RU2403910C1 - Method of treating diabetes in animals - Google Patents

Abstract

FIELD: medicine, veterinary science. ^ SUBSTANCE: invention concerns veterinary. The pharmaceutical composition for treating diabetes containing tripeptide Pro-Gly-Pro as an agent dissolved in such proportion that the amount of the intranasal tripeptide is 1 mg/kg of body weight. The method of treating diabetes involves the intranasal introduction of the described composition. ^ EFFECT: invention allows faster recovery, lower toxic effects without deterioration of clinical effectiveness, and simplified introduction procedure. ^ 2 cl, 2 tbl, 3 ex

Description

The invention relates to veterinary medicine and relates to medicines used in the treatment of diabetes.

As a medicine, the tripeptide Pro-Gly-Pro is used. This tripeptide in small amounts is constantly present in animals and humans as a product of the metabolism of larger peptides and proteins (collagen, elastin), which allows us to consider it as an endogenous compound.

It is known that this tripeptide has anticoagulant, fibrinolytic and fibrindepolymerization activity (V.E. Pastorova, L.A. Lyapina, T.Yu. Smolin, I.P. Ashmarin. Anticoagulant and fibrinolytic effects of some proline-containing peptides. Izvestiya AN, Biological Series , 1998, No. 3, p. 390-394). The antiulcer properties of the tripeptide Pro-Gly-Pro are known (Samonina EG and other Sechenov Russian Physiological Journal. 2001, November, 87 (11): 1488-1492). From RU 2252778 it is known to use this tripeptide as an agent used for the prevention and treatment of diabetes, as well as a pharmaceutical composition containing it. In the described source, solutions of the tripeptide Pro-Gly-Pro are administered orally and used as a means for the prevention and treatment of diabetes in the following concentrations - 0.04-0.2%. In the method for the prevention and treatment of diabetes, presented in RU 2252778, these solutions are taken in doses of 0.61-0.63 ml daily for 10 days.

The problem solved by the present invention is to increase the effectiveness of the treatment of diabetes and the convenience of using the drug in a pharmaceutical composition containing an effective amount of a Pro-Gly-Pro tripeptide suitable for intranasal use. In this case, also solved by the present invention, is the provision of such a pharmaceutical composition that would eliminate undesirable side effects. Therefore, the problem solved by the present invention is to increase the safety of treatment using the Pro-Gly-Pro tripeptide.

These tasks are solved by using the Pro-Gly-Pro tripeptide as a medicine in the following amount - 1 mg / kg body weight, which is administered intranasally. With this method of treatment, the drug is administered daily for 5 days.

The inventors of the present invention have unexpectedly found that lowering the amount of the Pro-Gly-Pro tripeptide administered to the animal by intranasal administration and shortening the treatment days does not lead to a deterioration in the quality of treatment. In addition, this reduces the possibility of developing unwanted side effects. It was also found that with a decrease in the amount of Pro-Gly-Pro tripeptide administered, the effectiveness of the treatment remains the same while increasing its safety.

The pharmaceutical composition according to the invention, in addition to the active principle represented by the Pro-Gly-Pro tripeptide, may also contain any suitable neutral carriers known to the person skilled in the art.

The method for treating diabetes according to the invention differs from the method presented in RU 2252778 by 5-day administration of the drug (instead of 10 days) and, at the same time, reduction of a single administered amount of active substance and the method of administration.

The invention is illustrated by the following examples.

Example 1

Take 1 mg of the preparation of the tripeptide Pro-Gly-Pro, dissolve in 0.25 ml of physiological saline and injected daily every 24 hours for 5 days intranasally to rats weighing 200-220 g of 0.05 ml (200 μg), which is marked by hypercoagulation in connection with the development of diabetes mellitus. Control animals with developed diabetes mellitus were injected in the same way and by the same procedure into a 0.85% NaCl solution in a volume of 0.05 ml per 200 g of body weight daily. Then, blood is taken 1 hour after the last injection of drugs, 8 and 20 days after drug withdrawal and centrifuged at 1000 rpm for 5 minutes to obtain platelet-rich plasma (for analysis of platelet aggregation), then additionally centrifuged blood for 10 min at 2000 rpm and receive a platelet poor plasma. In platelet-rich plasma, platelet aggregation in experimental samples was 74.5%, while in control it was 100%, i.e. the tripeptide Pro-Gly-Pro had antiplatelet activity. In platelet-poor plasma, an increase in enzymatic fibrinolytic activity was detected due to an increase in the activity of tissue plasminogen activator by 4.4 times, total fibrinolytic activity by 1.75 times, fibrin-depolymerization activity by 2.4 times, anticoagulant activity (according to the APTT test) - by 1, 9 times compared with the control (administration of physiological NaCl). At the same time, the blood sugar level in the experimental samples was lower than in the blood of control rat diabetes patients, against the background of the injected saline solution, by 2.23 times and corresponded to the normal initial value. Eight days after discontinuation of the peptide drug administration to rats with advanced diabetes, the sugar level decreased even more - 3 times as compared to the control rats with advanced diabetes, after 20 days it was 1.7 times lower in the experiment compared to control. By the 20th day of the experiment, the animal survival in the experiment was 87%, in the control - 50%. Therefore, intranasal repeated administration of the Pro-Gly-Pro peptide in a daily dose of 1 mg / kg of rat body weight led not only to an improvement in the rheological properties of blood, but also to a therapeutic antidiabetogenic effect.

Example 2

Take 0.2 mg of the preparation of the tripeptide Pro-Gly-Pro, dissolve in 0.25 ml of physiological saline and injected intranasally to each rat weighing 200-220 g of 0.05 ml (40 μg) daily every 24 hours for 5 days . Control animals in the same way and according to the same method of introducing a 0.85% solution of NaCl in a volume of 0.05 ml per 200 g of body weight daily. Then proceed as described in example 1. As a result of analysis of the parameters of hemostasis, the following is detected. In platelet-rich plasma, platelet aggregation in experimental samples is 89.5%, while in control it is 100%, i.e. the tripeptide exhibits weak antiplatelet activity when used in a given dose. In platelet-poor plasma, an increase in enzymatic fibrinolytic activity was detected due to a 2-fold increase in plasminogen activator activity, a 1.1-fold increase in total fibrinolytic activity, a 1.6-fold increase in fibrin-depolymerization activity, and a 1.5-fold increase in anticoagulant activity compared to the control (administration of saline). At the same time, the blood sugar level in the experimental samples was lower than in the control (patients with diabetes on the background of the injected saline solution), 1.6 times and corresponded to the normal initial value. Eight days after the cancellation of the peptide, the rats with advanced diabetes decreased even more — 2.6 times more than in control rats with advanced diabetes; after 20 days it was 1.7 times lower in the experiment compared to with control. By the 20th day, the survival rate of animals in the experiment was 75%, in the experiment - 49%.

Thus, intranasal repeated administration of Pro-Gly-Pro at a dose of 40 μg / kg of rat body weight led to a corrective fibrinolytic effect in the bloodstream of animals with hypercoagulation and to a tendency to have a therapeutic antidiabetogenic effect on rats, but to a much lesser extent than the use of the peptide at a dose 5 times higher (i.e. 1 mg / kg body weight).

Example 3

Take 2 mg of the preparation of the tripeptide Pro-Gly-Pro, dissolve in 0.25 ml of physiological NaCl and intranasally injected into each rat weighing 200-220 g of 0.05 ml (400 μg) daily every 24 hours for 5 days. Control animals in the same way and according to the same method of introducing 0.85% NaCl solution in a volume of 0.05 ml per 200 g of body weight daily. Then proceed as described in example 1. As a result of analysis of the parameters of hemostasis, the following is detected. In platelet-rich plasma, platelet aggregation in experimental samples is 76%, while in the control it is 100%, i.e. the tripeptide exhibits antiplatelet activity when used in a given dose. In platelet-poor plasma, an increase in enzymatic fibrinolytic activity was detected due to an increase in plasminogen activator activity by 4.1 times, total fibrinolytic activity by 1.8 times, fibrindepolymerization activity by 1.6 times, anticoagulant activity by 2 times compared with control (administration of saline).

At the same time, the blood sugar level in the experimental samples was lower than the control (patients with diabetes against the background of the injected saline solution) - 2.2 times and corresponded to the normal initial value. Eight days after the cancellation of the peptide administration to rats with developed diabetes, the sugar level decreased even more — 2.7 times as compared to the control rats with developed diabetes; after 20 days it was 1.8 times lower in the experiment. compared to control. By the 20th day after drug withdrawal, animal survival in the experiment was 84%, in the control - 50%. Therefore, when applying this dose of the peptide (2 mg / kg body weight), therapeutic antidiabetogenic and anticoagulant effects are also established. However, it is more advisable to use a lower dose of the peptide - 1 mg / kg body weight, since the effect is the same (Tables 1 and 2).

The above examples are illustrated in the following tables.

Table 1 The study of the rheological properties of blood (according to the APTT tests - activated partial thromboplastin time, SFA - total fibrinolytic activity, FDPA - fibrindepolymerization activity, t-PA - activity of tissue plasminogen activator in rat blood plasma, platelet aggregation in the body with hypercoagulation 1 hour after the last 5 hours after the first administration of the Pro-Gly-Pro peptide intranasally (PGP) at doses of 1 mg / kg (PGP-1), 0.2 mg / kg (PGP-2), 2 mg / kg (PGP-3) body weight of animals with developed diabetes mellitus (M ± m) Groups of rats administered drugs APTT (s) SFA (mm ² ) PDPA (mm ² ) t-PA (mm ² ) Platelet Aggregation,% PGP-2 n = 8 59.3 ± 6.1 * 21.0 ± 2.0 * 14.5 ± 1.3 * 10.0 ± 1.1 ** 89.5 PGP-1 n = 8 71.7 ± 9.9 ** 33.0 ± 6.3 ** 21.7 ± 3.4 ** 22.0 ± 4.4 ** 74.5 PGP-3 n = 9 75.4 ± 7.7 ** 34.0 ± 2.1 ** 18.3 ± 1.1 ** 20.8 ± 1.7 ** 76.0 NaCl (i / n + diabetes) n = 8 37.8 ± 1.8 18.8 ± 3.1 9.0 ± 3.3 5.0 ± 2.5 100.0 Note: statistical indicators are calculated in relation to the corresponding control with 0.85% NaCl solution. * - p <0.05, ** - p <0.01

table 2 The blood sugar level (mg%) in rat blood at different times after administration of the Pro-Gly-Pro (PGP) peptide at doses of 1 mg / kg (1), 0.2 mg / kg (2) and 2 mg / kg for 5 days daily (3) against the background of diabetes Rat groups Experience Conditions Statistical indicators Sugar Level (mg%) Initial value (before the introduction of alloxan) 4 days after the administration of alloxan 1 h after 5-fold administration of the PGP peptide After drug withdrawal day one PGP (2) M ± m 140.0 ± 5.1 300.0 ± 28.6 190 ± 16.7 127.2 ± 11.5 228.0 ± 17.8 n 8 8 8 5 5 p > 0.5 > 0.5 <0.01 <0.01 <0.01 p1 <0.01 > 0.5 > 0.5 > 0.5 2 PGP (1) M ± m 118.0 ± 6.9 372.3 ± 31.6 142.0 ± 12.8 110.4 ± 9.2 232 ± 17.2 n 8 8 8 5 5 P > 0.5 > 0.5 <0.01 <0.01 <0.01 p1 <0.01 <0.05 > 0.5 <0.01 3 PGP (3) M ± m 138.2 ± 3.6 304.1 ± 25.4 143.3 ± 8.9 121.2 ± 15.3 219.7 ± 16.7 n 8 8 8 8 8 p > 0.5 > 0.5 <0.01 <0.01 <0.01 p1 <0.01 <0.01 <0.01 <0.01 four NaCl M ± m 123.0 ± 4.8 331.8 ± 24.2 318.3 ± 29.0 340.0 ± 38.8 400 ± 13.3 n 8 8 6 four 3 Note: p - significance of differences between experience and control values p1 - significance of differences between experience and baseline

Claims (2)

1. A method of treating diabetes mellitus in animals, comprising intranasal administration of a Pro-Gly-Pro tripeptide to a subject in need thereof, characterized in that said tripeptide is administered in an amount of 1 mg / kg body weight daily for 5 days. 2. A pharmaceutical composition for treating diabetes mellitus in animals, containing Pro-Gly-Pro tripeptide as an active principle, characterized in that said tripeptide is contained therein in such a dilution that the amount of tripeptide administered is 1 mg / kg body weight.