolic syndrome and encephalopathy due to the use of the tetrapeptide Pro-Gly-Pro-Leu.

5 cl, 1 dwg, 6 tbl, 4 ex

Description

The invention relates to medicine, pharmacology and relates to a peptide drug, its pharmaceutical composition and method of treatment and prevention and disorders of carbohydrate lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy.

Currently, the prevalence of metabolic syndrome, i.e. a complex of metabolic, hormonal and clinical disorders, which are based on insulin resistance and compensatory hyperinsulinemia, ultimately leading to the development of obesity, hypertension, impaired carbohydrate and lipid metabolism, stroke, myocardial infarction, type II diabetes mellitus (insulin-independent) , violations of the function of the anticoagulant system of blood and other serious diseases. Excessive accumulation of fat in adipose tissue occurs when carbohydrates prevail in food. The increased formation of fat from carbohydrates is facilitated by activation of the pentose and glycolytic pathways under the influence of insulin and prolactin (Rajesh et al, 2005). Insulin inhibits the release of fat from the depot and facilitates the accumulation of glycogen in the liver. By lowering blood glucose levels, insulin boosts appetite. Violation of this entire complex system of neurohumoral regulation underlies excess fat deposition in adipose tissue - obesity (GM Kronenberg et al., 2010). Violations of carbohydrate and lipid metabolism in combination with other causes can lead to degenerative changes in brain tissue, i.e., in turn, to the development of encephalopathy.

It is known that a number of regulatory proline-containing peptides (Pro-Gly-Pro, Pro-Gly, Gly-Pro, Phe-Pro-Gly-Pro) are constantly formed in the body during the degradation and synthesis of collagen and other proteins (Ulyanov et al., 2005) , which allows us to consider such peptides as endogenous compounds. In experiments and clinical studies, the extreme multifunctionality of regulatory peptides, including the simplest oligopeptides, has been shown (Gerstein et al., 1998).

Peptides play an important role in regulating the state of the central nervous system. By acting on brain activity, peptides are able to change behavior, emotional status, motivation level, processes of remembering and storing information (see, in particular, RF Patent No. 2206573). Glyprolins Pro-Gly-Pro, Pro-Gly, Pro-Gly-Pro-Arg, Pro-Gly-Arg, Gly-Pro-Arg possess neuroprotective properties (Watchdogs T.P. et al., 2007), ensure the preservation of normal function insular and anticoagulant blood systems against the background of the development of experimental alloxan diabetes (see, in particular, Ashmarin I.P. et al., 2008).

A number of amino acids (proline, lysine, arginine, methionine, glutamine, etc.), including the essential amino acid - leucine, are involved in normalizing triglycerides and lipoprotein balance, and prevents the deposition of low density lipoproteins on the walls of arteries; reduce the risk of a wide range of diseases, including diabetes, atherosclerosis, atherothrombosis, etc. (Mazurov V.I., 1984). So, leucine has the ability to burn glucose (Nishitani et al., 2005) by stimulating the process in the glucose-alanine cycle, due to which the body maintains a stable level of sugar and maintains the necessary muscle mass in a low-calorie diet.

With the continuous process of collagen proteolysis and other related proteins in the body, the formation of ultimately simple peptides containing amino acids such as glycine, proline, hydroxyproline (Molinero et al, 2008), including Pro-Gly-Pro, Pro- Gly, Pro-Gly-Pro-Arg, Pro-Gly-Arg, Gly-Pro-Arg, which ensure the maintenance of the normal function of the insular and anticoagulant blood systems (see, in particular, RF Patent No. 2206573). Given that hyperglycemia contributes to the accumulation of fat in fat depots, and the aforementioned glyprolins have a hypoglycemic effect, we can assume that these same short peptides, especially including leucine in their structure, are able to participate in the metabolism of not only carbohydrates, but also fats.

Thus, peptides that are practically devoid of side effects, possessing neuroprotective properties, the ability to positively affect the insular system and lipid metabolism, can be included in the prevention and treatment of disorders of carbohydrate and lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, and diabetic encephalopathy.

Various drugs are available to treat metabolic disorders.

However, none of them simultaneously combines high efficiency, duration of action, the speed of the onset of the effect and the absence of negative aftereffect with prolonged use. Natural peptides are currently not used for the prevention and treatment of metabolic disorders.

The problem solved by this group of inventions is to create a highly effective drug with a wide spectrum of action, convenient to use and with minimal side effects, while the result achieved in solving this problem is to increase the effectiveness of prevention and treatment of disorders of carbohydrate and lipid metabolism with metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy.

To achieve the result, a method is proposed for the prevention and treatment of disorders of carbohydrate and lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy, including intranasal administration of a drug based on the tetrapeptide of the general formula Pro-Gly-Pro-Leu.

To achieve the result, it is also proposed to use the tetrapeptide of the general formula Pro-Gly-Pro-Leu as a means of preventing and treating disorders of carbohydrate and lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy. In addition, in the framework of the present invention, in order to achieve the result, a pharmaceutical composition is proposed for the prevention and treatment of disorders of carbohydrate and lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy, containing as active substance a tetrapeptide of the general formula Pro-Gly-Pro- Leu however, the composition can be performed for intranasal administration, for example, of the following composition: tetrapeptide in an amount of 1-10 g / l, nipagin in an amount of 0.9-1.1 g / l as a preservative, distilled water - the rest.

The invention is illustrated by the graph of the effect of various doses of Pro-Gly-Pro-Leu (PGPL) on the survival of cerebellar granular cells upon hyperstimulation of glutamate receptors induced by 300 μM NMDA (N-methyl-D-aspartate) in the presence of 5 mM Ca ²⁺ .

The following are examples illustrating the invention. Research conducted on experimental animals.

Example 1. The study of the effect of the PGPL tetrapeptide on the parameters of lipid metabolism and the accumulation of new fatty deposits in the body of rats on a fatty diet

The experiments were conducted on 30 experimental animals, 20 of which were kept for 15 days on a fat diet enriched with saturated fatty acids and cholesterol. The determination of the effect of PGPL tetrapeptide on lipid metabolism was carried out as follows. Male rats weighing 270-400 g were administered intranasally for 11 days with a PGPL tetrapeptide of 175 μg / kg body weight in a volume of 0.02 ml per 200 g of body weight or a 0.85% NaCl solution in the same volume ( control group 1). 10 normal healthy rats that were on a normal laboratory diet and did not receive any drugs made up control group 2. One hour after the last administration of the tetrapeptide PGPL, animals were bled for analysis. Indicators of fat metabolism in blood plasma were investigated by enzymatic colorimetric method. The concentration of total cholesterol (OH), the concentration of cholesterol (Ch) of high density lipoproteins (HDL) and the concentration of triglycerides (TG) were determined. The total concentration of low density lipoprotein cholesterol (LDL) and very low density (VLDL) was determined by the following formula:

Xc (LDL + VLDL) = OH-Xc (HDL).

The obtained blood test data are shown in table 1.

Table 1 Experience Conditions OH, mmol / l Xc (LDL + VLDL), mmol / l Xc (HDL), mmol / l TG, mmol / l Control group 2 (healthy rats) n = 10 0.84 ± 0.025 0.16 ± 0.018 0.68 ± 0.04 0.44 ± 0.023 Control group 1 n = 10 1.14 ± 0.03 0.33 ± 0.03 0.81 ± 0.03 0.56 ± 0.04 (one hundred%) (one hundred%) (one hundred%) (one hundred%) Experience n = 10 0.76 ± 0.04 0.14 ± 0.01 0.62 ± 0.05 0.46 ± 0.029 (67%) ** (42%) ** (77%) ** (82%) *

The significance of differences with respect to the corresponding parameters of the control group, taken as 100%: ** p <0.01, * p <0.05

As can be seen from the table, the PGPL tetrapeptide improves the parameters of fat metabolism. The studied peptide restored normal values of the lipid profile while eating foods high in animal fats enriched with cholesterol, saturated fatty acids and carbohydrates, which ultimately leads to an increase in blood levels of total cholesterol and atherogenic lipoproteins of low and very low density, as well as triglycerides. The latter is associated with the anticholesterol effect of the studied peptide, which was first discovered as a result of the studies.

Along with the determination of the lipid profile in rat blood, rats were weighed, the results of which are presented in Table 2.

table 2 Experience Conditions Experience (n = 10) Control (n = 10) Before the experience, (g) 396.4 ± 13.9 414 ± 9.78 (one hundred%) (one hundred%) After the 11th introduction peptide on the background of fat 407.0 ± 16.2 465 ± 18.3 diet, (g) (103%) (112%) Weight gain after 15 days being on fat 11.4 ± 1.4 ** 51.0 ± 4.81 diet, (g)

Summing up, after 11 days of using the PGPL tetrapeptide (or NaCl) against the background of a fatty diet, the experimental rats recovered by an average of 11.4 g, and the control rats by 51 g compared to the initial weight values, i.e. weight gain in the control group was almost 4.5 times greater than in the experimental group. Consequently, the PGPL peptide (in particular, in a daily dose of 175 μg / kg) with the simultaneous intake of fatty foods in the body over 2 weeks contributed to a decrease in body weight of animals by almost 4.5 times compared to the control when the animals were on the same fat diet and instead of the peptide, NaCl was obtained.

Thus, the leucine-containing PGPL gliprolin can be attributed to lipid-lowering drugs that reduce weight and block the accumulation of new body fat.

Example 2. A study of the effect of PGPL tetrapeptide on glycemia in rats on a carbohydrate and fat diet

The experiments were carried out on 60 outbred white male rats with a body weight of 250-340 g contained in a natural laboratory diet. Experimental persistent hyperglycemia in animals was caused by daily intragastric administration of a 40% glucose solution at a dose of 0.7 ml per 300 g of body weight for 7 days. Then the animals were divided into 2 groups (experimental and control), which continued to receive glucose. At the same time, a test group of rats was intranasally injected with a PGPL peptide solution (200 μg / kg in a volume of 0.02 ml) four times every 24 hours, and a 0.85% NaCl solution was administered in the same way and in the same volume in the same volume. On the 5th day on the day of the experiment, the rats were first injected with a glucose solution, then after 30 minutes a peptide solution (experiment) or a 0.85% NaCl solution (control). Blood was taken for analysis 1 h after administration of the peptide (or NaCl) and drug administration was then canceled. The next blood test was performed 5 days after the cancellation of all drugs.

Experimental rat hypercholesterolemia was performed with a fatty diet enriched with saturated fatty acids and cholesterol for 15 days. At the same time, the experimental animals received intranasally daily (1 time per day) for 11 days a preparation based on the PGPL peptide in a dose of 175 μg / kg in a volume of 0.02 ml. Blood was taken for analysis 1 h after the last injection of the peptide (experiment) or 0.85% NaCl solution (control).

The experimental results are presented in tables 3 (the dynamics of changes in blood parameters with intranasal administration of PGPL peptide at a dose of 1 mg / kg to rats 1 time per day for 5 days on the background of sugar load) and 4 (blood sugar level of animals 1 hour after 11 - daily administration of PGPL (experiment) or NaCl (control) while animals were on a cholesterol diet for 15 days).

Table 3 Experience Conditions Experience (introduced by PGPL) n = 10 Control (NaCl introduced) n = 10 Norm (healthy animals) n = 10 Sugar level 1 h after 5-fold administration of PGPL or NaCl, mmol / L 3.42 ± 0.21 ** 9.83 ± 1.0 ^## 5.0 ± 0.65 * Sugar level 5 days after drug withdrawal, mmol / l 3.79 ± 0.36 ** 9.83 ± 1.0 ^## 5.0 ± 0.65 **

Table 4 Experience Conditions Blood sugar level (mmol / L) (at the end of the experiment) Norm, n = 10 3.75 ± 0.15 Control, n = 10 6.0 ± 1.2 (100%) Experience, n = 10 4.3 ± 0.3 (71%) ** Note. Statistical indicators are calculated relative to the corresponding norm indicators - ^## p <0.01, ^# p <0.05 or control ** p <0.01, * p <0.05

An animal blood test 1 hour after the last 5th intranasal administration of the studied PGPL peptide to rats against the background of simultaneous administration of a glucose solution showed that the blood sugar level of the experimental rats was 65% lower than the control values.

5 days after the cancellation of all injected drugs, the blood sugar level of the experimental group of animals remained significantly reduced in relation to the control (table 3), therefore, the use of the PGPL peptide (in particular, five times 1 time / day at a dose of 200 μg / kg) per background sugar load has a protective hypoglycemic effect, helping to maintain normal blood sugar levels in experimental animals.

From table 4 it can be seen that when animals were kept on a fat diet with simultaneous intranasal administration of the PGPL peptide (in particular, at a dose of 175 μg / kg), in the experimental group of rats, the sugar level 1 hour after the last 11th administration was significantly lower than the values sugar in the control group and almost corresponded to the sugar level in normal rats. Thus, the leucine-containing PGPL gliproline, used during the period of intake of fatty diets in rats, protected the animals from hyperglycemia caused by fatty and sugary foods.

Example 3. The study of the effect of PGPL tetrapeptide on calcium homeostasis and the survival of brain neurons during hyperstimulation of glutamate receptors

The experiments were conducted on 7-9-day primary cultures of rat cerebellum granular cells with expressed glutamate receptors and suppression of glial cell proliferation. A one-hour exposure to 100-200 μM glutamate was used. The survival of neurons subjected to the toxic effects of glutamate was evaluated by a biochemical method.

The results of the effect of various doses of PGPL on the survival of cerebellar granular cells during hyperstimulation of glutamate receptors induced by 300 μM NMDA (N-methyl-D-aspartate) in the presence of 5 mM Ca ²⁺ are shown in the graph. In the presence of the preservative B-27 (growth factors and insulin) and a high concentration of K ⁺ (25 mM), the PGPL tetrapeptide at a dose of 200 μM had virtually no effect on the death of cereal granular cells. At the same time, the introduction of PGPL in doses of 5-200 μM reduced the death of neurons. Thus, 300 μM NMDA in the presence of 5 mM Ca ²⁺ caused the death of approximately 80% of neurons, preincubation of neurons with PGPL for 60 min, then it was present during the entire experiment, and in a dose-dependent manner it increased neuron survival by 10 to 35%. The most significant protection of cells against excitotoxicity was revealed at a dose of 200 μM PGPL.

The effects of PGPL on disturbances in calcium homeostasis and, in particular, on the development of delayed calcium dysregulation (OKD) in cerebellar neurons with excitotoxicity caused by NMDA, were studied in experiments on sister cell cultures on the same day as in experiments on neuron survival. The results of the experiment are presented in table 5.

Table 5 Sum the number of cells that had OKD Sum the number of cells that had OKD,% total number of cells the control 135 62 150 PGPL 25 25 149

As can be seen in table 5, PGPL at a dose of 200 μm delayed the development of OKD. If in control cultures, NMDA caused the development of OKD in 62% of neurons, then preincubation of neurons with PGPL and its presence in buffer solution together with NMDA reduced the number of neurons with OKPL to 25%.

Example 4. Investigation of the effect of PGPL tetrapeptide on the survival of cultured rat pheochromocytoma cells of PC ₁₂ under oxidative stress

The cells were incubated for 30 minutes in a medium with 1 mM H ₂ O ₂ . Such an effect led to the development of necrotic processes, in particular, to the disruption of the integrity of the cell membrane, which was detected several hours after the induction of oxidative stress. As a comparison substance, the peptide Pro-Gly-Pro was used at a concentration of 100 μM.

The results of the protective effect of the PGPL peptide on PC12 cells under oxidative stress conditions of the experiment are presented in table 6.

Table 6 The content of necrotic cells (%) Control (no oxidative stress) 7.9 ± 1.9 Oxidative Stress: + H ₂ O ₂ 70.2 ± 5.0 ^### + PGP 100 μM 34.5 ± 8.1 *** + PGPL 10 μM 43.8 ± 4.7 *** + PGPL 100 μM 31.7 ± 5.5 *** + PGPL 300 μM 39.6 ± 11.0 ** Data are presented as mean ± standard deviation. ^### p <0.001 relative to control; *** p <0.001, ** p <0.01 relative to cells subjected to oxidative stress (t-student test).

The introduction of Pro-Gly-Pro-Leu (at concentrations of 10, 100 and 300 μM) in the medium after washing the cells from H ₂ O ₂ led to a nearly twofold decrease in the content of necrotic cells. Moreover, the greatest positive effect was observed at a peptide concentration of 100 μM. A comparison of the cytoprotective effects of Pro-Gly-Pro-Leu and Pro-Gly-Pro at a concentration of 100 μM did not reveal significant differences in the effectiveness of these peptides in the test system used. These data indicate that the Pro-Gly-Pro-Leu peptide has the neuroprotective potential characteristic of a number of glyprolines.

To summarize, the tetrapeptide Pro-Gly-Pro-Leu is a unique peptide drug for the prevention and treatment of disorders of carbohydrate and lipid metabolism in metabolic syndrome and encephalopathy of various origins. A pharmaceutical composition based on it and a method for the treatment and prevention of disorders of carbohydrate and lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy meets the requirements of efficiency and safety.

Claims (5)

1. A method for the prevention and treatment of disorders of carbohydrate and lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy, including intranasal administration of a drug based on the tetrapeptide of the general formula Pro-Gly-Pro-Leu. 2. The use of the tetrapeptide of the general formula Pro-Gly-Pro-Leu as a means of prevention and treatment of disorders of carbohydrate and lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy. 3. A pharmaceutical composition for the prevention and treatment of disorders of carbohydrate and lipid metabolism in the metabolic syndrome and encephalopathy of atherosclerotic, vascular origin, diabetic encephalopathy, containing as active substance a tetrapeptide of the general formula Pro-Gly-Pro-Leu. 4. The composition according to claim 3, made with the possibility of intranasal administration. 5. The composition according to claim 3 or 4, made as follows: tetrapeptide in an amount of 1-10 g / l, nipagin in an amount of 0.9-1.1 g / l as a preservative, distilled water - the rest.

Images