RU2403909C1 - Method of treating diabetes in mammals and intranasal pharmaceutical composition for treating diabetes in mammals - Google Patents

Abstract

FIELD: medicine, veterinary science.

SUBSTANCE: invention concerns veterinary. There is described a method of treating diabetes in a mammal with the use of peptide Arg-Pro-Gly-Pro dissolved in such proportion that the amount of the introduced peptide is 1 mg/kg of body weight. Said substance is introduced 3 times intranasally.

EFFECT: invention allows faster recovery, lower toxic effects without deterioration of clinical effectiveness.

2 cl, 3 tbl, 1 ex

Description

The invention relates to veterinary medicine and relates to a method for treating mammals other than humans, as well as drugs used in the treatment of diabetes in mammals.

A known method of treating diabetes using the tripeptide Pro-Gly-Pro (RU 2252778). A method of treating diabetes, presented in RU 2252778, includes the administration of the Pro-Gly-Pro tripeptide to the patient in an effective amount. A solution of tripeptide in a dose of 0.61-0.63 ml was used for 10 days. However, this method does not provide for intranasal administration of a substance.

The known peptide Arg-Pro-Gly-Pro, which has antithrombic, anticoagulant, fibrindepolymerization and fibrinolytic activity (RU 2286166). However, it is not known from the specified document that the indicated peptide can be used for the treatment of diabetes mellitus, moreover, an effective intranasal route of administration of the indicated substance is not known.

According to statistics, currently the incidence of diabetes in mammals (type II diabetes) has increased significantly. The number of patients annually increases by 5-10%. Type II diabetes can be due to various reasons: from predominant insulin resistance with relative insulin deficiency to a secretory defect with or without insulin resistance. For the treatment of type II diabetes, oral hypoglycemic drugs of various groups are usually used: 1) sulfonylurea derivatives while maintaining the functional activity of the beta cells of the islet pancreatic apparatus. The drugs of this group bind to specific high-affinity receptors that are localized on ATP-sensitive potassium channels on the membranes of beta cells; 2) biguanides, irrespective of the functioning beta cells of the pancreas, providing a direct stimulating effect on glycolysis in peripheral tissues, increase the binding of insulin to tissue receptors and reduce the level of glucagon in the blood; 3) acarbose, which impedes the absorption of carbohydrates in the intestine; 4) glitazones, which reduce insulin resistance and increase the sensitivity of peripheral tissues to insulin; 5) starlix (a derivative of D-phenylalanine), which, like arginine, stimulates the secretion of insulin by the beta cells of the pancreas (Fisenko, 2001).

The problem solved by the present invention is to increase the effectiveness of the treatment of diabetes by providing a pharmaceutical composition containing an effective amount of the Arg-Pro-Gly-Pro peptide, and by more effective specific administration of the composition. In this case, the problem also solved by the present invention is the provision of such a pharmaceutical composition that would eliminate undesirable side effects, and the development of such a treatment method that would be safer without loss of effectiveness. Therefore, the problem solved by the present invention is to increase the safety of treatment using a short peptide.

These problems are solved by using the peptide Arg-Pro-Gly-Pro in the amount of 1 mg / kg of body weight as a medicine. Moreover, the specified substance is administered intranasally according to a certain scheme.

Intranasal administration of the drug has several advantages:

- the presence of a central action (due to the fact that the shells of the olfactory nerves are devoid of the blood-brain barrier, substances from the nasal cavity can immediately enter the brain);

- high bioavailability;

- the absence of the effect of the first passage through the liver and the associated adverse reactions;

- convenience and ease of use, which leads to improved patient compliance;

- the speed of development of the systemic effect.

However, drugs are usually administered intranasally, predominantly having a local effect, mainly to alleviate the symptoms of rhinitis. In recent years, it has been shown that in the nasal cavity active absorption of a number of medicinal substances can occur, which are also split in the gastrointestinal tract, in particular, hormones and substances of a protein-peptide nature. The modest use of the intranasal route of administration is associated with a number of limitations:

- irritation or contact sensitization of the nasal mucosa is possible, the cause of which is the adverse interaction of active or inactive components with the mucosa;

- only a small percentage of the drug can enter the systemic circulation. This means that an excess amount of the drug must be manufactured and introduced into the aerosol can, which, ultimately, leads to an increase in the cost of the drug;

- the intranasal drug delivery system can be used for drugs with certain physicochemical properties, for penetration into the systemic circulation in a therapeutically effective amount;

- the inability to ensure a constant, without hesitation, concentration of the drug in the blood and the associated adverse reactions.

Thus, despite the attractiveness of the intranasal route of administration, its use for each specific substance requires careful research and clinical trials, since the reaction from the mucosa can be unpredictable. It is impossible to say without conducting appropriate experiments whether the necessary therapeutic effect on the patient's body will be achieved.

The authors of the present invention unexpectedly discovered the possibility of using intranasal administration to a mammal of the Arg-Pro-Gly-Pro peptide in a low concentration, without leading to a deterioration of the systemic response. In addition, this reduces the possibility of developing unwanted side effects.

The method of treatment according to the invention uses a pharmaceutical composition containing, as an active principle, an Arg-Pro-Gly-Pro peptide, which may also contain any suitable neutral carriers known to one skilled in the art for intranasal administration.

The invention is illustrated by the following examples.

Examples.

The Arg-Pro-Gly-Pro peptide used in this work was synthesized at the Institute of Molecular Genetics, Russian Academy of Sciences (Moscow).

The experiments were conducted on 48 outbred white male rats at the age of 10 months. with a body weight of 270-350 g contained in a natural laboratory diet.

Type II diabetes mellitus in animals was caused by repeated intragastric administration of a 40% glucose solution at a dose of 0.5 ml per 200 g of body weight for 7-8 days.

2 series of experiments were carried out. In the first series, experimental animals were pre-prophylactically injected with a solution of the Arg-Pro-Gly-Pro peptide intranasally every 4 hours every 24 hours (at the rate of 0.05 ml / 200 μg per 200 g of body weight, i.e., in an amount 1 mg / kg body weight). Starting from the 5th day and up to the 7th day, the peptide was continued to be administered at the indicated dose and the sugar load was simultaneously carried out. Instead of the peptide, the control animals were injected with the same volume (0.05 ml / 200 g body weight) of a 0.85% NaCl solution. Blood was taken for analysis 1 hour after the last injection.

In the second series of experiments, rats were first injected intragastrically with a 40% glucose solution in a volume of 0.5 ml per 200 g of body weight during the first 5 days and from 6 days to 8 days (i.e. 3 days) while continuing to administer glucose, 30–40 min after glucose administration, the ArgPGP peptide was intranasally administered (at the rate of 0.05 ml / 200 μg per 200 g of body weight). The control animals, as well as the experimental ones, were injected with glucose for 8 days, and starting from the 6th day, the same volume (0.05 ml / 200 g body weight) of a 0.85% NaCl solution was additionally intranasally administered. Blood was taken for analysis 1 h after the last 8th injection of the peptide (experimental rats) or 0.85% NaCl solution (control rats).

Blood was taken from v. jugularis in an amount of 2 ml using 3.8% sodium citrate solution as a preservative in a blood: preservative ratio of 9: 1.

The following biochemical parameters of blood plasma were determined: total (SFA) and non-enzymatic (NF) fibrinolytic activity (Kudrjashov, Lyapina, 1986), tissue plasminogen activator (TAP) activity (Astrop, Mtillertz, 1952), activated partial thromboplastin time (APTT) ( Dolgov et al., 1996). In platelet-rich blood plasma, platelet aggregation was determined by the method of Berkovsky et al. (2003) using a 2-5 μM ADP solution as an aggregate; determination was carried out on an aggregometer (production of Moscow State University, Moscow). In addition, coagulogram parameters were recorded (T _{1 is the} beginning of clot formation, T ₂ is the end of clot formation, T is the coagulation time) on a domestic coagulometer (Russia). Blood sugar concentration was measured by the modified Hagedorn-Jensen method (Kantorovich, 1963). Blood samples were centrifuged in different modes: to determine platelet aggregation - at 1000g for 5 min (received platelet-rich plasma), and to conduct coagulological tests and blood sugar levels in platelet-poor plasma - at 3000g for 10-12 minutes.

All data were processed statistically by the student method.

In the first series of experiments, after intranasal administration of the studied peptide for 7 days with an additional sugar load in the last 3 days, it was found that under these experimental conditions the peptide had an activating effect on the SFA and TAP levels of the blood of experimental animals. Moreover, in the experiment, SFA increased by 1.5 times, TAP - by 1.4 times compared with the same indices in control rats. The indicators of NF, platelet aggregation of experimental and control rats were similar. However, the APTT indicator of experimental rats tended to decrease compared to the control (Table 1).

Table 1.
Rat hemostasis in the case of intranasal administration of Arg-Pro-Gly-Pro at a dose of 1 mg / kg body weight (7 days) with simultaneous intragastric administration of 40% glucose in a volume of 0.5 ml per 200 g of weight during the last 3 days body. Experiment Conditions SFA, mm ² NF mm ² TAP, MM ² Platelet Aggregation,% APTT, s Glucose level, mg% Control (0.85% NaCl + glucose) n = 6 26.4 ± 2.1 20.0 ± 1.3 28.8 ± 3.1 215 ± 4.5 ^## 32.8 ± 2.5 151.6 ± 10.7 Experience (Arg-Pro-Gly-Pro + glucose) n = 6 38.3 ± 2.2 ** 23.8 ± 1.7 40.0 ± 3.6 ** 228 ± 6.5 ^## 24.7 ± 1.9 98.0 ± 7.3 ** Norm 34 ± 1.4 24.6 ± 1.0 33.3 ± 1.7 100 ± 2.0 31.7 ± 1.9 68.0 ± 5.47 Note: statistical indicators are calculated relative to the corresponding control samples (** - p <0.01) or normal ( ^##- p <0.01) (for Tables 1-2).

When measuring the level of glucose in the blood of experimental rats, its significant decrease was found to be 1.5 times lower than that in the control rats.

Since in the first series of experiments platelet aggregation increased very sharply (by more than 100%) during the development of type II diabetes in both control and experimental rats, the use of these conditions to identify the anticoagulant effect of the peptide required changes in the experiments.

And in the second series of experiments, it was found that 1 hour after 3-fold intranasal administration of the peptide against the background of the development of type II diabetes (after 5 days of glucose administration and the subsequent continuation of its administration together with the peptide), the function of the anticoagulation system (PSS) is activated and reduced blood sugar (table 2).

As can be seen from the data in Table 2, with type II diabetes, the administration of the Arg-Pro-Gly-Pro peptide intranasally for 3 days against the background of a constant influx of glucose into the bloodstream significantly increased the SFA in the blood plasma of experimental animals by 1.2 times; 1.4 times, TAP - 2.9 times, APTT-1.18 times, T ₁ coagulograms - 2.3 times, T ₂ - 1.3 times.

As follows from the experiments, this method of introducing the peptide is the most effective for the manifestation of anticoagulant-fibrinolytic and antiplatelet activity.

It turned out that for the manifestation of the hypoglycemic effect of the peptide, the conditions of the second series of experiments are most acceptable.

As can be seen from table 3, the blood sugar level in experimental rats has undergone the following changes. One hour after a single injection of Arg-Pro-Gly-Pro, the sugar level in rats increased by 24.8 mg% compared to the norm against the background of sugar loading, in the control - by 22.4 mg%.

Table 3. The sugar level (in mg%) in the blood of rats at different times after the administration of the Arg-Pro-Gly-Pro peptide after administration against the background of the development of type II diabetes. Experience Conditions Rat blood sugar in mg% 1 h after a single injection of the peptide on the background of sugar load 1 hour after triple administration of the peptide against a background of sugar load 5 days after cancellation of peptide administration NaCl + glucose control 102.4 ± 4.2
p <0.01 172.0 ± 6.1
p <0.01 80.8 ± 3.1
p> 0.1 Experience Arg-Pro-Gly-Pro + glucose 114.8 ± 6.1
p <0.01
p ₁ > 0.1 124.0 ± 4.6
p <0.05
p ₁ <0.01 81.0 ± 23.9
p> 0.1
p ₁ > 0.1 Norm 80.0 ± 2.7 92.0 ± 5.5 61.3 ± 33.1

One hour after the peptide was administered three times to experimental rats on the background of sugar load, the sugar level remained practically unchanged, however, it exceeded normal values by 26 mg%. In control rats, it was significantly increased to 172 mg% and exceeded normal values by 89 mg%.

5 days after discontinuation of the use of the peptide, a decrease in blood sugar level to 80-81 mg% was found both in the experiment and in the control, moreover, these values were significantly different from normal values.

Thus, only a 3-day use of the Arg-Pro-Gly-Pro peptide against the background of the development of type II diabetes (against the background of sugar load) led to an unreliable increase in blood sugar, while in the absence of a peptide (administration of physiological saline instead of the peptide) significant hyperglycemia was noted, and type II diabetes continued to develop. In this case, hemostasis indicators activated partial thromboplastin time, coagulogram parameters, total and non-enzymatic fibrinolytic activity, tissue plasminogen activator activity and platelet aggregation in experimental rats corresponded either to hypocoagulation against the background of the introduced peptide, or increased blood coagulability - against the background of physiological saline used instead.

Consequently, the peptide RPGP even with a single application against the background of sugar load exerts an anticoagulant and fibrinolytic effect of an enzymatic and non-enzymatic nature, reduces platelet aggregation, and its triple use simultaneously prevents the development of type II diabetes mellitus.

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Claims (2)

1. A method for the treatment of diabetes in mammals, comprising intranasal administration of the Arg-Pro-Gly-Pro peptide in the form of a pharmaceutical composition, wherein said peptide is contained in it in such a dilution that the amount of peptide administered is 1 mg / kg of mammalian body weight, wherein the specified composition is administered daily for three days. 2. An intranasal pharmaceutical composition for treating diabetes in mammals containing Arg-Pro-Gly-Pro peptide as an active principle, wherein said peptide is contained in such a dilution that the amount of the administered peptide is 1 mg / kg body weight a mammal.