Anti-ishemic and antihypoxic agent

FIELD: medicine.

SUBSTANCE: invention refers to pharmaceutical industry, namely to medicated products normalising cardiac function, particularly being perspective for treatment of coronary heart disease (CHD). Peptide of general formula CH3CO-Lys-Lys-Arg-Arg-NH2 is disclosed as an anti-ischemic and antihypoxic agent.

EFFECT: declared invention ensures apparent anti-ischemic, antihypoxic and adaptive action and is being available in medical practice.

1 cl, 3 ex, 5 tbl

 

The invention relates to the pharmaceutical industry, namely, the treatment-and-prophylactic means, normalizing the function of cardiac activity, in particular promising for the treatment of coronary heart disease (CHD).

The problem of treatment of coronary heart disease is extremely relevant, because according to the who working group (1997) in Russia 87.5% of deaths among men and 85% among females 45-74 years of age accounted for CHD and stroke, and their share in the total mortality exceeds 41%.

There are a large number of pharmaceutical drugs aimed at the treatment of coronary artery disease. These include antianginal drugs, money, dilates blood vessels (menthol, methyl valerate, Corvalol, etc.), sedatives, androgen and vitamin preparations, adenosintriphosphate, antispasmodics etc. (Mashkovsky PPM Medicines. - M.: Medicine, part 1. p.64-89, 173-175, 293, 347-349 part 2, 1986, p.6-9, 88, 141-186, 511; UA 96124897, 1999; EN 97119808, 1999; EN 2144922, 2000). In particular, for these purposes, use derivative diamancel or their mixtures with inderal (EN 2155036, 1998), derivatives of adenosine-5'-triphosphate (EN 2190411, 1999) in the injectable form, derived trimethyl-3-hydroxypyridine (EN 2276138, 2004), salt solutions echinochrome (EN 2137472, 1998). As a rule, the use of synthetic chemical and pharmaceutical products has a large number of constraints. While drugs with antisemites the m action as a rule, are not antihypoxants. So, one of the widely used tools with antiischemic activity is obzidan (inderal) (Mashkovsky PPM Medicines. Kharkov: Torsing, 1997, vol. 1, s-264). However, the application of this tool is limited in patients with obstructive peripheral vascular disease because of the presence of a negative inotropic effect. Antihypoxic activity of this tool is not described.

Relatively harmless to the body are considered drugs of natural origin. These include, in particular, are meliloti based on an extract of Melilotus officinalis (RU # 2002127568, 2004, CL AC 35/78), leutar based tinctures aralia and extract Eleutherococcus (EN 2005481, 1995), cardiofit containing ingredients 14 plants (RU 2137496, 1998), etc.

The disadvantages of this group of drugs are relatively low efficiency, limited market of raw materials, lack of antihypoxic effect.

The most famous antihypoxic agent is piracetam (Mashkovsky PPM Medicines. - M.: Medicine, part 1, pp.109-111). It has a positive effect on metabolism and blood circulation. Data is available and its positive effect in patients with coronary heart disease, but only in elderly and senile age (Laishevsky, L.G. Imanov, Vshadow. therapeutic use of piracetam in patients with myocardial infarction. // Cardiology. 1987, 2, p.46-49).

It is known that succinic acid and its salts possess adaptogenic ability and have antihypoxic, anti-stress and neurotropic effect, normalizes energy metabolism. It is also known cardioprotective and anti-ischemic action of succinic acid and its salts (Succinic acid in medicine, food industry, agriculture. Collection of scientific articles. Pushchino. 1997; EN 2108095, 1998).

The closest efficiency to the present invention is a tool that has anti-ischemic and antihypoxic activity (EP 199900877, 2001), includes m-carbarnoyl-4-phenyl-2-pyrrolidone (CFP) and two - or trehosnovnoy carboxylic acid. As a dibasic acid is used or oxalic acid, or malic or succinic acid, as trehosnovnoy lemon. Funds for efficiency exceed the effectiveness of m-carbarnoyl-4-phenyl-2-pyrrolidone (CFP) and the number of well-known drugs used as reference Comparators. The tool containing CFP and succinic acid, showed the highest activity in all studies. However, these tools are poorly soluble in water, their fluids have a low pH value, which limits the ways of their introduction, biodot Pesti and this consequently reduces efficiency.

The technical challenge faced by the authors, was the creation of medicines, combining high antiischemic and antihypoxic activity with minimal negative effects on the body.

The technical problem was solved in the course of research in the field of peptides included in the composition of the natural hormones that regulate the functioning of the human body. As a result of these studies was established peptide CH3CO-Lys-Lys-Arg-Arg-NH2with combined anti-ischemic and hypoxic activities and homology in primary sequence fragment of adrenocorticotropic hormone (ACTH). The product does not contain non-natural amino acid residues, or any Deputy, is not peculiar to endogenous regulatory peptides, therefore, in the body it is decomposed to individual amino acids, which are involved in natural metabolism.

The drug is rapidly eliminated from the bloodstream, however, experiments show that the results of his actions are evident even after 24 hours after injection. It is established that in the concentration range from 0.001 to 100 μg/ml, it does not have cytotoxic properties. According to the results of the radioligand analysis of the drug with high affinity and specificity associated with ACTH receptors on the membranes of adrenal cortex of rats. On Asano, that it inhibits the extinction of conditioned reflexes after stopping reinforcements.

The peptide synthesis was performed on an automatic synthesizer (Applied Biosystems, model A and "Vega Coupler, model C250, USA), and the purification was performed by preparative reversed-phase chromatography (GC "Gilson, France)column Prep Nova-Pak HR C18, 6 um, 60Å, (19×300 mm) (Waters, USA). The synthesized material okharakterizovali data analytical reversed-phase HPLC (Gilson chromatograph, France), column Waters Delta Pak C18, 5 um, 100Å, (Waters, USA), amino acid analysis (hydrolysis of 6M HCl, 24 h, 110°; amino acid analyzer LKB 4151 Alpha Plus (Sweden)) and mass spectral analysis (mass spectrometer PerSepetive BioSystems Voyager-DE Biospectrometry WorkStation, USA).

The drug was applied intranasally. One drop of solution contains 50 micrograms of the active substance. Single dose of 150-1500 mcg (2-20 µg/kg). If you want to use high dosages introduction is carried out in several stages at intervals of 10-15 minutes. Daily dose of 400-4000 mcg (6-60 mg/kg). The drug is usually prescribed daily for 3-5 days, if necessary, the treatment extended to 14 days.

Experiments on animals have shown that the drug has anti-ischemic, cardioprotective and antihypoxic, as well as significant stress-protective and adaptogenic activity.

P is apart significantly improves coronary blood flow and increases contractile activity of the myocardium in the postinfarction period, normalizes disorders of atrioventricular conduction in the myocardium, reducing the intensity of cytolysis of the myocardium, the intensity of lipid peroxidation.

The product is shown (according to preliminary data) with the following pathologies:

- intellectual-mnestic disorders in the vascular lesions of the brain;

- status after traumatic brain injury and neurosurgical operations;

- chronic heart failure with circulatory disorders;

- for the prevention and treatment of post anesthesia disorders of various origins, including after ionizing radiation;

- to increase the adaptive capacities of the human body in extreme situations.

Was shown the effectiveness of intranasal applications of the drug laboratory animal models heat, cold shocks, hypobaric hypoxia and acute haemorrhage. One of the resulting effects is to reduce the concentration of glucocorticoids (11-oxicorticosteroids) in the adrenal glands and blood, stabilization of the level of adrenaline and noradrenaline in the adrenal and plasma, reactivation of diaminooctane in the myocardium with a simultaneous decrease in the content of histamine.

Example 1. Obtaining the drug and its characteristics.

Synthesis of peptide CH3CO-Lys-Lys-Arg-Arg-NH2what was b held by a solid phase method using Boc/Bzl strategy for the synthesis of peptides Coupler-250. For the temporary protection of the α-AMINOPHENYL used tert-butyloxycarbonyl group. For blocking of side radicals of arginine and lysine used mesitylenesulfonyl and 2-chlorobenzenesulfonyl groups, respectively.

As insoluble matrix was used 4-methyl-benzylaminopurine (m-BHA-resin) with an initial capacity of 1 mmol/g 2.9 g BHA-polymer was placed in the reaction vessel of the synthesizer and conducted capacity polypeptide chain according to the following program attach a single amino acid residue (table 1). Removal of temporary protective groups were triperoxonane acid, neutralization with 5% solution of diisopropylethylamine in dimethylformamide. Condensation was carried out by the method of activated esters, using a three-fold excess of the corresponding component is enabled. Preliminary activating carboxyl components was carried out for 30 minutes using oxybisethanol and diisopropylcarbodiimide. To monitor the completeness of the reaction of condensation used ninhydrin test. After joining of the amino acid residues corresponding to the sequence of the synthesized peptide was performed acetylation of N-terminal amino group using 40 equivalents of acetic anhydride (5 ml) in 25 ml of dimethylformamide. Then peptidyl-poly the EP was dried in a desiccator to constant weight. The output is protected peptidyl-polymer 96%. Under this treatment removed all side protection group, and the peptide was tsalala from high-molecular matrix, the time of release was one hour.

Table 1
Program join one amino acid residue
N p/pOperationReagentsThe rate of recurrenceTime minThe volume of reagents ml
1.Removal of the BOC-protection (release)Triperoxonane acid1225
2.Re-releaseTriperoxonane acid1225
3.FlushingMethylene chloride1225
4.FlushingDimethylformamide3125
5.Neutralization5% R-R DIEA in DMFA3125
6.FlushingDimethylformamide3125
7.Condensation9.0 mmol oxibendazole ether corresponding derived amino acids120 h25
8.FlushingDimethylformamide3125
9.FlushingMethylene chloride3125
10.Ninhydrin test

Remove the side of the protective groups and cleavage of the peptide from the insoluble matrix was performed under the action of anhydrous liquid hydrogen fluoride in the presence of scavengers. 3.5 g of peptidyl-polymer was treated with 27 ml of hydrogen fluoride and 3 ml of m-cresol at 0°C and stirring on a magnetic stirrer for 1 hour. Then hydrogen fluoride was evaporated and suspended residue in 50 ml of 50% solution of acetic acid in water. The solution was filtered and was extracted 3 times with 10 ml ether. After lyophilization of an aqueous solution obtained 1.4 g of a coarse product.

Synthesized peptide product was purified using preparative reversed-phase liquid chromatography on a column of Prep Nova-Pak HR C18, 6 um, 60 Å, (19×300 mm, Waters, USA and characterized by amino acid analysis data (amino acid analyzer Alpha Plus, LKB, Sweden) and mass spectrometry (mass spectrometer Per Sepetive Biosystems Voyager-DE Biospectrometry Workstation"). The amino acid composition of the final product corresponded to theoretical. Purity according to analytical HPLC was at least 98%. The yield of the final product is 50%.

Translation of the peptide in the acetate form was carried out according to the following procedure: 450 mg of the obtained peptide in the form triptoreline salt RA who worked in 45 ml of deionized water, added resin IRA - 68 in the acetate form and was stirred for 30 minutes. Then the polymer was filtered, washed with water, the filtrate and wash water were combined and liofilizirovanny. Received 350 mg NP-4 in the acetate form (74%).

Example 2. In fsri Institute of toxicology of the FMBA of Russia was estimated efficacy (conditional designation NP-4)obtained in example 1, in experimental myocardial infarction (aim)caused by ligation of the left coronary artery, in comparison with known drug-antioxidant Riboxin.

The experiments were performed on the nonlinear white rats-males weighing 180-230 g, age 3.5 - 4 months. (Rapolano, St. Petersburg). Modeling of myocardial infarction was performed by ligation of the left coronary artery by way Hijack and Gaylatino. Registration of ECG was performed on generalizirovanny animals with polygraph RM-6000 (Japan).

Just been formed 5 experimental groups (each experimental group consisted of 15 animals).

1. Intact animals (linopirdine);

2. Animals with aim without treatment;

3. Animals with aim therapy drug Riboxin (10 mg/kg);

4. Animals with aim therapy drug NP-4 (2.0 mg/kg);

5. Animals with aim therapy drug NP-4 (20.0 mg/kg).

Study drug NP-4 was administered intranasally once a day rate 7 days after aim. Riboxin who drove intragastric once a day rate 7 days after aim. Intact animals, lineameriloan, was injected solvent is distilled water. Animals with aim without treatment was also introduced solvent. Comparative efficacy was evaluated by several groups of indicators:

- mortality and clinical picture;

- functional - ECG (1 h, 3 days and 7 days);

biochemical blood and tissue of the heart (24 hours);

- histological - (24 hours, 3 days and 7 days).

Experimental modeling of myocardial infarction was accompanied by approximately 20-26% mortality; thus, the study was observed 11-12 animals from each group. As evidenced by the data obtained after experimental myocardial infarction was not observed significant differences in the frequency of occurrence of THEM in the experimental groups. The frequency of THEM averaged 83.2% in each group, without electrocardiographic signs of THEM - 16.8%.

The magnitude of ST-segment elevation was not significantly different between groups. The decrease in ST below contours (depression) is characterized by post-infarction ischemic changes and indicates a failure of blood supply, hypoxia of the myocardium. The severity of ST-segment depression 3 days after aim significantly differed from the control (1.5 times lower (p<0.05)on therapy with drugs Riboxin and NP-4. And during therapy NP-4 in a dose of 20 mg/kg the value of the depot is Essie was 2 times smaller than in controls (p<0.05).

The magnitude of ST depression in experimental animals with THEM 7 days after aim in the control group significantly increased 1.2 times compared to the period of day 3 (p<0.05). During therapy drugs Riboxin and NP-4 (2 μg/kg) amount of ST depression remained at the same level, and on therapy NP-4 (20 mg/kg) decreased significantly 2-fold (p<0.05), 4 times less expressed in comparison with the control. This fact indicates the presence of anti-ischemic effect of drugs Riboxin and NP-4, increasing the resistance of the myocardium to hypoxia on the background of this therapy, and there is a direct linear dozozavisimoe for drug NP-4.

In the control group showed marked persistent negative chronotropic effect (decrease in the heart rate significantly by 32%) (table 2); reliable pronounced decrease in the amplitude of R-wave in the observation period of 7 days in 2 times in comparison with the norm and with the observation period of 1 h, which characterizes the inhibition of contractile activity of the myocardium in the postinfarction period; persistent progressive disorders of atrioventricular and intraventricular conduction (elongation PQ and QT 1.5-2 times compared with the period before EIM).

On the background of drug therapy Riboxin was also observed persistent significant reduction of heart rate by 30%; reliable pronounced decrease in the amplitude of R-wave on Wed the ke observation 7 days in 2 times in comparison with the norm and with a period of 1 hour; persistent violations of intraventricular conduction (lengthening of the QT 1.5 times in 1 hour and 2 times 7 days compared with the period before aim); normalization of atrioventricular conduction.

The drug is NP-4 in a dose of 2 µg/kg and at a dose of 20 mg/kg had no effect on heart rate (bradycardia comparable with control). The amplitude of the R-wave was reduced in the subacute post-infarction period (significantly higher than in 2 times in comparison with control). Also there was a significant (2-fold) increase in the amplitude of the T wave after 7 days. These characteristics indicate the increase in contractile activity of the myocardium during therapy with the drug NP-4, and this effect was equally evident in both doses of the study drug. Observed persistent violations of intraventricular conduction (lengthening of the QT 1.5 times in 1 hour and 7 days compared with the period before aim); normalization of atrioventricular conduction.

td align="center"> S
Table 2
The influence of the studied drugs on the dynamics of heart rate and ECG parameters in rats in experimental animals under conditions of acute myocardial infarction, M±m
IndicatorsHRPRTPQQT
Control
1 hour323±27•0.022±0.0012.01±0.430.048±0.020.26±0.1330±564±9•
7 days274±20*•0.020±0.0080.80±0.19*•0.038±0.010.22±0.1780±8*•90±15•
Riboxin
1 hour315±36•0.022±0.0012.02±0.450.049±0.010.27±0.1339±561±9•
7 days334±14•0.017±0.0030.97±0.20*•0.046±0.010.28±0.1043±770±13•
NP-4 2 ág/kg
1 hour316±16•0.022±0.0032.02±0.420.054±0.020.26±0.13•35±760±9•
7 days277±14*•0.018±0.0021.94±0.400.043±0.010.57±0.21•*28±1072±7•
NP-4 20 mg/kg
1 hour320±34•0.022±0.00091.93±0.460.047±0.0090.25±0.12•28±563±8•
7 days314±21•0.033±0.0021.81±0.410.039±0.010.58±0.20*•55±1*70±6•
* p<0.05 compared with period 1 hour
• p<0.05 compared to normal - until theM

The results of morphometric and biochemical studies on the background of experimental myocardial infarction are presented in table 3.

2.85±0.12
Table 3
Morphometric and biochemical study on the background of aim
IndicatorsThe intactControlRiboxinNP-4 2 ág/kgNP-4 20 mg/kg
Body weight, g185±10180±10200±10205±10195±5
The relative weight of the heart, mg/100 g of body weight3.5±0.14.9±0.33.5±0.1*3.1±0.1*3.3±0.2*
ALT, blood, µkat/l0.22±0.032.46±0.451.19±0.12*0.94±0.270.88±0.16
ACT, blood, µkat/l0.65±0.051.175±0.17*0.96±0.22*0.80±0.10*
KF, blood, µkat/l0.71±0.101.96±0.160.95±0.11*0.97±0.13*0.79±0.11*
LDH, blood, mmol/h/l4.90±0.328.92±0.366.77±0.276.85±0.284.95±0.32*
Catalase, blood, mg·ml/min445±20252±28260±35300±49302±49
The serum MDA, nmol/mg protein1.65±0.254.82±0.334.80±0.303.15±0.203.18±0.22
Restored glutathione, heart, mg%85±1040±545±550±1035±10
Glycogen, heart, mg%2450±11085±90 1350±100*1850±110*1550±150*
Lactic acid, heart, mg%60±10185±15170±1090±5*140±15
ATP, heart, µmol/g2.30±0.110.55±0.101.23±0.14*1.57±0.16*1.20±0.12*
SDG, heart, ug formazan/g protein/h145±1575±1055±1580±1090±15
The intensity of tissue respiration, heart μl of O2/100 mg/HR61±638±438±536±553±5
HMM, heart, nmol/mg protein3.57±0.357.50±0.454.50±0.35*4.15±0.25*5.15±0.30
Catalase, heart, mg·ml/min475±20 260±35252±38305±40293±42
*reliable differences from control at p<0.05

It was shown that aim characterized by significant increase of the relative weight of the heart. In addition there was a significant signs of cytolysis in myocardium: an increase in ALT, AST, KF and LDH in plasma. Recorded violations of the processes of tissue respiration and metabolism in myocardium: decreased reserves of ATP, glycogen; increased content of oxidized products of metabolism - lactic acid; observed activation of free-radical reactions (increased MDA) and reduced antioxidant (catalase, SDG, VG).

The medications had a cardioprotective effect, and the drug is NP-4 was more effective than drug Riboxin, and the effects of the two investigated doses NP-4 did not differ among themselves.

24 hours after surgery in left ventricular myocardium of rats of the control group was observed common disorder of blood circulation in the form of hyperemia, slight bleeding, blood stasis in the capillaries, edema. Mainly under the epicardium were visible muscle fibers, intensively stained with eosin or picric acid.

3-and the ducks after the operation in the zone of ischemia was observed the formation of a heart attack. Disorders of the circulatory wore not already shared, and local character. The necrotic cardiomyocytes were deprived of transverse iscertainly cores, homogeneous stained by eosin. A large part of necrotic fibers were painted a very pale, being able lysis. In the zone of necrosis were hemolyzed erythrocytes, polymorphonuclear leukocytes. Leukocyte infiltration was observed mainly around the hearth necrosis. The stroma of the myocardium outside the zone of necrosis was swollen.

On day 7 was observed organization of a heart attack, resorption of necrotic fibers and replacement of areas of necrosis young connective tissue, rich in cellular elements predominantly fibroblastic series. At a later date on the location of these foci of cell proliferation in fibroblast number was formed fibrous scar.

With the introduction of drugs Riboxin, NP-4 2 µg/kg, and NP-4 20 mg/kg histological pattern was somewhat different. In rats these experimental groups 24 hours after surgery common disorder of blood circulation in the myocardium of the left ventricle were less pronounced compared with the control. In the area of ischemia was observed eosinophilia muscle fibers and slight swelling.

On the 3rd day was observed the formation of myocardial infarction, mainly under the epicardium of the anterior wall of the left Zheludok is. The size of the lesion necrosis were significantly lower than those of control rats. Necrotic fibers were pale colored, deprived of scarceness and nuclei. In the hearth of necrosis was observed swelling, a small amount of hemolyzed erythrocytes and leukocytes. Leukocyte infiltration around the hearth necrosis was poorly expressed. Interstitial edema out of ischemia was absent.

On the 7th day at the site of necrotic fibers were observed proliferative reaction of the stroma with a large number of fibroblastic cells of the row. The size of the plot proliferation were lower compared to control.

The obtained experimental data allow us to draw the following conclusions:

1. Experimental myocardial infarction is characterized by the formation of the center of necrosis in 82,3%, insufficient coronary blood flow in acute and subacute periods, inhibition of contractile activity of the myocardium and disorders of atrioventricular and intraventricular conduction; breach of tissue respiration and metabolism, activation of lipid peroxidation and reduced antioxidant defenses.

2. The drug Riboxin has a pronounced anti-ischemic effect: improves coronary blood flow in 1.5 times in the postinfarction period compared to the control. The drug has no effect on the contractile activity of the myocardium in portinfer the fair period, helps to normalize impaired atrioventricular conduction; and cardioprotective effect reduces the intensity of free radical oxidation and increases the level of antioxidant protection.

3. The drug is NP-4 has a pronounced anti-ischemic effect: significantly improves coronary blood flow in 1.5 times in the postinfarction period at a dose of 2 µg/kg and 4 times at a dose of 20 mg/kg compared with control. The drug is NP-4 significantly increases the contractile activity of the myocardium in the postinfarction period 2 times, contributes to the normalization of disorders of atrioventricular conduction in the myocardium, and this effect was achieved at a dose of 2 µg/kg and does not change during subsequent increase in dosage. The drug is NP-4 has a pronounced cardioprotective effect reduces the intensity of cytolysis of the myocardium, the intensity of lipid peroxidation, increases the level of antioxidant protection, and more efficient than the comparison drug Riboxin.

Example 3. Study of the influence of the drug NP-4 on the body in models of acute hemorrhage and hypobaric hypoxia in rats in vivo.

In our experiments we used adult male Wistar rats weighing 180-200 g Model of acute haemorrhage was 1-hour arterial hypotension. To create it in anesthetized rats (Nembutal 40 mg/kg) through the catheter in the tail artery was made to the patient to achieve a mean arterial pressure of 40 mm Hg Before bleeding the animal was intravenously injected heparin (0.5 ml, 50 U/ml) to prevent clotting during the long experiment. After a time hypotension in rats resuscitated by intraarterial administration of blood. Immediately after replacement of blood loss was performed by intravenous infusion of the studied peptides (1 and 10 µg/kg/min for 10 min) or a similar volume (0.8 ml) of saline solution through the jugular vein. Since, as a rule, regulatory peptides have a cascading mechanism of action and their influence may be affected after a long time after the introduction, the decapitation of the animals and the taking of material for biochemical studies were performed on the 1st and 7th day. The control group of animals was conducted in full operation without hemorrhagic shock. Values of biochemical parameters in this group was taken as 100%. The control and experimental group contained 10 rats.

To create conditions for the development of acute hypobaric hypoxia in rats was placed in a pressure chamber with a pressure of 154 mm Hg, corresponding to the atmospheric pressure at the altitude of 11500 feet above sea level, and was kept in these conditions before making their lateral position. Study drugs to rats of the experimental group were administered intranasally (0.1 and 1 μg/kg animal body weight) for 1 and 24 hours before "recovery", computers is the super rats of the control group was injected with saline. The decapitation of the animals and the taking of material for biochemical studies were performed on the 1st and 7th day after "recovery". Values of biochemical parameters in the control group was taken as 100%. Each group contained 10 animals.

Determination of glucocorticoids (11-oxicorticosteroids) in the adrenal glands and blood was carried out according to the method, the principle of which is based on the ability of 11-oxicorticosteroids to fluorescamine after treatment with a mixture of sulfuric acid and ethanol (3:1, V/V). Hormones of tissue was extracted with chloroform. The content of the fluorescent product was measured on fluorimetry "Hitachi 850 (Japan) with λ (wavelength) 530 nm (λ excitation light = 470 nm). Number 11-oxicorticosteroids were determined by calibration curve and expressed in micrograms per 1 g of tissue. For statistical processing of results was using t-student test.

The content of catecholamines in the adrenal glands was determined by the following method. Extraction of catecholamines from tissues was performed by the sequential processing of the homogenate acid n-butanol, a mixture of n-heptane and water (4,5:1, by volume). Then the aqueous phase was added 2 M sodium acetate containing 0.2% EDTA and aluminum by Brockmann I. After centrifugation (5 minutes at 2000g) sorbent was washed with water, was transferred to a 0.5 M phosphate buffer, pH 6.0, containing 0,75% EDTA, and is emali for 15 minutes in a shaker for elution of catecholamines, and then again centrifuged. Fluorescent products of catecholamines were obtained using trihydroxybenzophenone reaction. Measurement of fluorescence was performed on fluorimetry "Hitachi 850 (Japan) for adrenaline at λ=500 nm and exciting light with λ=410 nm and norepinephrine at λ=485 and λ=385 nm, respectively. The amount of adrenaline and noradrenaline were determined by calibration curve and expressed in micrograms per 1 g of tissue. The significance of differences between experimental data control and the experience was assessed by t-criterion of student.

Table 4 presents data on the effect of the drug NP-4 contents 11-oxicorticosteroids and catecholamines in the adrenal glands and blood plasma of rats in the 1st and 7th day of postresuscitation period after hemorrhagic shock. It is seen that after 1 day after the shock level 11-oxicorticosteroids in the adrenal and plasma increased by 59%. After 7 days the content 11-oxicorticosteroids in the adrenal and plasma was decreased, remaining above the reference level by 19%. Introduction peptide at a dose of 1 mg/kg resulted in a decrease in the content of 11-oxicorticosteroids in the adrenal and plasma as after 1 and 7 days, increase the dose to 10 mcg/kg was accompanied by a decrease in the level of hormones in all cases studied to control values. As can be seen from table 2, after 1 day after the shock of concentration hell is analine and noradrenaline in the adrenal glands was decreased compared to the control level by 38%. Through 7 days after the shock, these figures were close to control values. In contrast, after 1 hour after the shock of the adrenaline in plasma increased by 79%, and after 7 days exceeded the control level by 24%. A sharp decrease in the concentration of epinephrine in the adrenal within 1 hour after the shock and the simultaneous increase in plasma indicates an increase in hormone secretion from the adrenal glands into the blood. Introduction peptide at doses of 1 and 10 µg/kg prevented this effect: the level of adrenaline in the gland and plasma after 1 and 7 days after the shock was close to the control. Thus, the study drug in doses of 1 and 10 mg/kg has positive corrective action on the organism of rats subjected to hemorrhagic shock.

Table 4
The influence of the drug NP-4 on level 11-oxicorticosteroids (COP) and catecholamines in the adrenal glands and blood plasma of rats in the postresuscitation period after hemorrhagic shock
HormoneThe group of animalsHormone levels (%)
After 1 dayAfter 7 days
CA in plasma Control
GSH+FR***
NG+NP-4 (1 μg/kg)
NG+NP-4 (10 µg/kg)
100±26*
(0,38±0.1 ág/ml)
148±8*
118±9*
99±6**
100±28*
(0,28±0.08 µg/ml)
131±10*
109±6*
94±8*
The adrenaline in the adrenal glandsControl
GSH+FR***
NG+NP-4 (1 μg/kg)
NG+NP-4 (10 µg/kg)
100±10*
(422±42 mg/g)
62±9*
90±6**
98±5**
100±13*
(440±58 μg/g)
110±15*
102±10*
99±9*
The adrenaline in
plasma
Control
GSH+FR***
NG+NP-4 (1 μg/kg)
NG+NP-4 (10 µg/kg)
100±6*
(39,9±2.4 µg/ml)
179±12*
111±10*
100±6**
100±7*
(27,0±1.9 µg/ml)
124±11*
110±10*
98±9*
Noradrenaline in the adrenal glandsControl
GSH+FR***
NG+NP-4 (1 μg/kg)
NG+NP-4 (10 µg/kg)
100±10*
(200±20 µg/g))
78±8*
111±10*
100±6**
100±13*
(192±25 µg/g)
105±12*
102±10*
100±4**
*P<0.02, **P<0.001
*** Hemorrhagic shock+saline

Table 5 shows the results of the study drug and NP-4 contents 11-oxicorticosteroids and adrenaline in the above is ocajnicko and blood plasma of rats in the 1st and 7th day after hypobaric hypoxia. It is seen that the character of changes in the content of hormones in the adrenal glands and plasma after "recovery" was the same as in the case of hemorrhagic shock. Intranasal introduction of peptides in doses of 0.1 and 1 mg/kg for 1 and 24 hours before the "rise" normalized content 11-oxicorticosteroids and adrenaline in the adrenal glands and blood plasma of rats subjected to hypoxia.

Table 5
The influence of the drug NP-4 on level 11-oxicorticosteroids (COP) and catecholamines in the adrenal glands and blood plasma of rats after hypobaric hypoxia
HormoneThe group of animalsHormone levels (%)
After 1 dayAfter 7 days
The COP in the adrenal glandsControl
GG+FR***
GG+NP-4 (0.1 mg/kg twice)
GG+NP-4 (1 mg/kg twice)
100±17*
(35±6 μg/g)
164±12*
123±11*
102±8*
100±14*
(36±5 µg/g)
111±8*
108±9*
104±10*
CA in plasmaControl
GG+FR***
GG+NP-4 (0.1 mg/kg twice)
GG+NP-4 (1 mg/kg twice)
100±33*
(0,3±0,1 µg/ml)
148±8*
118±9*
99±6**
100±33*
(0,3±0,1 µg/ml)
131±10*
109±6*
94±8*
Adrenaline
in the adrenal glands
Control
GG+FR***
GG+NP-4 (0.1 mg/kg twice)
GG+NP-4 (1 mg/kg twice)
100±8*
(410±35 µg/g)
84±10*
98±5**
98±5**
100±13*
(418±56 µg/g)
107±12*
102±10*
99±9*
The adrenaline in plasmaControl
GG+FR***
GG+NP-4 (0.1 mg/kg twice)
GG+NP-4 (1 mg/kg twice)
100±10*
(30,8±3.1 µg/ml)
156±13*
104±10*
102±4**
100±8*
(28,0±2,2 µg/ml)
114±12*
107±10*
99±8*
*P<0.02, **P<0.001 ***Hypobaric hypoxia + saline

The results showed that the drug NP-4 provides corrective normalizing effect on the body of rats under such forms of stress as hemorrhagic shock and hypobaric hypoxia.

In these examples, the data show that the claimed product has a pronounced anti-ischemic, hypoxic and adaptation effects on the body and is promising for use in medical practice.

The peptide of General formula CH3CO-Lys-Lys-Arg-Arg-NH2as among the STV, with antiischemic and antihypoxic activity.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to DNA sequences coding recombinant chimeric proteins, which inhibit angiogenesis; coded by chimeric proteins; their therapeutic application; medicinal composition, containing chimeric proteins. Claimed invention provides medications blocking VEGF and inhibiting angiogenesis, and medicinal compositions containing chimeric proteins and suitable chimeric carriers, and their medicinal form.

EFFECT: efficient therapeutic application of medicinal composition.

13 cl, 6 dwg, 11 ex

FIELD: medicine; cardiology.

SUBSTANCE: inventionrepresents medication, which contains biologically active base, namely bromide 1-(β-phenylethyl-4-("п"-dimethylaminobenzylidenamino) 1,2,4-triazol and shape-forming component, namely water for injections.

EFFECT: increase of treatment efficiency, in comparison with use of traditional medications.

3 ex, 3 tbl

FIELD: pharmacology.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to production of medications used in case of upper airways diseases (rhinitis, pharyngitis, laryngitis, tracheitis); in case of neuralgia (myalgia, artralgia, migraine, cardioneurosis), stenocardia (light forms), itching dermatosis. Medication contains: menthol, eucalyptus oil, lump sugar, sodium salt of carboxymethylcellulose, talc, stearic acid or its salts, additionally containing black mint oil and aerosyl with definite component ratio. Also claimed is method of obtaining said medication.

EFFECT: increase of tablet strength, increase of tablet disintegration time.

2 cl, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel atropoisomers of formula , in which R and R1 each independently represents hydrogen or methyl; R2, R3 and R4 each independently represents hydrogen or trifluoromethyl, on condition that R2, R3 and R4 all do not represent hydrogen; and R5 represents bromine, chlorine; or to its non-toxic pharmaceutically acceptable salt, solvate. Invention also relates to pharmaceutical composition, as well as to method of treatment.

EFFECT: obtaining novel biologically active compounds, possessing properties which open calcium-activated potassium channels of high conductivity.

12 cl, 6 ex, 2 tbl, 7 dwg

FIELD: chemistry.

SUBSTANCE: invention refers to new compounds of formula (I): , where: each R0 independently stands for -H, -COOH, -OR', -SO3H, where R' stands for -H, lower alkyl, or, if x=2, both Ro are taken together to form 1,3-dioxolyl ring, or each Ro independently represents lower alkyl, each R1 and R2, is independent standing for hydrogen, G stands for (CR"2)p where R" stands for -H or acetamido and where p is equal to 0-3, Ar stands for aryl or 6-merous heteroaryl with one N atom as heteroatom, and each x and y is independent, equal to 1-4.

EFFECT: compounds I can be used in pharmaceutical compositions with vasculostatic activity, reducing overflow from blood vessels, for treating cancer, a heart attack and similar.

72 cl, 7 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: claimed is medication including 2-[4-[2-(benzimidasol-2-ultio)ethyl]piperasin-1-yl]-N-[2,4-bismethyltio)-6-methyl-3-pyridyl]acetamide(further mentioned as compound 1), its pharmaceutically acceptable salt or its hydrate and pitavastatin and pharmacologically acceptable carrier, medication being intended for simultaneous introduction or separate introduction (versions); also claimed are versions of said medication application and pharmaceutical composition. Synergism of claimed compounds activity in reduction of plaque area size in aorta is shown. Processed with said composition plaques were most stabilised (i.e. 5.3% of plaque area contained macrophages, the remaining plaque area was occupied by collagen) in comparison with independent application of each of the components.

EFFECT: reduction of lipid-rich plaque, stabilisation of lipid-rich plaque and/or prevention of rupture of lipid-rich plaque in atherosclerotic affection.

13 cl, 2 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: claimed is application of 4-methyl-2,6-diisobornylphenol as mediation increasing brain blood flow.

EFFECT: it is demonstrated that effect of 4-methyl-2,6-diisobornylphenol in terms of expression does not yield to effect of vinpocetine, but does not cause reduction of systemic arterial pressure.

2 tbl, 4 ex

FIELD: gene engineering.

SUBSTANCE: invention concerns gene engineering, particularly immunogenic conjugates, and can be applied in treatment and prevention of states related to angiotensin system activated by renin. Virus-like part of RNA bacteriophage is conjugated with angiotensin peptide component by non-peptide covalent link. Obtained conjugate of angiotensin peptide component with carrier is applied in immunisation.

EFFECT: conjugate of angiotensin peptide component with carrier.

29 cl, 8 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine and can be used in case of brain tissue ischemia occurrence, a myocardium and other tissues. For this purpose parenteral injection of a preparation, having a halogenic anesthetic in sub-anesthetic doses is carried out. In quality of halogenic anesthetics dezflurane, isoflurane, halothanum or sevoflurane are administered.

EFFECT: invention allows providing the accelerated induction of protection against an ischemia at the expense of oxygen consumption depression with an ischemic tissue, and also at the expense of fast and convenient introduction halogenic volatile anesthetics, allowing avoiding the by-effects bound to their use at a traditional way of injection.

20 cl, 5 dwg, 9 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: present invention pertains to new compounds with formula (I): where R1 and R2 each independently represents a hydrogen atom, C1-8 alkyl or a halogen atom; R3 represents C1-8 alkyl, which can be substituted with 1-3 halogen atom(s) or phenyl; R4 represents a hydrogen atom or C1-8 alkyl; R5 and R6 each independently represents a hydrogen atom; X represents a sulphur atom or oxygen atom; ring A is 4-(trifluoromethyl)piperidin-1-yl, 2,2-difluoro-1,3- benzodioxol-5-yl or 3,4-dihydro-1H-isoquinolin-2-yl. The invention also relates to salts or solvates of this derivative, as well as medicinal preparation, pharmaceutical composition, method of preventing and/or treating diseases, caused by PPAR, and use of this derivative.

EFFECT: obtaining new biologically active compounds, which can be used for preventing and/or treating diseases caused by PPARδ.

8 cl, 39 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: present invention refers to compounds of Formula II and to methods of immune response suppression, e.g. by inhibition of indirect MHC type II of T-cells activation. Compounds under invention can be applied to treatment or prevention of derangements, such as rheumatoid arthritis and/or multiple sclerosis.

EFFECT: production of compounds which can be used for immune response suppression.

25 cl, 19 dwg, 4 tbl, 22 ex

FIELD: medicine.

SUBSTANCE: invention can be used for medical treatment of secondary hypothyroid state accompanied by low synthesis of thyrotrophic hormone by hypophysis and of iodine hormone by thyroid gland. Substance of invention implies application of peptide Lys-Glu-Asp-Gly as a medicine stimulating synthesis of thyrotrophic hormone by hypophysis and of thyroid hormone by thyroid gland.

EFFECT: high specific activity of introduced peptide and decrease of side effect risk.

4 tbl, 1 ex

FIELD: medicine; pharmacology.

SUBSTANCE: releasing peptides of growth hormone are described with formula (I): R112345-R2, where:А1 designates Aib, Apc or Inp; А2 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl) or D-Тrp; А3 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-2Ser(Bzl) or D-Trp; А4 designates 2Fua, Orn, 2Pal, 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, 3Thi, Thr(Bzl); А5 designates Apc, Dab, Dap, Lys, Orn or deleted; R1 designates hydrogen; and R2 designates NH2; and their pharmaceutically acceptable salts.

EFFECT: pharmaceutical compositions and the methods of their application are presented.

25 cl, 1 tbl, 2 ex

FIELD: medicine, peptides.

SUBSTANCE: invention relates to osteogenic growth oligopeptides used as stimulators of hemopoiesis. Invention proposes using an oligopeptide of molecular mass in the range from 200 to 1000 Da, comprising one of the following sequence: Tyr-Gly-Phe-Gly-Gly, Met-Tyr-Gly-Phe-Gly-Gly used in preparing a pharmaceutical composition and enhancing mobilization of hemopoietic stem cells from many differentiation line into peripheral blood, in particular, CD34-positive hemopoietic stem cells. Advantage of the invention involves expanding field in using oligopeptides used in stimulation of hemopoiesis.

EFFECT: enhanced and valuable properties of oligopeptides.

34 cl, 2 tbl, 7 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: method involves applying composition based on receptor antagonist P substance and magnesium salt.

EFFECT: reduced hematoencephalic barrier permeability; reduced risk of vasogenic brain edema; prevented water accumulation in brain; smoothing consequences caused by reduced cognitive abilities.

23 cl, 2 dwg, 3 tbl

FIELD: medicine, chemistry of peptides, amino acids.

SUBSTANCE: invention relates to novel biologically active substances. Invention proposes the novel composition comprising peptides of the formula: H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys. The composition shows ability to inhibit proliferative activity of mononuclear cells, to induce suppressive activity and their ability for secretion of cytokines TNF-1β (tumor necrosis factor-1β) and IL-10 (interleukin-10 ).

EFFECT: simplified method for preparing composition, valuable medicinal properties of composition.

4 cl, 16 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: peptide of the following formula: X-Pro-Gly-P, where X = Thr-Lys-Pro-Arg-; Lys-pro-Arg-; pro-Arg-; Arg-, being of untiulcerous activity. They should be applied at intraperitoneal injection at the dosage of 0.58-3.20 mcM g/kg for preventing and treating ulcers of gastro-intestinal tract.

EFFECT: higher efficiency and prophylaxis.

4 dwg, 5 ex

FIELD: organic chemistry, medicine.

SUBSTANCE: invention represents ligands MC-4 and/or MC-3 of the formula (I): , wherein X means hydrogen atom, -OR1, -NR1R1' and -CHR1R1' wherein R1 and R1' are taken among the group: hydrogen atom, (C1-C6)-alkyl and acyl; (1) each R2 is taken independently among the group: hydrogen atom, (C1-C6)-alkyl; or (2) (a) R2 bound with carbon atom that is bound with X and Z1 and substitute R5 can be optionally bound to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; or (b) R2 bound with carbon atom that is bound with ring Ar can be bound with R7 to form ring condensed with ring Ar; each among Z1, Z2 and Z3 is taken independently from the following groups: -N(R3e)C(R3)(R3a)-, -C(R3)(R3a)N(R3e)-, -C(O)N(R3d)-, -N(R3d)C(O)-, -C(R3)(R3a)C(R3b)(R3c)-, -SO2N(R3d)- and -N(R3d)SO2- wherein each among R3, R3a, R3b and R3c, R3d, R3e when presents is taken independently among hydrogen atom and (C1-C6)-alkyl; p is a whole number from 0 to 5 wherein when p above 0 then R4 and R4' are taken among hydrogen atom, (C1-C6)-alkyl and aryl; R5 represents 5 substitutes in phenyl ring J wherein each R5 is taken among hydrogen atom, hydroxy-, halogen atom, thiol, -OR12, -N(R12)(R12'), (C1-C6)-alkyl, nitro-, aryl wherein R12 and R12' are taken among hydrogen atom and (C1-C6)-alkyl; or two substitutes R5 can be bound optionally to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; q = 0, 1, 2, 3, 4 or 5 wherein when q above 0 then R6 and R6' are taken among hydrogen atom and (C1-C6)-alkyl; Ar is taken among the group consisting of phenyl, thiophene, furan, oxazole, thiazole, pyrrole and pyridine; R7 are substitutes at ring Ar wherein each R7 is taken among hydrogen, halogen atom, -NR13R13', (C1-C6)-alkyl and nitro- wherein R13 and R13' are taken among hydrogen atom and (C1-C6)-alkyl; r is a whole number from 0 to 7 wherein when r is above 0 then R8 and R8' are taken among hydrogen atom and (C1-C6)-alkyl; B is taken among -N(R14)C(=NR15)NR16R17, -NR20R21, heteroaryl ring and heterocycloalkyl ring wherein R14-R17, R20 and R21 are taken independently among hydrogen atom and (C1-C6)-alkyl; s = 0, 1, 2, 3, 4 or 5 wherein when s is above 0 then R and R9' are taken among hydrogen atom and (C1-C6)-alkyl; R10 is taken among the group consisting of optionally substituted bicyclic aryl ring and optionally substituted bicyclic heteroaryl ring; D is taken among hydrogen atom, amino- and -C(O)R11 wherein R11 is taken among the following group: hydroxy-, alkoxy-, amino-, alkylamino-, -N(R19)CH2C(O)NH2 wherein R19 represents (C1-C6)-alkyl, -NHCH2CH2OH and -N(CH3)CH2CH2OH, or its isomers, salts, hydrates or biohydrolysable ester, amide or imide.

EFFECT: valuable medicinal properties of compounds.

18 cl, 107 ex

FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

The invention relates to medicine and can be used as a means of regulating the level of glucose for the prevention and treatment of diabetes

FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

Up!