WO2011053191A1

WO2011053191A1 - Agent for treating stress-related diseases and disorders in humans and animals, and treatment and/or prevention method using said agent

Info

Publication number: WO2011053191A1
Application number: PCT/RU2010/000615
Authority: WO
Inventors: Инэса Ивановна МИХАЛЕВА; Владислав Борисович ВОЙТЕНКОВ; Игорь Аркадьевич ПРУДЧЕНКО; Вадим Тихонович ИВАНОВ
Original assignee: Общество С Ограниченной Ответственностью "Исследовательский Центр "Комкон"
Priority date: 2009-10-26
Filing date: 2010-10-18
Publication date: 2011-05-05
Also published as: RU2450823C2; RU2009140209A

Abstract

The present invention relates to medicine and concerns an agent for treating and preventing stress-related diseases and disorders in humans and animals which corresponds to KND nonapeptide of the following formula: ТrрLуsGIуGlуАsnАlаSеrGlуGlu. The agent for treating and preventing stress-related diseases and disorders in humans and animals consists of a family of oligopeptides that comprises KND nonapeptide analogues and derivatives. The invention also relates to treatment methods using said agents, characterized in that they involve using the agents per se or as antigens in order to produce antibodies for medical use.

Description

An agent for the treatment of stress-related diseases and disorders in humans and animals, as well as a method of treatment and / or prophylaxis using this agent.

This invention relates to oligopeptides with positive pharmacological properties and the use of these oligopeptides for the treatment and / or prevention of stress-related diseases and disorders in humans and animals. The invention also encompasses the use of these peptides for the manufacture of pharmaceutical compositions.

The present invention relates to oligopeptides with pharmacological activity, which are suitable substances for the treatment and prevention of stress-related pathological changes that apply to the central nervous system (CNS), but are not limited to it.

Abbreviations

ADH - antidiuretic hormone

WHO - World Health Organization

DSIP - delta-sleep inducing peptide

SDSIP - family of delta-sleep inducing peptide

KND - KN-DSIP, ΚΝ-DSIP, (which corresponds to (Lys, Asn) -DSIP. According to the one-letter amino acid code, Lys is denoted by K, and Asn is denoted by N)

CKND - ΚΝ-peptide family

USP = US Pharmacopoeia

CNS - central nervous system

BBB - blood-brain barrier

MAO - monoamine oxidase

MDA - malondialdehyde

GABA - gamma-aminobutyric acid

NADP - nicotinamide adenine dinucleotide phosphate Definitions

Peptide analogues

Peptides described in the general formula (I) are called analogues - see below, which differ from the peptide with the main claimed amino acid sequence by replacing some amino acid residues with others, including non-proteinogenic residues, or by converting peptides of the general formula (I) to the corresponding cyclic peptides or (retro-inverse) analogs or by replacing amide bonds between residues with the corresponding Ν-alkylamide or ester bonds (N-alkylated and depsipeptide analogues).

Derivatives of peptides of formula (I) are essentially also analogues of peptides of formula (I). Here (and usually in the literature), peptide derivatives are more chemically consistent with products of modification of the main peptide chain or side chains of amino acid residues, for example, by their Ν-acylation with fatty acid residues or by conversion of the C-terminal carboxyl groups of peptides to ester, amide or> T- alkyl (aryl) amide groups.

Multimers correspond to structures consisting of several identical peptide units combined by covalent bonds in one structure directly through functional groups of monomer units or by using various known branching or bridging agents.

Proteinogenic (protein) amino acids are called amino acids, which are the structural elements of the proteins of living organisms.

Pharmaceutically acceptable salts are salts described in the United States Pharmacopeia or any other accepted pharmacopeia. These salts include, but are not limited to, salts of mineral or organic acids such as carbonic, hydrochloric, sulfuric acid, phosphoric acid, as well as lactic, benzoic, citric, oxalic acids and other acids.

Carriers and auxiliary fillers

Appropriate carriers and auxiliary ingredients provide the possibility of medical use of the pharmaceutical composition and, if necessary, contribute to the preservation of the oligopeptide (s) protected by the patent during storage and production of finished dosage forms. Such substances are well known in the art and can be found in standard literature. Suitable, but not limited to the following, are, for example, calcium carbonate / phosphate, various mono- and disaccharides, polysaccharides, fats, biopolymers such as human albumin, as well as biocompatible polymers, for example polyethylene glycol.

Stress is one of the main factors threatening human health. Stress is the physiological response of an organism to damaging factors of various origins or the threat of exposure to such factors. Stress can be caused by a number of factors, the so-called stressors: in addition to physical and mental injuries caused by severe accidents or infectious diseases, chemical irritations or radiation can also contribute to stress. The given sources of information (1-26) illustrate both the nature of stress and are the previous level of knowledge regarding the claimed inventions.

Acute and chronic stress can disrupt or weaken the homeostasis of the body, which translates into a deterioration in the ability to maintain the physiological balance of vital functions, such as maintaining cardiac activity or respiration.

The loss of the body's ability to maintain homeostasis can lead to a permanent disruption of the functioning of major organs and systems, such as neuroendocrine and cardiovascular. Typical consequences of impaired ability to adequately respond to prolonged stress can be: psychosomatic or immunological disorders, and even damage to organs, such as myocardial infarction and, for example, stress-induced stomach ulcers.

Homeostasis of the nervous system and the body as a whole is largely supported by neuroregulatory factors - neurohormones, neuromodulators and neurotransmitters, which can be directly secreted by hypothalamic cells (1), as well as various secretory cells, including those outside the central nervous system, for example, in the thin intestines.

Neuroregulatory factors cause changes in the functioning of the mammalian organism, some of which contribute to the maintenance of homeostasis. Often, neuroregulatory factors have a peptide nature (2).

Nonapeptide ADH (antidiuretic hormone, vasopressin), which regulates the reabsorption of water in the kidneys as a “thirst hormone,” is an example of such. Endogenous regulatory peptides of the modulator type include the so-called delta-sleep inducing peptide (DSIP), which is the object of comparison closest to the present invention and differs from the molecules described in this patent, described below, by their primary structure, namely the nature of the two amino acid residues of the molecule . The structure of DSIP was established in 1977. It was shown that in rabbits during low-frequency electrostimulation of the thalamus in the delta phase of sleep, a neuropeptide with the amino acid sequence Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu ( 3-4).

A subsequent detailed study of this factor by various groups of scientists around the world showed that the main possible field of its application is not only the treatment of sleep disorders (5-7), but the area of preventing the development of stress-induced pathology of the body and the treatment of the consequences of the development of pathology. Studies of the therapeutic properties of DSIP have shown its increased therapeutic effect in endo-and exogenous disorders of the central nervous system homeostasis (8-11).

In EP 1949906 A1 (12), DSIP is described as a treatment and prophylaxis against stress-induced massive damage to nerve cells caused by stress-inducing factors such as radiation, infection or trauma. DSIP stabilizes the basic functions of the central nervous system and as a result has a corrective effect on disorders of the homeostasis of the body as a whole.

The various effects of DSIP on the endocrine regulation of the body are not limited to improving its resistance to stressors: more fundamental properties of the peptide are described, such as interaction with the corticotropin system, antioxidant, immunomodulating effects and membranotropic properties (13-15).

The presence of a whole family of peptides, to which DSIP belongs, was supposed back in the 1990s, since it is known that endogenous DSIP (as an immunoreactive factor) can be detected both in the initial and in phosphorylated or glycolized form (16).

Patent EP 0065747 B1 (23), which describes DSIP as an agent suitable for controlling the symptoms of withdrawal symptoms, patent RU 2099078 C1 (24), which describes the medical use of an anti-stress agent, is also prior art for this invention. based on DSIP and patent RU 2115660 C1 (25), which describes analogues of DSIP as anti-stress and anticonvulsant agents.

The present invention was made taking into account the previous level of knowledge, with the aim of introducing a new family of peptides that regulate body functions, with preferably emerging therapeutic, especially stress-protective and adaptogenic properties.

The inventive means - family of peptides - include analogues and derivatives of the main claimed peptide shown in the General formula (I), which is the acronym of the new family of peptides:

(I) Χ-Υ'-Υ ² -Υ ³ -Υ ⁴ -Ζ

where Z = Y ⁵ -Y ⁶ -Y ⁷ -Y ⁸ - Y ⁹ -R, or Z = Y ⁵ -Y ⁶ -R, or Z = R;

X = H or an acyl residue of formic, acetic, propionic or other fatty acids (e.g. palmitic, stearic), as well as their alkyl or

(aryl) alkyl derivative;

Y ¹ = aromatic amino acid residue Tgr or Tug, Phe or their corresponding Ν-methyl derivative of the L- or D-configuration;

Y ² = amino acid residue of Lys or Arg, His, Orn or their Ν-methyl derivative of the L- or D-configuration;

Y ³ = Y ⁴ = residue Gly or Ala, Thr, Ser, Asn, L- or D-configuration;

Y ⁵ = Asn or Gin residue, their Ν-alkylamide derivative, Asp, Glu, their ester derivative, or M ^a methyl derivative of the indicated residues of the L- or D-configuration;

Y ⁶ = Ala residue or β-Ala, Gly, Ser, Thr, Phe, Val, L or D configuration;

Y ⁷ = residue Ser or residue Thr, Asn, Gin L- or D-configuration;

Y ⁸ = Gly residue or Ala, Ser, Thr, Asn residue of the L or D configuration;

Y ⁹ = residue Glu, Asp, Asn, Gin L- or D-configuration;

R = —OH, —ORi, —NH ₂ , —NHR], or —N (Rj) ₂ (where Ri is alkyl or arylalkyl).

A new family of regulatory peptides related to DSIP but previously unknown structure that differ from the current level of knowledge (DSIP) by the nature of the two amino acid residues of the peptide chain corresponding to the known structure of DSIP is presented. Such changes in the structure of 2 amino acid residues lead to an increase in the therapeutic effect, in particular, in prevention and therapy stress related diseases in humans and animals. The inventors show in an experiment with forced swimming in rats that the subject of the invention increases the stress resistance of animals when compared with DSIP.

The present invention relates to the prevention and treatment of pathological and stress-related disorders of homeostasis in humans and animals.

Regarding the mechanism of action of CKND, the inventors suggest that the peptides have a modulating effect on the level of excitatory and inhibitory neurotransmitters, including GABA, serotonin or glutamate. Like SDSIP, the subject of the present invention can also modulate the activity of key membrane mitochondrial enzymes, as well as enzymes that control metabolism in the central nervous system and peripheral tissues, such as monoamine oxidase A, hexokinase, NADP-dependent malate dehydrogenase, creatine kinase, respiratory chain enzymes, hypothalamic glutamine superoxide dismutase (8, 9, 13). .

A computer search for amino acid sequences homologous to the subject of the invention using the BLAST search program (PubMed, Basic Local Alignment Search Tool) revealed a 100% match to the human nuclear protein 1B site (called the “Jumonji domain containing 1B”) located on chromosome 5 human genome, and consisting of 1551 amino acid residues (17).

JMJD1B is a member of the family of histone demethylases type JHDM1 and JHDM2, including the JmjC domain. JmjC-domain-containing histone demethylases capable of demethylating the lysine residues of histones are conserved in different species from yeast to humans. Histone methylation-demethylation is an important mechanism of post-translational modifications and plays an exceptional role in the regulation of chromatin structure and gene transcription. These enzymes are ubiquitously present in various tissues. The first studies of the JMJ base gene suggest its important role in the development of the nervous system and some organs, such as the liver, spleen, and thymus (18, 19).

EP 1950286A1 (22) closes the use of a mixture of Jumonji domain enzymes with demethylating properties, especially for the treatment of prostate cancer.

This invention is made taking into account the previous level of knowledge, in order to present a new family of peptides that regulate body functions, with preferably emerging therapeutic, especially stress-protective and adaptogenic properties.

A new family of oligopeptides with a peptide chain length of 4, 6 or 9 amino acid residues, as described above, corresponds to the following general formula:

(I), X-Y'-Y ² -Y ³ -Y ⁴ -Z

(aryl) alkyl derivative;

Y is the amino acid residue of Lys or Arg, His, From or their Ν-methyl derivative of the L- or D-configuration;

= residue of Gly or Ala, Thr, Ser, Asn, L- or D-configuration;

Y ⁶ = Ala residue or β-Ala, Gly, Ser, Thr, Phe, Val, L or D configuration;

Y ⁷ = residue Ser or residue Thr, Asn, Gin L- or D-configuration;

Y ⁸ = Gly residue or Ala, Ser, Thr, Asn residue of the L or D configuration;

Y ⁹ = residue Glu, Asp, Asn, Gin L- or D-configuration;

R = -OH, -OR}, -NH ₂ , -NHR _1? or -N (Ri) 2 (where

-alkyl or arylalkyl).

The subject of the invention is the claimed K D peptide from the group of peptides of the general formula (I) has the following structure: TrpLysGlyGlyAsnAlaSerGlyGlu.

The peptides of the above general formula (I) can be synthesized by classical methods of peptide synthesis, both the traditional method of synthesis in solution and the solid-phase method of synthesis on a polymer carrier. Peptides are purified by chromatographic methods. Analysis of the structure and degree of individuality of synthetic peptides is carried out by modern physicochemical methods. These methods are well developed and described in the relevant literature (20, 21).

The group of the claimed peptides may also include the following analogues and derivatives of peptides of the family (I): 1. cyclic analogs formed using known methods for the synthesis of cyclopeptides by closing the (4-16)-membered ring through the N- and C-terminal or side functional groups, including when additional low molecular weight bridges weighing up to 500D are included in the cycle.

2. (retro-inverse) analogs of peptides of the general formula (I) obtained by the synthesis of peptides from the corresponding D-amino acid derivatives by changing the direction of acylation, for example, of the following form: ABCDEF (some peptide, all residues of the L-configuration) and FEDCBA ( its (retro-inverse) -analogue or synonym (retro-enantio) -analogue, all the remnants of the D-configuration). The latter analogues are mirror symmetric to the first and can be equally recognized by biological targets.

3. multimers of peptides corresponding to the general formula (I), formed by synthesis of constructs, where several monomer units (from 2 to 6) are connected directly through covalent bonds through their functional groups or by the inclusion of low molecular weight branching bridges between monomers of amino acid or other nature for example, agents such as derivatives of lysine or other polyamines or polyacids.

4. peptides attached (including adsorbed or encapsulated) to various pharmacologically acceptable polymer carriers, for example, polyacrylates, polylactates, polysaccharides, gelatin, etc.

The general formula can be expanded by adding additional peptide fragments to one or both ends of the amino acid chain of type (I) nonapeptides, so that the extension fragments from the C- and / or N-terminus include from 1 to 5 protein amino acid residues each. The N and C ends of any elongated peptides as well as all peptides of formula (I) may be free and have no substituents at the N and C ends of the amino acid chain, or be blocked by X and / or R.

Also to the above family of peptides are those peptides that exist in whole or in part in the form of compositions with pharmaceutically acceptable carriers. ι

This invention also relates to mixtures of the peptides of the present invention with other neuropeptides, for example DSIP, its analogs and their pharmaceutically acceptable salts and pharmaceutically acceptable solvents and / or solutions and / or excipients, for example, distilled water, physiological solution, suitable buffers, calcium carbonate / phosphate, various mono- and disaccharides, polysaccharides, fats, etc. The present invention also relates to mixtures with other known neuroprotective agents, such as, for example, piracetam. It can be assumed that the combined use of the peptides of this invention with piracetam will enhance the protective effect of each component separately, as described previously for a mixture of DSIP and piracetam (26). These mixtures can be used in the manufacture of pharmaceutical compositions in the preparation of dosage forms suitable for the treatment and / or prevention of diseases, especially those associated with stress-induced diseases and disorders of the body in humans and animals.

Other pharmaceutical compositions of peptides of the KND family specified in the above general formula (I) and other synergistically active components such as protein amino acids (e.g. neurotransmitters glycine, arginine and other amino acids), natural antioxidants, e.g. carnitine, carnosine, homocarnosine or their precursors, for example, acetyl derivatives.

The ratio of the main components of the above pharmaceutical compositions by weight is as follows: peptide component: amino acid component: natural antioxidant component 1: (0-250): (0-250).

The present compositions may also include various other neuroprotective metabolically active substances (one or more), such as phospholipids (lecithin), unsaturated fatty acids, choline and its derivatives, trimethyl glycine, vitamin C, vitamin A and / or its precursors, nicotinamide, vitamin E , B vitamins, folic acid, lipoic acid, eicosapentenoic and docosahexaenoic acids, bioflavonoids, coenzyme Q 10, taurine, terpenes, polyphenols, extracts of plant origin (garlic, onion, green tea, gingko beluba, female nya, licorice, ginger, gotu kola, conifers), succinic acid, riboxin, macro and trace elements (magnesium, calcium, potassium, chromium, selenium), etc.

Compositions may include other acceptable ingredients commonly used in the preparation of medicaments such as fillers, flavoring and flavoring agents, sweeteners, preservatives, etc. The present compositions are based on natural substances, which can also be included in parapharmaceutical biologically active additives, homeopathic remedies and baby food. A subject of the invention includes a therapeutically effective amount of the peptides of the invention for use in a dose range from a few μg / kg to 1-10 mg / kg or in homeopathic doses in the case of homeopathic dosage forms.

It is known that substances of a peptide nature exert their effect not only when administered as various injections, but also using other delivery routes, in particular, intranasal (11).

The inventive tool can be administered intranasally in the form of nasal drops, in the form of a spray, nebulized powder, aerosol, sublingually in the form of absorbable tablets, capsules and powders; in the form of various injections (intravenous, intramuscular, subcutaneous and intradermal); intralumbally and intracisternally (intraventricularly), percutaneously using devices for therapeutic electrophoresis; in the form of suppositories, ointments, creams, lotions, and also be included in baby food.

The subject of the invention also relates to the use of oligopeptides of the KND family as peptide antigens for the production of specific anti-antibodies for medical purposes, the introduction of which into the body can lead to the same effects as the use of the basic starting substance.

The present invention relates to agents that have pharmacological activity and are substances suitable for the treatment and prevention of stress-induced pathological changes. These changes relate mainly to the central nervous system, but are not limited to it. Exogenous and endogenous factors, such as intoxication, trauma, serious illnesses, cause stress and can provoke a violation of the homeostasis of the body as a whole. If the body is not able to cope with these disorders of homeostasis, due to various diseases or chronic disorders, both functional and organic, so-called stress-induced diseases and disorders can develop.

Often there are not only functional, but also morphologically visible organic damage to the central nervous system, characterized by massive death of neurons.

The described agent, and in particular the preferred formula, as well as its derivatives, analogues and physiologically acceptable salts, is able to prevent the development of deficiency of endogenous regulatory peptides, for example, DSIP and others, which can occur in case of severe or chronic stressful situations. Animal experiments showed a similar focus of DSIP and KND, with KD even superior to DSIP in terms of effectiveness in some respects.

The following describes by way of example the effect of the subject invention. In order to better present the data, a comparison is made between the DSIP, KND and the control group.

Examples:

1. Materials and methods.

An experimental assessment was made of the biological activity of the KND oligopeptide family, as well as some variants of compositions and mixtures of oligopeptides of this family with other pharmaceutical agents.

270 Wistar male rats at the age of 3 months were divided into 18 groups and received in each group 5 rats subcutaneously for 2 periods of administration for 4 days for 2 weeks, doses of 100 μg / kg of peptides of the family, except for two cases (see below) in which peptides of the KND family were administered at higher dosages (250 and 400 μg / kg); 100 μg / kg DSIP dissolved in water for injection in a volume of 0.2 ml, or water for injection in a volume of 0.2 ml per rat. The introduction was carried out in the morning.

The following variants of peptides of the KND family and their compositions with other pharmacological agents were introduced:

KND-1: a substance with an amino acid sequence

TrpLysGlyGlyAsnAlaSerGlyGlu;

KND-2: a substance with an amino acid sequence

TyrLysGlyGlyAsnAlaSerGlyGlu,

KND-3: a substance with an amino acid sequence

TrpMeLysGlyGlyAsnAlaSerGly Glu,

KND-4: a substance with an amino acid sequence

TrpLysGlyGlyAsnAlaSerAlaGlu,

KND-5: substance with amino acid sequence

TrpArgAlaGly AsnTyrSerGlyGlu,

KND-6: substance with amino acid sequence

TrpLysGlyGlyAspAlaSerGly Glu, KND-7: substance with amino acid sequence

TrpLysGlyGlyGluAlaSerGly Glu,

KND-8: substance with amino acid sequence

TrpOrn Gly GlyAsnAlaSerGly Glu,

KND-9: substance with amino acid sequence

TrpLysAla GlyGlnAlaSerGlyGlu,

KND-10: substance with amino acid sequence

TrpHisAlaGlyAspAlaSerGlyGlu,

KND-11: a substance with an amino acid sequence

TrpLysGlyGlyAsnAlaThrGlyGlu

KND-12: a substance with an amino acid sequence

TrpLysGlyGlyAsnAlaSerGlyGlu in a mixture with the synergistically active amino acid glycine,

KND-13: a substance with an amino acid sequence

TrpLysGlyGlyAsnAlaSerGlyGlu in a mixture with the synergistically active amino acid glycine and the natural antioxidant carnosine in a ratio of 1: 25: 30,

KND-14: substance with amino acid sequence

TrpLysGlyGlyAsnAlaSerGlyGlu in a mixture with the synergistically active amino acid glycine and the natural antioxidant carnosine in a ratio of 1: 50: 50,

KND-15: substance with amino acid sequence

TrpLysGlyGlyAsnAlaSerGlyGlu in a mixture with the synergistically active amino acid glycine and the natural antioxidant carnosine in a ratio of 1: 100: 100,

KND-16: substance with amino acid sequence

TrpLysGlyGlyAsnAlaSerGlyGlu in a mixture with the additional neuroprotective metabolically active component riboxin,

KND-17: substance with amino acid sequence

TrpLysGlyGlyAsnAlaSerGlyGlu, administered at a dose of 250 μg / kg,

KND-18: a substance with an amino acid sequence

TrpLysGlyGlyAsnAlaSerGlyGlu, administered at a dose of 400 mcg / kg.

2. Toxicity study. The body mass of adult rodents is a common parameter for studying the toxicity of various substances. The increase in body weight between the days of the first and last injections was comparable in all groups (table 1).

KND-1 (see claim 1)

K D-3 (see claims 1.2) Increase Control DSIP KND-3 body weight, g

Average 21.20 22.14 20.01 value

Standard 6.70 4.96 4.14 deviation

N 5 5 5

KND-4 (see claims)

KND-5 (see claims 1.2)

KND-6 (see claims claims invented]

KND-7 (see claims 1.2)

Increase Control DSIP KND-7 body weight, g

Average 20.19 23.02 18.00 value

Standard 6.71 9.90 10.14 deviation

N 5 5 5

KND-8 (see claims 1.2)

Increase Control DSIP KND-8 body weight, g

Average 18.20 19.00 21.00 j value

Standard 7.90 8.60 8.10 deviation

N 5 5 5

KND-9 (see claims 1.2)

KND-10 (see claims 1.2)

KND-11 (see claims 1.2)

KND-12 (see claim 3) Increase Control DSIP KND-12 body weight, g

Average 23.10 22.00 24.00 value

Standard 6.79 3.90 6.13 deviation

N 5 5 5

KND-13 (see claim 3)

KND-14 (see claim 3)

KND-15 (see claim 3)

iGSiD-16 (see claim 3)

KND-17 (see claims 8)

Increase Control DSIP KND-17 body weight, g

Average 20.2 21.4 19.5 value

Standard 2.4 3.5 1.9 deviation

N 5 5 5

K D-18 (see claim 8)

Thus, the toxicity of none of the investigated substances is not registered.

3. Forced swimming test

Two hours after the last injection of KND 1-18, DSIP or rat water, the test was conducted in a forced swimming test, which allows you to evaluate both the physical strength and endurance of the animal, as well as the emotional state.

The test was carried out in a pool of water, the temperature of which was controlled by a thermometer and was 30 ± 1 ° С throughout the experiment. A weight consisting of metal elements, which was calculated based on the weight of the rat, was always attached to the neck of the rats on a soft, not restricting the animal’s movements and not obstructing the air flow collar, which was always 20% of it, the rat was weighed and the weight of the metal elements were calculated immediately before the animal was immersed in water.

After the rat was immersed in water, the swimming duration was estimated, which was recorded by a stopwatch and measured in seconds. At the end of the swimming time, the moment was taken when the rat stopped swimming, sank to the bottom of the pool and for at least 10 seconds made no further attempts to surface. At this point, the animal was removed from the water for further research.

While in the group receiving DSIP, only a slightly longer swimming time was observed than in the control group, in KND groups 1-18 the swimming time was most often significantly longer than in the DSIP and control groups (table 2). Table 2: Rat performance in the Forced Swimming Test

KND-1 (see claim 1)

KND-4 (see claims 1.2) Duration of control of the DIP KND-4 of the swimming, s average 314.02 353.09 457.00 standard 76.87 83.74 47.55 deviation

N 5 5 5

KND-5 (see claims 1.2)

KND-6 (see claims 1.2)

KND-7 (see claims)

swimming, average 318.09 317.53 469.07 standard 100.50 129.02 98.78 deviation

Ν 5 5 5

KND-8 (see claims 1.2)

KND-9 (see claims 1.2)

KND-10 (see claims) duration control DSIP KND-10 swimming, s

ICND-11 (see claims 1.2) Duration of control KIP of KND-11 swimming, average 392.45 321.41 399.09 standard 122.56 107.43 97.06 deviation

N 5 5 5

KND-12 (see claim 3) Duration of control of the KND-12 DSIP of swimming, with an average of 356.21 344.40 418.04 standard 100.21 132.12 88.86 deviation

N 5 5 5

KND-13 (see claim 3) Duration of control of the DIP KND-13 I swimming, s average 314.00 300.52 388.03 standard 110.41 87.52 74.44 deviation

N 5 5 5

KND-14 (see claim 3)

KND-15 (see claim 3) Duration of control KND-15 DSIP of swimming, with an average of 299.12 292.20 319.03 standard 91.31 127.99 77.73 deviation;

N 5 5 5

KND-16 (see claim 3)

KND- 17 (see claims 8)

KND-18 (see claim 8)

Based on the results obtained, it can be stated that when using KND in various variations and mixtures, not only did not mainly a deterioration of physical strength, endurance and psycho-emotional state, but there was a clear improvement of these parameters compared with both control groups and groups receiving DSIP. Not all KND analogues and mixtures thereof with various pharmacological agents showed the same activity, however, its orientation in all cases was similar.

3. Assessment of the effect of stress and substances on the state of the antioxidant system of the body.

Immediately after the forced swimming test was completed, the animals were sacrificed by guillotination and an autopsy was performed. Blood was taken for analysis of the content of malondialdehyde (MDA). It was shown that the use of both DSIP and KND 1-16 did not lead to a significant change in the level of oxidative stress in rats under forced swimming test conditions (Table 3).

Table 3: Blood MDA

KND-1 (see claim 1)

K D-2 (see claims 1.2)

KND-3 (see claims 1.2)

KND-4 (see claims 1.2)

KND-5 (see claims 1.2)

Activity Control DSIP KND-5 MDA, unit average 9.98 9.87 11.23 standard 0.85 0.98 0.42 deviation

N 5 5 5 KND-6 (see claims 1.2)

KND-9 (see claims 1.2)

KND-10 (see claims 1.2)

KND-11 (see claims 1.2)

K D-12 (see claim 3) Activity Control DSIP KND-12 MDA, units average 9.91 10.04 10.91 standard 0.82 1.98 0.23 deviation

N 5 5 5

KND-13 (see claim 3)

Activity Control DSIP KND-13 MDA, unit average 10.80 12.00 11.37 standard 2.07 1.91 1.31 deviation

N 5 5 5

KND-14 (see claim 3)

KND-15 (see claim 3) Activity Control DSIP KND-15 MDA, units average 9.90 11.52 10.81 standard 1.11 1.97 0.94 deviation

N 5 5 5

KND-16 (see claim 3) ^'

KND-17 (see claims 8)

KND-18 (see claim 8) Activity Control DSIP KND-18

MDA, unit average 9.13 10.13 09.99 standard 1.76 1.73 1.12

deviation

N 5 5 5

Group averages were compared to show trends.

As shown in the described example, rats treated with 100 μg / kg of the drug most often had greater endurance in terms of swimming in water than control animals. This indicates greater physical strength, as well as better stress resistance.

A similar trend with DSIP was shown by indicators of changes in the weight of immunocompetent organs, as well as MDA activity.

In general, an increase in MDA activity can be considered a consequence of chronic stress. However, past experiments have shown that DSIP in the first minutes after administration, as well as in the first minutes after stress exposure, has a slight prooxidant effect, then replaced by a reliable antioxidant 14).

The results of the forced swimming test impressively demonstrate the potential of the subject invention to support physical strength under stress, an extremely important characteristic in severe diseases.

The superiority of KND in this indicator not only over the control group, but also over the DSIP, is also evident.

In addition to the main group of experiments, an additional assessment of the effectiveness of KND was carried out at various dosages, or in a mixture with the synergistically active amino acid glycine under hypokinetic stress.

The experiment was carried out on white rats of both sexes weighing 150-200 g. Hypokinetic stress was caused by placing rats in special chambers restricting the mobility of animals for a period of 6 hours. 1 hour before the onset of stress, KND was administered in various dosages, either in a mixture with glycine or saline.

The stress level was judged by the accumulation of MDA in the animal brain. The following results were obtained (table 4):

Table 4 (see paragraphs of formulas 3 and 8).

* - significance of differences compared with control p <0.01

An additional group of experiments was conducted to evaluate the detoxification effect of KND in various doses in mice that received a semi-lethal dose of the cisplatin cytotoxic drug. K D was administered intraperitoneally in a single dose of 60 μg / kg or 120 μg / kg 24 hours before and after administration of the cytostatic agent.

Cytostatic cisplatin was administered once intravenously in a single dose of 15 mg / kg, causing 50% death of animals. Studies were performed on 50 mice, BDF1 hybrids, and females.

The death of animals from acute cytostatic toxicity was recorded. The introduction of KND led to a twofold decrease in the death of animals from acute toxicity of cisplatin.

The results obtained in additional experiments indicate that the use of KND does not reduce the activity of the body's anti-stress defense system, but also suppresses the intensification of oxidative processes caused by hypokinetic effects. A pronounced antistress and antitoxic effect of KND is shown.

In general, the results indicate that the claimed agent can affect the ratio of excitatory and inhibitory neurotransmitters in the body, such as GABA, glutamic acid, aspartic acid and serotonin, and also exhibits antioxidant and antitoxic effects.

Among the possible targets of KND exposure may be mitochondrial enzymes that control the metabolism of the central nervous system and peripheral nerve tissues, e.g. MAO A, hexokinase, NADP-dependent malate dehydrogenase, creatine kinase, as well as respiratory chain enzymes, hypothalamic glutamine synthetase, SOD, catalase, etc.

Means - a family of peptides - is therefore suitable for the treatment of a wide range of stress-related, especially neurological pathological processes. Therefore, CKND can be used for diseases both in the form of monotherapy, and in combination with various medications, herbal preparations, food additives, as well as for the treatment of various manifestations and consequences of impaired homeostasis.

These include, but are not limited to, neurohumoral and endocrine disorders caused by external and internal disorders. The mechanism of action of the proposed peptides can be described, for example, as stress-protective, adaptogenic, neuroprotective, immunomodulating, geroprotective, antioxidant, anticonvulsant, antitoxic.

KND is also obviously suitable for the treatment and prevention of primary drug cravings, withdrawal symptoms, sleep disturbances, encephalopathies, neurosis, autonomic disorders, cognitive impairment, attention deficit disorder, episodes of depression, cardiovascular disease, cerebrovascular disease, autoimmune and endocrinological diseases.

It is well known that the active ingredients of peptides, due to rapid enzymatic degradation in the digestive tract, are only relatively suitable for oral administration. The preferred form of application of the subject invention therefore becomes intranasal or sublingual administration.

These administration forms provide an opportunity to avoid the intestinal-blood barrier, thus allowing a significant reduction in the dose of the administered substance. In addition, when used along this path, the risk of infection is eliminated, the administration is painless, and also does not require the presence of specially trained medical personnel.

However, the agent may be administered by any of the administration routes.

The conviction of the inventors is that the substance, like DSIP, can easily cross the BBB.

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Annals of the New York Academy of Sciences. Vol. 739. Models of Neuropeptide Action. Ed. by F.L. Strand, B. Beckwith, B. Chronwall, C.A. Sandman. New York Academy of Sciences, New York, 1994, pp. 1-349.

Monnier M., Hosli L., Dialysis of Sleep and Waking Factors in Blood of the Rabbit. Science 146, 796-798 (1964)

Schoenenberger G.A., Monnier M. Characterization of a delta-electroencephalogram (6-sleep) -inducing peptide. Proc. Natl. Acad. Sci. USA 74, 1282-1286 (1977)

Schneider-Helmert D., Graf M., Schoenenberger G.A. Synthetic Delta Sleep-inducing Peptide Improves Sleep in Insominacs. Lancet 1, 1256-7 (1981)

Schneider-Helmert D., Gnirss F., Monnier M., Schenker J., Schoenenberger G.A. Acute and delayed effects of DSIP (delta sleep-inducing peptide) on human sleep behavior. Int. J. Clin. Pharmacol Ther. Toxicol. 19, 341-5 (1981)

SchoenenbergerG.A. 1984, Characterization, properties and multivariate functions of Delta-Sleep-Inducing Peptide (DSIP). Eur. Neurol. 23, 321-345

Khvatova, E.M., Rubanova, N: A. Prudchenko, I. A., Mikhaleva, I. I. Effects of 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, 367-369 (1995)

Khvatova E. M., Samartzev V. N., Zagoskin P. P. Delta sleep inducing peptide (DSIP): effect on respiration activity in rat brain mitochondria and stress protective potency under experimental hypoxia. Peptides, 24, 307-311 (2003).

Stanojlovic O., Ivanovic D., Mirkovic S., Mikhaleva I. Delta sleep-inducing peptide and its tetrapeptide analogue alleviate severity of metaphit seizures. Pharmacology Biochemistry and Behavior, 77, 227-234 (2004).

I.I. Mikhaleva, B.O. Voitenkov Delta-sleep peptide and deltaran: from chemical and biological research to medicine. New Drugs, vol. 3 pp. 6-20, (2007).

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Prudchenko I.A., Stashevskaya L.V., Shepel E.N., Mikhaleva I.I., Ivanov V.T., Shmalko Yu.P., Chaly A.P., Umansky V.Yu., Grzhevskaya S .N. Synthesis and biological properties of delta-sleep peptide analogues (DSIP). P. Antimetastatic action. // Bioorgan, chem., 1993.V.19, N12, S.1177-1190.

Rikhireva G.T., Makletsova M.G., Menzheritsky AM, Vartanyan L.S., Gurevich SM., Lozovskaya E.L., Kopylovsky S.A., Rylova A.V., Mikhaleva I.I., Prudchenko I.A. Changes in the intensity of free radical reactions in rat organs under hypokinetic stress and the protection of delta-sleep by an inducing peptide and its tyrosine-containing analogue. // Proceedings of PAH, JYo2, 243-256, (1993).

Mikhaleva 1. 1., Rikhireva, G. T., Prudchenko, I. A., Golubev, I. N. Interaction of delta sleep-inducing peptide and its analog with cellular membranes: A structure-function analysis. Russian Journal of Bioorganic Chemistry, 32, 160-165 (2006).

Prudchenko, I.I. Mikhaleva. The endogenous problem of the delta sleep peptide. Advances in Modern Biology Vol. 114, pp. 728-740, (1994).

Strausberg R.L. et al., Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc. Natl. Acad. Sci. U.S.A. 99 (26), 16899-16903 (2002).

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Claims

CLAIM

1. An agent for the treatment and prevention of stress-related diseases and disorders in humans and animals, which corresponds to the KND nonapeptide of the following formula: TrpLysGlyGlyAsnAlaSerGlyGlu.

2. The tool for the treatment and prevention of stress-related diseases and disorders in humans and animals, which is a family of oligopeptides of the general formula (I), including analogs and derivatives of the nonapeptide K D of the formula according to claim 1:

(I) Χ-Υ'-Υ ^ -Υ ^ Ζ,

(aryl) alkyl derivative;

Y ³ = Y ⁴ = residue Gly or Ala, Thr, Ser, Asn, L- or D-configuration;

Y ⁶ = Ala residue or β-Ala, Gly, Ser, Thr, Phe, Val, L or D configuration;

Y ⁷ = residue Ser or residue Thr, Asn, Gin L- or D-configuration;

Y ⁸ = Gly residue or Ala, Ser, Thr, Asn residue of the L or D configuration;

Y ⁹ = residue Glu, Asp, Asn, Gin L- or D-configuration;

R = -OH, -OR], -NH _2, -NHR], or -N (R ₂ (wherein R _\ alkyl or arylalkyl).

3. The tool according to any one of paragraphs 1, or 2, characterized in that the pharmaceutical composition comprises at least one synergistically active amino acid, such as glycine or a natural antioxidant, such as, for example, carnosine or homocarnosine or their natural precursors and derivatives, wherein the ratio of components by weight is: peptide component: amino acid component: natural

antioxidant 1: (0-250): (0-250).

4. The tool according to any one of paragraphs 1-3, characterized in that the pharmaceutical composition contains one or more pharmaceutically acceptable solvents and / or carriers and / or excipients, such as distilled water, saline, buffer solution, calcium phosphate / calcium carbonate, polysaccharides , fats, etc.

5. The tool according to any one of paragraphs 1-4, characterized in that the pharmaceutical composition includes at least one additional neuroprotective metabolically active component, for example: lecithin, unsaturated fatty acids, choline, B vitamins, folic acid, bioflavonoids, plant nutraceuticals, macro -and trace elements, etc.

6. The tool according to any one of paragraphs 1-5, characterized in that the composition may include other acceptable ingredients, usually used for the preparation of drugs as fillers, flavoring and perfumes, sweeteners, preservatives, etc.

7. The tool according to any one of paragraphs 1-6, characterized in that the compositions are based on natural substances, which can also be included in parapharmaceutical biologically active additives, homeopathic remedies and baby food.

8. The tool according to any one of paragraphs 1-7, characterized in that the compositions include a therapeutically effective amount of peptides for use in the dose range from several μg / kg to 1-10 mg / kg or in homeopathic doses in the case of homeopathic dosage forms.

9. The method of treatment using the tool according to any one of paragraphs 1-2, characterized in that the tool is used as antigens for the production of antibodies for use in medicine.

10. The method of treatment using the funds according to any one of paragraphs 1-8, characterized in that the form of application of these funds is the introduction in the form of nasal drops, in the form of a spray, sprayable powder, aerosol, sublingually in the form of absorbable tablets, capsules and powders; in the form of various injections; intralumbally and intracisternally (intraventricularly), in the form of suppositories, ointments, creams, lotions, transdermally using devices for therapeutic electrophoresis.

11. The method of treatment using the tool according to any one of paragraphs 1-10, characterized in that for the treatment of a wide range of stress-related, features of neurological pathological processes CKND can be used for diseases both in the form of monotherapy, and in combination with other well-known neuroprotective and antioxidant drugs, herbal remedies, and nutritional supplements.