Peptides having anti-stress, anticonvulsant and neuroprotective action

 

(57) Abstract:

Usage: in medicine, as compounds with anti-stress, anticonvulsant and neuroprotective effects. The inventive peptides of the General formula: Trp-X-Gly-Gly-Asp-R, where X is the residue of a hydroxyl-containing ainability L - or D-configuration, R-Ala-Ser-Gly-Glu or Ala-Ser-Gly or Ala-Ser, or Ala, and their pharmaceutically acceptable salts. Synthesis I implement a consistent extension of the peptide chain using tertbutyloxycarbonyl schema solid-phase peptide synthesis, synthesis is carried out in automatic mode on a peptide synthesizer. 7 table.

The invention relates to new biologically active compounds, specifically, the peptides of General formula: Trp-X-Gly-Gly-Asp-R, where X is the residue of the hydroxyl - containing amino acids L-or D-configuration, R-Ala-Ser-Gly-Glu or Ala-Ser-Gly or Ala-Ser, or Ala; and their pharmaceutically acceptable salts having anti-stress, anticonvulsant and neuroprotective effects. The proposed compounds may find application in medicine.

In the process of life the human body is subjected to different in strength and nature of the stimuli, which leads to the development of stress-Rea is participating in the organism under the influence of any impacts (Horizons P. D. General characteristics and the response value of the stress. -Bulletin of the Academy of medical Sciences of the USSR, 1975, No. 8, S. 30-35; Selye, Stress without distress. -M.: Progress, 1982. - 128 S.

In response to significant exposure to adverse environmental factors in humans and animals occur adaptive biochemical and physiological changes, which, however, in the case of excessive and prolonged stress become abnormal and cause or exacerbate many serious diseases. In this regard, the problem of human adaptation to the critical factors of the external environment and increase its resilience to their disturbing effect is extremely important.

Currently known endogenous neuropeptides (substance P, opinie peptides) with adaptogenic and anti-stress action (neurochemistry. Edited ashmarina I. P. and Stukalova P. In..- M.: Publishing house of Institute of biomedical chemistry of RA MN, 1996, S. 207-310).

But the spectrum of action of these peptides is wide enough, and along with anti-stress they exhibit undesirable side effects.

Known peptide Delta-sleep (DSIP), which possesses a strong ability to normalize or to limit stress biochemically delta-sleep inducing peptide (DSIP) and some analogues on the activity of monoamine oxidase type A in rat brain under hypoxia stress. FEBS Letters, 368, pp 367 - 369; Mendjerizky A. M., Lysenko A. C., Uskova N. I.-Biochemistry, 1995, T. 60, vol.4, S. 585-592; Mendjerizky A. M., Lysenko, A. C., I. Mikhaleva I. the Influence of DSIP on the intensity of proteolysis in the rat brain during hypokinesia. -Ukr. biochem.zhurn., 1992, 64, S. 47-51. Richeria, T, Makletsova Meters, and other changes in the intensity of free radical reactions in the organs of rats under hypokinetic stress and protection of Delta-sleep inducing peptide and its tyrosinaemia analogue. - Izvestiya, 1993, N 2, S. 243-256).

The invention relates to new biologically active compounds.

We offer the peptides of General formula:

Trp-X-Gly-Gly-Asp-R,

where

X is the residue of the hydroxyl - containing amino acids L-or D-configuration,

R-Ala-Ser-Gly-Glu or Ala-Ser-Gly or Ala-Ser, Ala,

are analogues of the peptide Delta-sleep (DSIP), containing 6-9 amino acid residues (table. 1) and have anti-stress, anticonvulsant and neuroprotective action.

Unlike natural peptide DSIP offer the peptides have a selective and significantly more pronounced effect. These peptides are produced using solid-phase or classical synthesis in solution and then purified and isolated in a homogeneous condition.

oC for 1 h After removal from the reaction mixture of hydrogen fluoride, the residue was washed with dry diethyl ether (CH ml) on a glass filter and then extracted with peptide 15% acetic acid (3x10 ml) and lyophilized. For the purification of peptides using gel chromatography on Sephadex G-15 in 0.1 M acetic acid. To confirm the 200 mm) firm Macherey-Nagel(Germany). Conditions of chromatography: A - 0,2 % TFA; B - MeCN : 0,2% TFA; iPrOH (80:25:20). Gradient: 0-1 min (100% A); 1-21 min (100% A - 50% A); 21-23 min (50% A -0% A); 23-26 min (0% A - 100% A). The rate of 1 ml/min, detection at 226 nm. Peptides characterized correct data of amino acid analysis of the NMR mass spectra. The purity, yield and physico-chemical characteristics of the peptides are given in table. 2.

It is known that the stress reaction is preceded by a state of sustainable adaptation and plays an important role in its formation. While the main effort of the organism is aimed at maintaining the function of vital organs and the mobilization of energy and structural resources. In response to stress is the release of catecholamines and subsequent activation of the sympatho-adrenal and hypothalamic-pituitary systems. In conditions of prolonged exposure to the stimulus is the exhaustion of the compensatory capacity and, consequently, the failure of adaptive mechanisms of the body. Due to excess catecholamine activation of lipases, phospholipases and lipid peroxidation of cell membranes leads not to a physiologically favorable changes in the composition of the lipid bilayer, and damage the ultrastructure of cells and different pathologyplant on white rats-males weighing 180-200 g Hypoxic hypoxia is created in the pressure chamber flow type at atmospheric pressure 196 mm RT.art., which corresponds to an altitude of 10 km; the exposure time 15 min Peptide 1 is administered to the animals intraperitoneally in a dose of 120 mg/kg for 20 minutes before lifting.

The mitochondrial fraction of the brain receive the generally accepted method of differential centrifugation. Environment selection: sucrose - 250 mm, Tris-HCl 10 mm, EDTA 1 mm, pH 7,4.

2.1. Determination of the activity of monoamine oxidase (MAO) type A.

MAO activity AS determined in the mitochondrial fraction. As the substrate used serotonin in saturating concentrations. Determination of enzyme activity carried out by the method Horkina. MAO activity AS judged by the release of ammonia after isothermal distillation. Protein determined by the method of Lowry.

2.2. The study of respiration and oxidative phosphorylation of mitochondria in the brain.

Oxygen consumption and mitochondrial suspension register polarographic method using an open platinum cathode. The incubation medium: sucrose - 250 mm Tris-HCl(OH) - 10 mm, KCl 15 mm, KH2PO4- 20 mm, EDTA 0.5 mm, a pH of 7.4. As the substrate breathing used a 15 mm solution glutamate is the brain of animals (Richeria, So, Makletsova Meters, and other changes in the intensity of free radical reactions in the organs of rats under hypokinetic stress and protection of Delta-sleep inducing peptide and its tyrosinaemia analogue. - Izvestiya, 1993, N 2, S. 243-256). For example, the activity of mitochondrial monoamine oxidase type a (MAO A) from the brain of rats subjected to hypoxia, reduced by an average of 52%. Under the proposed action peptide 1 (intraperitoneal administration to rats at a dose of 120 mg/kg) enzyme activity in animals under stress is reduced by only 14% as compared with intact animals. This result suggests that largely retains the ability of the enzymes of the brain to the Association with the mitochondrial membrane and thereby stabilizing the enzymatic characteristics of the membranes of mitochondria in hypoxia.

Hypoxia leads to disruption of energizing brain tissue, which is reflected, in particular, the decrease in the rate of ATP synthesis and the decrease in the respiratory control. So, speed is reduced phosphorylation of ADR when the concentration of this metabolite 50 and 75 µm (table. 3). The preliminary introduction of peptide 1 increases these figures to the level of intact animals. the control animals (4,010,14 Rel.ed).

Example 3. The study of the anticonvulsant activity of peptides.

Experiments performed in the acute experiment on mice BALB/C mice weighing 18-22, Generalized convulsive activity cause the introduction of corazol (60 mg/kg, intraperitoneally) in a volume of 0.2-0.3 ml of the introduction of the peptide at doses of 0.1 and 1.0 mg/kg (intraperitoneally) is carried out in a volume of 0.2-0.3 ml of physiological solution for 30 min before applying corsola. Within 30 min after application convulsant see convulsive reactions in individual transparent chambers (40 x 30 x 20 cm). Take into account the latent period of the occurrence of the first and clinicalevidence seizures, as well as the severity of seizures, which are evaluated according to a scale. (Kryzhanovsky, N., Shandra A. A., Makolkin R. F. , godlewsky HP Bulletin of the experimental. biology and medicine, 1985, T. 99, No. 5, S. 527-532).

Peptides 1-3 have a strong anticonvulsant effect. The introduction of these peptides in doses of 0.1 m 1.0 mg/kg (intraperitoneally) leads to a significant increase in the time of occurrence of the first and clinico-tonic seizures, as well as reduce the severity of seizures compared with the control animals (table. 4). The data show that the inventive peptides 1-3 show more is in leads to increased lipid peroxidation (LPO) and accumulation in the tissues of the products of this reaction, in particular malondialdehyde (MDA). This is one of the causes damage to cell membranes, changes in the activity of membrane-bound enzymes and disruption of intracellular metabolism (Shidlovskaya I.e., the Intensity of lipid peroxidation in rat tissues by hypokinesia. - Space. biology and aviakosm. medicine, 1985, T. 34, vol. 1, S. 19-22). In this regard, the intensity of the FLOOR is one of the criteria characterizing the stress response in mammals) (Goldman A. W. and Alexander Y. A. Psychopharmacotherapy of neurotic disorders. -M.: Medicine, 1987, S. 287).

Example 4. The study of lipid peroxidation and levels of GABA under stress.

Experiments carried out on outbred white rats of both sexes weighing 170-250 g

4.1. Hypokinetic stress.

Animals are placed in a special chamber, sharply limiting their mobility for a period of 1 h and 6 h (Belov, T. I., Petrova N. In., Jonson Y. Lows Geruleus: regulation and function of blood-brain barrier in normal conditions and in conditions of emotional stress. - Bulletin of the experimental. Biol. and med., 1986, so 101, No. 4, S. 395-397). As control animals using rats that are contained in the vivarium. Introduction peptide 1 is ecay the brain and determine the level of malondialdehyde (Steel I. D., Horseville Tons, Method for the determination of MDA with TBA. Modern methods in biology. -M.: Medicine, 1977, S. 66).

4.2. Hyperbaric oxygen therapy (HBOT).

Animals are placed in a special chamber and expose 0.3 MPa of oxygen for 2 hours as control animals using rats that are contained in the vivarium. Introduction peptide 1 exercise intraperitoneally at a dose of 150 mcg/kg 60 min before stress exposure. Animals decapitat, remove the brain and determine the level of malondialdehyde (ibid.).

4.3. The definition of the content of GABA in the brain of animals.

A portion of brain tissue (1 g) is homogenized in 10 ml of 0.4 perchloro acid and centrifuged at 3000 rpm for 20 min. From the obtained supernatant taken 2 ml) and evaporated to dryness on a water bath. The residue is dissolved in 0.1 ml of water and 0.03 ml apply the strips of chromatography paper brand "C" size 2 x 28 see the starting Line is separated from the cathode 14 see Electrophoresis carried out in 0.2 N sodium acetate buffer (pH 4.6) at a voltage of 200 V and a current of 12 mA for 2 hours Strips dried and stained with 1% ninhydrin solution in acetone at a temperature of 90oC. Spot on ForageMax corresponding GABA, were the standard use of synthetic GABA (Sigma, USA).

When gipoklikemicheskim stress is a significant increase in MDA level was 51% after 1 h of stress and 103% after 6 hours, a Similar effect is observed and on the other type of stress - hyperbaric oxygenation (HBO). The addition of peptide 1 at doses of 100-150 mg/kg (intraperitoneally) leads to a noticeable decrease in MDA level (table. 5).

The stress response involves activation of the GABAergic system in the brain of mammals and especially with an adaptive increase in the intensity of biosynthesis of GABA (Goldman A. W. and Alexander Y. A. Psychopharmacotherapy of neurotic disorders. -M. : Medicine, 1987, S. 287). However, in the future, when the development of stress reactions depletion occurs biosynthetic capabilities and a sharp decrease in the level of GABA in the brain of animals (PL. 6), leading to an unfavorable imbalance of inhibitory and excitatory neurotransmitter amino acids, which in turn causes changes in the regulation of the basic functions of the body. As can be seen from the above data, peptide 1 not only prevents the stage of depletion of GABA in animals under conditions of 6-hour stress, but also maintains the state of urgent adaptation wonders what about the factor.

In General, the proposed peptide 1 has a neuroprotective effect in the stress effects due to the positive effect of the regulation of the basic energy processes in the brain and restore the level of energization brain tissue. At the same time this peptide reduces the activity of the process of lipid peroxidation than increases the stability of the membranes and regulates their functional state.

According to modern ideas about the role of stress in the pathogenesis of diseases of the nervous system, stress is considered as etiopathogenetic factor poststructural neurosensorial and the subsequent development neuroimmunol conflict. (Stepanenko E. M., Vilkov, A., Kryzhanovsky, N. Bulletin of the experimental. Biol. and the honey. 1984, T. 98, No. 9, S. 36-338; Vilkov, A. , Stepanenko E. M., Kryzhanovsky, N. Bulletin of the experimental. Biol. and the honey. 1987, No. 9, S. 288-290).

It is established that one of the real mechanisms of induction of immune responses to neuroantigen when stress is the activation of free radical processes, leading to disruption of the structure of cell membranes and increased permeability hematoencephalic barrier that increases the risk of contact specific bellixedda systems to stress and different intensity of prooxidant systems of the body. So, in Guinea-pig model of experimental encephalomyelitis (EAE) easily develops neurosensorial after stress, while the white outbred rats it develops only after the depletion of natural antioxidant capacities of animals through the use of monthly lipid diet (ibid.). In artificial depletion of antioxidant potential in rats dramatically weakened their natural tolerance to EAE, and introducing them encephalitogenic serum caused 100% morbidity and death of animals. At the same time, the preliminary introduction of animal antioxidant agents, including indirect antioxidant actions - peptide Delta-sleep, significantly reduced the proportion of diseased animals and the severity of the disease.

Neuroprotective effect of peptide 1 was shown in rats that are on lipid diet, with a depleted antioxidant system and subjected to painful and emotional stress. The experiment was assessed by the following indicators: content of corticosterone as a marker of stress, the degree of activation of peroxidation processes and antioxidant systems in the brain and blood of the chemiluminescent method, and the level of vitamins A and E in blood, cholesterol, diene to the neigh norepinephrine, dopamine, serotonin and histamine in certain areas of the brain.

Example 5. Study of the anti - and Pro-oxidant processes in the brain and blood of rats subjected lipid diet and stress.

Experiments carried out on outbred white rats-males weighing 170-250 g Lipid diet (old fat with a small amount of grain) support during the month. Rats subjected to painful and emotional stress for 4 h (Steel I. D., Horseville Tons, Method for the determination of MDA with TBA. Modern methods in biology. - M.: Medicine, 1977, S. 66). Peptides injected 1 h before stress intraperitoneally at a dose of 120 mg/kg a day animals decapitat and examine the degree of activation of peroxidation processes and antioxidant systems of the chemiluminescent method, and the level of vitamins A and E in serum (Vilkov, A., Smirnova, O. B., Mezhova L. I. Bulletin of the experimental. Biol. and the honey. , 1993, No. 10, S. 364-366).

Evaluate the content of histamine in various structures of the brain - the hypothalamus, the thalamus and the hippocampus colorimetric method (Korobov L. N., Khodakova A. A. , Frenkel M. L. Lab. the thing, 1982, N 4, p. 7-10). As standard use histamine firm "Fluka (Switzerland).

The received data is an indicator of the level of antioxidants in the system (table. 7) significantly lower in the group with a preliminary introduction of peptide 1 in comparison with the control group. While the inventive peptide 1 shows greater effectiveness than the natural peptide Delta-sleep. At the same time, the peptide 1 significantly reduced the content of Schiff bases (40%) and diene conjugates (30%) in the cerebral cortex of rats subjected to lipid diet and emotional-painful stress that demonstrates a lower level of prooxidant processes in animals, protected peptide 1. These results are consistent with a higher content of vitamin E in the blood of experimental animals (30%) and with lower levels of corticosterone, which also indicates reduced expression of Pro-oxidant processes and anti-stress action of peptide 1. In addition, peptide 1 clearly shows the ability to reduce the amount of histamine in all investigated structures of the brain - the hypothalamus, the thalamus and the hippocampus on 76,43% and 38% respectively when comparing the control and stressed animals and experimental animals received a single dose of peptide 1 for 1 h before stress exposure.

The evidence is clearly pronounced antioxidant and antisteroidogenic diseases (encephalitis, schizophrenia, multiple sclerosis and others).

Example 6. The toxicity study of peptide 1 (in accordance with GF XI).

Experiments conducted on 10 outbred white mice of both sexes weighing 20-22, Peptides injected intraperitoneally at a dose of 3 g/kg (0.5 ml sterile saline). Monitoring should be performed within 72 h after injection, while mice are kept separately on full feed ration (the State Pharmacopoeia of the USSR.-M.: Medicine, 1987, vol. XI, I. 2).

In experiments to study the toxicity of peptide 1 has been shown that intraperitoneal injection of peptide 1 at a dose of 3 g/kg to mice did not cause any abnormalities in the behavior of animals and 72 h after injection of peptide 1 death is not recorded.

Thus, the above data strongly suggest the severity of the claimed peptide analogues of the peptide Delta-sleep, anticonvulsant, anti-stress and neuroprotective activity, which, taking into account their toxicity to animals, can be recommended as a potential neuroprotective drugs for the treatment and prevention of different forms of encephalopathy.

Peptides>/BR>R - Ala-Ser-Gly-Glu, or Ala-Ser-Gly or Ala-Ser, or Ala;

and their pharmaceutically acceptable salts having anti-stress, anticonvulsant and neuroprotective effects.

 

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The invention relates to peptides of formula (I): X - A1- A2- Thr - Ala - Val - Gly - His - Leu - psi - A9- Q, where X represents a hydrogen, a simple relationship linking the alpha-amino group of A1with gamma-carboxyl part 3-propionyloxy part of A2if A2is Glu[-], or a group of the formula R1CO-, where R1selected from the group comprising: hydrogen, C1- C10- alkyl, or phenyl C1- C10- alkyl, A1is a D - or L-amino acid residue selected from the group consisting of: Phe, p - Hl - Phe, pGlu, Nal, Pal, Tpi, unsubstituted Trp or Trp substituted in the benzene ring by one or more substituents from the group comprising C1- C3- alkyl, or A1represents a peptide bond linking the acyl part of R1CO with alpha aminocyclo A2if A2represents Gln, Glu/-/ Glu (Y) or His, where /-/ is a simple relationship linking the gamma-carboxyl group of A2with the alpha-amino group of A1if A2is Glu, where X represents a simple bond, Y represents - or SIG5where R5is hydrogen, C1- C3- alkyl or phenyl; Leu - psi - is a reduced form Lой adjacent A9- balance is pseudopeptides communication; A9is a TAS, Ista, or DМТас; and Q represents NH2or CQ1where Q1is hydrogen, and pharmaceutically acceptable acids or salts, and pharmaceutical compositions, which has antagonistic activity against bombezin and to a method of treating cancer in mammals on the basis of the peptides of formula (I)

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The invention relates to the field of natural physiologically active peptides, specifically to an improved method for producing a peptide-sleep formula I:

TrpAlaGlyGlyAspAlaSerGlyGlu

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AA1-AA2-AA3-AA4-AA5-AA6(I)

where AA1group D - or L-N-thioxanthine, D - or L-N-centerlized, D-5H-dibenzo(a, d)cycloheptanol, L - or D-10,11-dihydro-5H-dibenzo(a, d)(cyclohepten-5-yl)glycine or L - or D--amino-10,11-dihydro-5H-dibenzo(a, d)cyclohepten-5-acetic acid, the amino acids may have a protective group;

AA2leucine, arginine, ornithine, or glutamic acid;

AA3aspartic acid, N-metilparabena acid;

AA4isoleucine, phenylalanine;

AA5isoleucine, N-methylisoleucine;

AA6tryptophan, N-formylthiophene

or their pharmaceutically acceptable salts

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The invention relates to new chemical substances that have valuable biological properties, and more particularly to a derivative of a peptide of formula (I):

AA1-AA2-AA3-AA4-AA5-AA6(I)

where AA1group D - or L-N-thioxanthine, D - or L-N-centerlized, D-5H-dibenzo(a, d)cycloheptanol, L - or D-10,11-dihydro-5H-dibenzo(a, d)(cyclohepten-5-yl)glycine or L - or D--amino-10,11-dihydro-5H-dibenzo(a, d)cyclohepten-5-acetic acid, the amino acids may have a protective group;

AA2leucine, arginine, ornithine, or glutamic acid;

AA3aspartic acid, N-metilparabena acid;

AA4isoleucine, phenylalanine;

AA5isoleucine, N-methylisoleucine;

AA6tryptophan, N-formylthiophene

or their pharmaceutically acceptable salts

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FIELD: medicine, cardiology.

SUBSTANCE: the suggested method should be performed at the background of medicinal therapy with preparations out of statins group, tevetene, polyoxidonium and conducting seances of plasmapheresis by removing 800 ml plasma twice weekly with N 5 due to additional intramuscular injection of immunophan 0.005%-1.0 with N 10 and fluimucyl 300 mg intravenously daily with N 5-10, total course of therapy lasts for 2 mo. The method provides modulation of leukocytic functional activity, moreover, due to altered cytokine profile and, thus, through disintegration of protein-lipid complexes participating in the development of atherosclerotic platelets.

EFFECT: higher efficiency of therapy.

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