EA028069B1 - Peptide having nootropic action, compositions based thereon and methods of application thereof - Google Patents

Abstract

The invention relates to medicine, in particular to peptide agents having nootropic properties. The invention is directed to broadening the range of agents for protecting central nervous system having nootropic action, effective and safe both in using in adults and children including infants. For this purpose human beta-casomorphin-7 of formula Tyr-Pro-Phe-Val-Glu-Pro-Ile is used. The invention can be used for preparing a composition for administration to newborns, babies and preschool- and school-aged children, and also for application in adolescents and adults. The invention can also be used for preparing a pharmaceutical composition, a biologically active food additive, functional nutrition products or foods for particular nutritional uses.

Description

The present invention relates to medicine, namely to peptide agents having nootropic properties. The invention can be used for the manufacture of a composition for administration to newborn children, toddlers and preschool and school children, as well as for use in adolescents and adults. The invention can also be used for the manufacture of pharmaceutical compositions, biologically active food additives, functional foods or specialized foods.

State of the art

Neurological and behavioral disorders are currently receiving much attention. The nootropic concept has become the largest contribution to the development of psychopharmacology, both in applied and fundamental terms. Currently, a significant number of drugs have been created that are used, first of all, for the correction of impaired mnemonic functions, as well as with a decrease in the level of vital activity that occurs with many diseases and extreme effects. Nootropics are also indicated for impaired adaptation and mental activity during aging, including presenile and senile dementia. In addition, nootropics are used for disorders of cerebral circulation, intoxications (including alcohol), withdrawal symptoms, sleep deprivation, fatigue, asthenic and depressive states after craniocerebral trauma and neuroinfections, as well as for the correction of side effects of psychopharmacotherapy (in the treatment of neuroleptics, antidepressants, psychostimulants). In pediatric practice, nootropic drugs are used to treat cerebral growth, encephalopathy, intellectual deficiency, disorders that form in premature infants as a result of intrauterine hypoxia and other damaging effects. A feature of nootropics is the possibility of their use not only by patients, but also by healthy people in extreme situations to relieve mental fatigue, improve mental performance (Shtrygol S.Yu. et al. Side effects of nootropic drugs // Provizor, 2003, No. 11).

Despite the fact that at present there is a fairly wide range of nootropic drugs, mainly pyrrolidone derivatives (the so-called racetams), the creation and introduction of new drugs continues. This is due to the fact that for the treatment of diseases accompanied by cognitive impairment, it is necessary to use drugs that combine nootropic and pronounced neuroprotective (cerebroprotective) properties. But most neuroprotectors (antioxidants, calcium channel antagonists) lack direct beneficial effects on memory functions, and nootropics usually do not have pronounced neuroprotective properties. In addition, to achieve a nootropic effect, high doses of drugs are required. In addition, many nootropic drugs often interact with drugs, in particular, used in childhood (for example, with paracetamol, vitamin Ό, macrolides).

Therefore, one of the promising areas is the creation of drugs based on peptides. They regulate the integral function of the central nervous system, showing nootropic and pronounced neuroprotective activity in significantly lower doses than classic nootropics. This feature is inherent in endogenous neuropeptides. Known, in particular, the nootropic and neuroprotective properties of vasopressin and ACTH, as well as their fragments. For example, a semax preparation consisting of seven amino acid residues is a fragment of the ACTH4-10 molecule (RF patent No. 2045958). Semax is one of the first domestic peptide drugs developed at the Institute of Molecular Genetics, Russian Academy of Sciences. Semax exhibits adaptogenic, antioxidant, angioprotective, neurotrophic and pronounced nootropic effects. It acts on the functions of the forebrain: improves memory consolidation, increases learning ability. Already in small doses, Semax helps to improve the energy processes of the brain, increases resistance to hypobaric and vascular hypoxia, to stressful effects. For children under 7 years of age, a special dosage form has been developed - the drug minisem. The drug is usually well tolerated, but it is allowed to be taken only from 3 months, in addition, individual intolerance to the drug is possible (Radar Drug Reference, updating the description of the drug by the minisem manufacturer 07.07.2005).

Cerebrolysin is a complex of neuropeptides (15%) with a molecular weight of not more than 10,000 Da, free amino acids (85%) and trace elements obtained from the brain of young pigs. For more than 20 years, it has been used as a neuroprotector and nootropic. In general, cerebrolysin demonstrates good tolerance, however, individual intolerance to the drug is possible, and the use of the drug during pregnancy and during breastfeeding is possible only when the intended benefit to the mother outweighs the potential risk to the fetus / newborn. The need for intravenous or intramuscular administration in some cases also limits the use of cerebrolysin in children (Radar Drug Reference, updating the description of the drug cerebrolysin by the manufacturer on 03/11/2013).

Known drug cortexin, containing, in addition to a complex of neuropeptides obtained from the cerebral cortex of cattle or pigs, the molecular weight of which does not exceed 10,000 Da, also amino acids, vitamins and minerals. Cortexin for children - a nootropic drug intended for patients of pediatric age, is approved for use from birth (Studenikin V.M. et al. Peptide bioregulators and their use: from neonatology to gerontology // Attending physician, 2010, No. 6, p. 72-75). However, possible individual intolerance and the need for parenteral administration of this drug in some cases also impose their limitations.

Unfortunately, the need for the use of nootropic drugs can occur at any age, starting from the first days of life, as well as during pregnancy and lactation of the mother. In this regard, the problem of finding new compounds, which are effective nootropics, the use of which has no contraindications, in particular, related to pregnancy and the neonatal period, which are also the drugs of choice in case of individual intolerance or ineffectiveness of other drugs, remains an urgent task at present.

Open literature contains a lot of information about beta-casomorphins (BCM). In animal experiments, it was found that these peptides reduce pain sensitivity (Dubynin V.A. et al. Effect of beta-casomorphine-7 on rat nociception with systemic administration // Bull. Expert Biol. And Med., 1992, No. 9, pp. 284-286), regulate maternal and child behavior (Dubynin V.A. et al. Changes in the behavioral effects of beta-casomorphine-7 during the postnatal development of white rat pups // Journal of Higher Nervous Worker, 2001, No. 51 (3), pp. 386-389), have anxiolytic (Maklakova A.S. et al. Behavioral effects of beta-casomorphine-7 and its back-Tug analogues // B Yul.Expert Biol. and Med., 1993, No. 8, pp. 155-158) and cardiotropic effect (Lethal C. e! α1..Νοηορίοί6 есес! о о £ £ £еее11--5айайайайайайайайайайайайайайайайай: abegabank! o! be bie1a-abgeposer1og-O-rgo1esh sotr1ekh apb tjyyup o £ tuosagb1a1 gekropkek! o 18orgo1gepoe1 // Rurbbek, 1991, Mag-Arg; 12 (2), p. 265-270). It is known that HMCs are able to bind to mu- and delta-type opioid receptors (Texbetasbeg N. e! A1., Mycrho! Et-bepueb oryu geser! Pdaibk // Vyurobtegk, 1997, Uo1. 43 (2), p. 99-117), and a number of their biological effects are blocked by the opioid receptor antagonist naloxone, therefore, the effects of HSR are primarily associated with the action on the opioid system (§ip Ζ. Е! А1., And Rurbbe Roipb ίη 5> s1y / orbgesha apb Aibkt Saikek Webuyuga1 Sapdek ίη Ka! K // Aibkt, 1999, Uo1. 3 (1), pp. 85-95). Their ability to influence the serotonergic system was also shown (Sokolov O.Yu. et al. Interaction of betacazomorphins-7 with 5-HT2-serotonin receptors // Bull. Exp. Biol. Med., 2005, No. 11, p. 595-597) .

Despite the significant amount of published data, the presence of nootropic properties in human beta-casomorphine was not known. This invention substantially complements the spectrum of neurotropic properties of human beta-casomorphine.

SUMMARY OF THE INVENTION

The technical result of the present invention is to expand the arsenal of means of protection of the central nervous system with nootropic effects, effective and safe both in adults and children, including the first year of life.

The specified technical result is achieved by using a group of beta-casomorphins, namely human beta-casomorphins-7, of the formula Tug-Rgo-RbeUa1-O1i-Rgo-11e as a hepatapeptide.

The specified technical result is also achieved by the use of the heptapeptide Tug-Rgo-RbeUa1-O1i-Rgo-11e for the manufacture of compositions with nootropic activity, including pharmaceutical compositions, biologically active food additives, as well as specialized foods, including functional children's nutrition.

Another object of the invention are compositions having nootropic activity and containing human beta-casomorphin-7 (the heptapeptide Tug-Rgo-RbeUa1-O1i-Rgo-11e, human BCM-7) in a therapeutically effective amount. Such compositions include pharmaceutical compositions, dietary supplements, as well as specialized foods, including for functional baby food.

In some embodiments, the invention relates to the use of the heptapeptide Tug-Rgo-Rbe-Wa1-O1iRgo-11e as a means of having nootropic activity in children aged 0 months to 3 years, including children who are on artificial or natural feeding, as well as in children in a situation of feeding a mixed type. In some embodiments, the invention relates to the use of the heptapeptide Tug-Rgo-Rbe-Wa1-O1i-Rgo-11e in children aged 3 to 7 years or in patients aged 7 years, including adults.

In one embodiment, the invention relates to the use of the heptapeptide Tug-Rgo-Rbe-Wa1-O1i-Progo11e for the prevention and / or treatment of disorders of neuropsychic development in childhood, including developmental delays and borderline neuropsychic disorders of behavior and adaptation, such as hyperactivity, including attention deficit disorder, neurosis-like, autistic and affective disorders, minimal cerebral dysfunction.

In one embodiment, the invention relates to the use of the heptapeptide Tug-Prgo-Rbe-Va1-O1i-Prgo11e as a source of the peptide in order to support the nervous system and manifest its nootropic properties.

In some embodiments, the invention relates to the use of the heptapeptide Tug-Rgo-Rbe-Wa1-O1iRgo-11e for the manufacture of a composition having nootropic activity. In one embodiment, the invention relates to the use of the heutapeptide Tut-Prgo-Pb-Va1-C1i-Prgo-11e for the manufacture of a pharmaceutical composition. In some embodiments, the invention relates to the use of the heptapeptide TugRto-PbE-Va1-C1i-Prgo-11e for the manufacture of biologically active food additives, functional foods or specialized foods for children or adults. In some embodiments, the invention relates to the use of the heptapeptide Tut-Rgo-Rie-Wa1-S1i-Rgo-11e for the manufacture of baby food, including infant formula, cereal for infants, complementary foods, healthy nutritional products for needy individuals .

As a result of the experiments, it was found that the heptapeptide Tut-Rgo-Rie-Va1-S1i-Rgo-11e is able to exert a direct activating effect on learning processes, memory, mental activity, and increase brain resistance to aggressive influences. The experiments showed that the manifestation of the nootropic properties of the heutapeptide Tut-Prgo-Pb-Va1-C1i-Prgo-11e is due to its versatile effect on the central nervous system (CNS) and is associated with its ability to stimulate the central nervous system processes ensuring their survival (neurotrophic effect), the ability to increase the resistance of the central nervous system cells to damaging factors (neuroprotective effect) and the ability to stimulate the processes of their regeneration in case of damage (neuroplastic effect).

Human beta-casomorphine-7 is a substance of natural origin, as it is inherent in human breast milk. Compared with molecules of chemical origin, the presented agent is safer and does not exhibit side effects. In this regard, this peptide is characterized by wide practical applicability: it can be used both in children and in adults, including during pregnancy and lactation, in various compositions.

Brief Description of the Drawings

FIG. 1 is a chemical diagram of the synthesis of the heptapeptide Tut-Prgo-Pb-Va1-C1i-Prgo-11e.

FIG. 2 - test results an open field with a change in illumination: along the vertical axis - the number of peeps in minks. Legend: in white - the group VPK + N ₂ O (n = 16); black - the group of military-industrial complex + high-speed lines (n = 14); * - difference from the control group with p <0.05 according to the Student's t-test.

FIG. 3 - test results elevated cross-shaped labyrinth: along the vertical axis - the number of exits to the illuminated sleeves. Legend: in white - the group VPK + N ₂ O (n = 16); black - the group of military-industrial complex + high-speed lines (n = 14); # - difference from the control group with p <0.05 according to the chi-square criterion.

FIG. 4 - forced swimming test results: along the vertical axis - time of the first episode of active swimming, sec. Legend: in white - the group VPK + N ₂ O (n = 16); black - the group of military-industrial complex + high-speed lines (n = 14); # - difference from the control group with p <0.05 according to the chi-square criterion.

FIG. 5 - test results forced swimming: along the vertical axis - the number of acts of immobilization. Legend: in white - the group VPK + N ₂ O (n = 16); black - the group of military-industrial complex + high-speed lines (n = 14); * - difference from the control group with p <0.05 according to the Student's t-test.

FIG. 6 - hot plate test results: along the vertical axis is the average duration of the latent period of paw licking, sec. Legend: in white - the group VPK + N ₂ O (n = 16); black - the group of military-industrial complex + high-speed lines (n = 14); * - difference from the control group with p <0.05 according to the Student's t-test.

FIG. 7 - the results of the passive avoidance reaction: along the vertical axis - the duration of the latent period on the day of passive avoidance reaction test, sec. Legend: in white - the group VPK + N ₂ O (n = 16); black - the group of military-industrial complex + high-speed lines (n = 14); * - difference from the control group with p <0.05 according to the Student's t-test.

FIG. 8 - results of the passive avoidance reaction: along the vertical axis - the difference (delta) between the latent periods during training and passive avoidance reaction testing, p. Legend: in white - the group VPK + N ₂ O (n = 16); black - the group of military-industrial complex + high-speed lines (n = 14); * - difference from the control group with p <0.05 according to the Student's t-test.

FIG. 9 - results of measuring the volume of the hippocampus during an MRI scan: along the horizontal axis on the left is the volume of the left hemisphere; on the right is the volume of the right hemisphere. The vertical axis is the volume of the hippocampus in mm ³ . Legend: in white - the group VPK + N ₂ O (n = 7); black color - the group of military-industrial complex + high-speed lines (n = 7); * - difference from the control group with p <0.05 according to the Student's t-test.

FIG. 10 - test results Ktdyyp teyekh (reflex of coup from back to paws): along the vertical axis - time of coup from back to paws, sec. Legend: in white - group H ₂ O + H ₂ O (n = 16); in black - the group H ₂ O + HSR (n = 17); * - difference from the control group with p <0.05 according to the Student's ΐ-criterion.

FIG. 11. - test results CaP teieh (children's motor activity): along the vertical axis, reaction time, sec. Legend: in white - group H ₂ O + H ₂ O (n = 16); in black the group Н2О + ВСМ (n = 17); # - difference from the control group with p <0.05 according to the chi-square criterion.

FIG. 12 - results of the passive avoidance reaction: along the vertical axis - the duration of the latent period on the day of passive avoidance reaction test, sec. Legend: in white - group H ₂ O + H ₂ O (n = 16); in black - the group H ₂ O + HSR (n = 17); # - difference from the control group with p <0.05 according to the chi-square criterion.

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FIG. 13 - the results of the passive avoidance reaction: along the vertical axis - the difference (delta) between the latent periods when learning and checking passive avoidance reaction, p. Legend: in white - group H ₂ O + H ₂ O (n = 16); in black - the group H ₂ O + HSR (n = 17); # - difference from the control group with p <0.05 according to the chi-square criterion.

DETAILED DESCRIPTION OF THE INVENTION

Human beta-casomorphine of the general formula Tug-Rgo-Rye-Ya1-C1i-Rgo-11e is obtained by classical methods of peptide synthesis. Various synthesis methods differ in the physical state of the phase in which the synthesis proceeds, namely, whether it is a liquid phase or a solid phase. The invention can be implemented using both a solid-phase synthesis technique and a liquid-phase synthesis technique, as well as their hybrid variant. One of the possible schemes for the chemical synthesis and production of the peptide is shown in Example 1 and in FIG. one.

In one aspect, the invention can be used in children of various age groups. The inclusion of the peptide in the diet of young children (from 0 months to 3 years), for example, as part of adapted infant formula or complementary foods, as well as in the diet of toddlers and preschool children (from 3 to 7 years), for example, in the form of a biologically active food supplement or specialized food. The inclusion of the peptide in the diet of newborns and children under the age of 3 years can be used both in the case of fully artificial feeding, and in the case of natural feeding, as well as in a mixed-type feeding situation. The use of human beta-casomorphine is also promising for the creation of therapeutic food products. In children's practice, human beta-casomorphine-7 can be used to treat cerebral growth, encephalopathy, intellectual deficiency, disorders that form in premature babies as a result of intrauterine hypoxia, and other damaging effects.

In another aspect, the invention relates to a method for the prevention and / or treatment of disorders of neuropsychic development in childhood, including developmental delays and borderline neuropsychic disorders of behavior and adaptation, such as hyperactivity (including attention deficit), neurosis-like, autistic and affective violations.

In another aspect, human beta-casomorphine-7 can be used in patients over the age of 7 years, including in adults.

Beta-casomorphine-7 can be used in case of asthenic and depressive conditions after traumatic brain injuries and neuroinfections, as well as for the correction of side effects of psychopharmacotherapy (in the treatment of antipsychotics, antidepressants, psychostimulants) in patients at any age. Beta-casomorphine-7 of a person can be used not only in the presence of pathological changes, but also by healthy people in extreme situations to relieve mental fatigue, improve mental performance. In one aspect, the invention can be used in patients of any age as a source of peptide in order to support the nervous system and manifest its nootropic properties.

Human beta-casomorphine-7 is promising for the creation of pharmaceutical compositions based on it, including compositions for oral or intranasal administration. The concept of a pharmaceutical composition means various dosage forms containing human betacazomorphine-7, which can find prophylactic and / or therapeutic use in medicine as a means with a nootropic effect. Pharmaceutical compositions intended for oral administration can be either in the form of a solid dosage form (i.e., in the form of capsules, tablets, pills, and the like), or in the form of a liquid dosage form (as a solution). Pharmaceutical compositions intended for intranasal administration may be in the form of a solution or drops for intranasal administration. Dosages and administration of the pharmaceutical composition depend on the route of administration, age, condition and weight of the patient.

Beta-casomorphine-7 is promising for the creation of biologically active additives, functional foods and specialized foods for children and adults, which can positively affect the neuropsychic state of a person in both children and adults.

The concept of a biologically active food supplement (food supplement) means a composition of biologically active substances intended for direct intake with food or the introduction of food products. In most cases, biologically active food additives belong to the class of natural food components and have pronounced physiological and pharmacological effects on the basic regulatory and metabolic processes of the human body.

The concept of functional foods includes foods of systematic consumption, which preserve and improve health and reduce the risk of developing diseases due to the presence of functional ingredients in their composition. Functional foods have a positive effect on the body, preventing the onset of certain diseases, contributing to the growth and development of the body.

The concept of specialized food products touches upon such areas of application as sports nutrition, dietetic nutrition, therapeutic and prophylactic nutrition in case of harmful

- 4 028069 working conditions, nutrition of pregnant and lactating women, nutrition of young children, school and child nutrition, enteral nutrition and parenteral nutrition. Features of any type of specialized nutrition are determined, first of all, by age, body weight, individual characteristics of food tolerance and further by sports, the presence of a specific disease, and the characteristics of harmful production.

The concept of an effective amount implies the use of such an amount of active principle, which, in accordance with its quantitative indicators of activity and toxicity, as well as on the basis of specialist knowledge should be effective in this form in the patient.

In order to manifest its nootropic properties, human beta-casomorphine-7 can be used in an amount of from 0.01 to 100 mg. More preferably, human beta-casomorphine-7 may be used in an amount of from 0.01 to 10 mg. Preferably, 0.01-0.1 mg is used in the intranasal pharmaceutical composition of human beta-casomorphine-7. Preferably, in the composition of the nutrition for young children (from 0 months to 3 years), including for newborns, human betacazomorphine-7 is used in an amount of 0.15-0.3 mg. Preferably in the composition of biologically active additives and functional foods for children aged 3-7 years and for patients over the age of 7 years, including adults, beta-casomorphine-7 is used in an amount of 1.1-1.5 mg . Preferably, the intranasal pharmaceutical composition of this invention is administered 2-3 times a day at intervals of 3-4 hours daily; the duration of the course application can be up to 14 days, preferably 3-5 days. Preferably, the specialized foods, functional foods, and nutritional supplements of this invention are administered daily 1-3 times a day for 3-6 weeks, more preferably 1 month.

To obtain the pharmaceutical compositions of the invention, an effective amount of the heptapeptide Tug-Prgo-Rie-Va1-O1i-Prgo-11e, as an active principle, is mixed with a pharmaceutically acceptable carrier, excipient and / or diluent according to the pharmaceutical compounding methods. Moreover, the pharmaceutical composition as a diluent may preferably contain a pharmacopoeial buffer solution and excipients, including high molecular weight compounds, stabilizers, preservatives and solubilizers. The composition is administered orally or more preferably intranasally.

In yet another aspect, human beta-casomorphine-7 is used to produce biologically active food additives (food additives) and specialized food products for children aged 3-7 years and older patients, including adults. The nutritional supplement may further comprise additional peptides with a molecular weight of less than 10 kDa. It may additionally contain amino acids. Suitable amino acids are, for example, asparagine (Ν), methionine (M), glutamic acid (E), valine (V), proline (P), arginine (K), alanine (A), cysteine (C), phenylalanine ( P), glutamine (O), glycine (O), threonine (T), isoleucine (I), tryptophan (XV). tyrosine (Υ), threonine (T), serine (§), histidine (H), aspartic acid (Ό), lysine (K), leucine (b), etc. Amine additives are preferably used in their optically active form, most preferably the use of b-amino acids.

The nutritional supplement may additionally contain vitamins such as vitamin A, various B vitamins, vitamin C, vitamin Ό, E and / or K.

Additionally, it may contain minerals and / or trace elements, such as calcium, magnesium, iron, copper, sodium, zinc, manganese, iodine, potassium, selenium, chromium, molybdenum, fluorine, chlorine, phosphorus.

Additionally, the components may be caffeine and taurine, fatty acids such as ω-fatty acids, alpha lipoic acid, phospholipids, phosphatidylserines, plant extracts such as ginkgo biloba, huperzine, neurotransmitter precursors like ΌΜΑΕ (dimethylaminoethanol), etc.

The food supplement may further contain flavoring agents, colorants like titanium dioxide, iron oxides, etc., and / or preservatives, etc. Preservatives may be ethyl paraben (p-hydroxybenzoic acid ethyl ester), benzalkonium chloride, benzetonium chloride, benzoic acid, butyl paraben (p-hydroxybenzoic acid butyl ether), methyl paraben (p-hydroxybenzoic acid methyl ester), potassium sorbate, propionic acid, propyl paraben (p-hydroxybenzoic acid propyl ester), sodium benzoate, sodium propi tension, sorbic acid.

Additionally, the composition may contain acceptable additives, fillers and / or neutral fillers, such as microcrystalline cellulose, maltodextrin, magnesium stearate, colloidal silicon dioxide, silicon dioxide, lactose, maltose, sodium carboxymethyl cellulose, modified cellulose, plant cellulose, calcium phosphate, sodium phosphate, vegetable glycerin, sodium starch, polyvinylpyrrolidone, polyvinylpolypyrrolidone, cellulose resin, stearic acid, gelatin, mannitol, sodium ascorbate, glycerin, rice flour, maltodextrin disodium phosphate, etc.

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The food supplement is administered orally in the form of tablets, coated tablets, capsules, pastes, chewable tablets or solutions for drinking. If a coated tablet is used, the preparation may be resistant to gastric juices, therefore an enteric coated dosage form may be used. The nutritional supplement of this invention can be used in an oral dosage form.

In yet another aspect of the invention, human beta-casomorphine-7 can be used to make specialized foods.

In yet another aspect, human beta-casomorphine-7 is used to produce foods for young children, including infant formula, cereal for infants, other complementary foods, or healthy nutritional products for needy individuals.

Nutrition for young children (from 0 months to 3 years) of the present invention can advantageously be used as a balanced diet for young children. Such a food product includes lipids, proteins, and carbohydrates, and is preferably administered in liquid form.

Preferably, the nutrition for infants and / or young children of the present invention includes lipids. Preferably, nutrition for young children (0 months to 3 years) of the present invention includes plant lipids. Preferably, the lipid component is a combination of plant lipids and at least one oil selected from the group consisting of fish oil, animal oil, algae oil and bacterial oil. Preferably, the lipid comprises more than 50 mg / 100 kcal (preferably more than 1 wt.% Of all fatty acids) of α-linolenic acid (AL). Preferably, the nutrition of young children (from 0 months to 3 years) of the present invention includes long chain polyunsaturated fatty acids (DC-PUFA), more preferably eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA) and / or arachidonic acid ( ARC).

Preferably, nutrition for young children (0 months to 3 years) of the present invention includes proteins. The proteins used in the nutritional composition are preferably selected from the group consisting of animal proteins (albumin-dominant products are preferred), vegetable proteins (such as soy protein, wheat protein, rice protein, potato protein, pea protein), hydrolysates (partial and / or complete), free amino acids and mixtures thereof.

Preferably, nutrition for young children (0 months to 3 years) of the present invention includes digestible oligosaccharides. The digestible oligosaccharides used in the nutritional composition are preferably selected from the group consisting of sucrose, lactose, maltose, galactose, glucose, fructose, solids of corn syrup, starch and maltodextrins, and mixtures thereof, more preferably lactose.

Preferably, nutrition for young children (0 months to 3 years) of the present invention includes minerals, trace elements and vitamins, choline, taurine, carnitine, inositol, biotin and / or mixtures thereof. Preferably, the nutrition includes sodium, potassium, chlorides, calcium, phosphorus, magnesium, iron, iodine, copper, zinc, selenium, manganese, as well as vitamins A, Ό, E, K, C, B1, B2, PP, B6, B12, pantothenic and folic acid.

Preferably, nutrition for young children (0 months to 3 years) of the present invention includes nucleotides. Preferably, the composition includes cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate and inosine 5'-monophosphate. Preferably, the nutrition for infants and / or young children of the present invention includes both DC-PUFA and nucleotides.

Preferably, nutrition for young children (from 0 months to 3 years) of the present invention has an osmolality of 50 to 500 mOsmol / kg, more preferably 100 to 500 mOsmol / kg.

From the above description, a person skilled in the art can easily recognize the main features of the present invention and, without deviating from the essence and scope, make various changes and modifications of the invention to adapt it to various applications and conditions. Therefore, other embodiments are also included in the scope of the present invention.

The possibility of an objective manifestation of a technical result when using the invention is confirmed by reliable data given in the examples containing experimental information obtained in the process of conducting research using methods adopted in this field. The invention is illustrated by drawings.

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Example 1. A method of producing a peptide with the formula Tug-Prgo-Pbe-Va1-C1i-Prgo-11e by liquid-phase synthesis (see Fig. 1).

The following example is provided to illustrate the method according to the present invention and should not be construed as in any way limiting the scope of the invention.

The peptide Tug-Rgo-PbE-Va1-C1i-Rgo-11e is obtained by synthesis in solution by the carbodiimide method with β-hydroxybenzotriazole. As carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is used.

Ethyl acetate is used as a solvent. The release of the BOC-protecting group is carried out with trifluoroacetic acid, and the Ζ-protecting groups with catalytic hydrogenation (a more detailed description is presented in Fig. 1).

Purification of the peptide is carried out by preparative HPLC; the peptide was characterized using a mass spectrometer.

Example 2. Assessment of the nootropic activity of the heptapeptide Tug-Rgo-Rbe-Va1-C1i-Rgo-11e against the background of cerebral dysfunction.

To evaluate the properties of the heptapeptide, a model of fetal valproate syndrome in laboratory animals was used. In this model, the state of the central nervous system of rats was assessed using behavioral assessment techniques, as well as MRI studies of the brain.

Prenatal administration of high doses of valproic acid (VPA) is a model of the pathology of the development of the central nervous system and brain dysfunction and can serve as a way to assess the preventive and corrective effects of drugs of various groups, including peptide substances. To develop fetal valproate syndrome in the offspring, females were administered valproic acid at a dose of 600 mg / kg on the 12.5th day of pregnancy. The dry valproic acid salt preparation was dissolved in distilled water (1 mg / ml). The injection was performed intraperitoneally with an insulin syringe. An equivalent volume of solvent was administered to the control group.

The following behavioral features are observed in rat pups whose mothers received an injection of VPA on the 12.5th day of pregnancy: impaired nociception and increased sensitivity to non-painful stimuli, motor hyperactivity and stereotype combined with low research motivation, increased anxiety levels, and a depleted repertoire of social behavior and its increased latency. In addition, in newborns, there is a lag in development, weight loss, delayed motor maturation and poor learning (5> s1teeg T. e1 a1., 2005).

The introduction of a large dose of valproic acid in the antenatal period, in fact, is a non-specific negative effect on the development of the fetal nervous system. Unfortunately, a similar situation is also possible in real clinical practice - pharmaceutical preparations, injuries, even stressful environmental conditions can have a similar non-specific damaging effect. Moreover, some of these reasons are also relevant for adults (for example, traumatic brain injuries, the effects of toxins / chemicals). Such effects can lead, in the General case, to neuropsychiatric diseases.

The impact of human beta-casomorphine-7 on the manifestations of fetal valproate syndrome was assessed for the first time. 3 broods of pups were used after the prenatal injection of MIC, each brood was divided into 2 halves:

human valproic acid + beta-casomorphine-7 group (MIC + BCM): calves underwent intraperitoneal administration of beta-casomorphine-7 at a dose of 1 mg / kg (peptide dilution - 1 mg / ml) from the 1st to the 14th day of life , 14 rats of both sexes;

VPK + H ₂ O group: received an equivalent volume of solvent, 16 rats of both sexes.

In the first part of the work, standard behavioral techniques were used, such as an open field (OP), an elevated cruciform labyrinth (PCL), forced swimming (Porsolt test), a hot plate, and the development of a conditional passive avoidance reaction (passive avoidance reaction).

Test An open field with a change in illumination.

An open field (OP) is a round plastic arena with a diameter of 100 cm with high walls (42 cm). The arena floor is drawn by two concentric circles located at an equal distance (20 cm) from each other and from the edge of the arena, as well as by straight lines dividing the field by 19 areas approximately equal in area. In addition, minks (13 pieces) are located on the lines and in the center of the arena. The arena is located on a stand at a height of 55 cm from the floor.

At the beginning of the experiment, the rat was placed in the center of the arena. Each animal was tested for 5 min, of which the first 3 min in bright light, then 1 min in red light and the last 1 min again in bright light. Red (15 W) and bright (60 W) lamps were suspended at a height of 80 cm from the center of the field; when you turn on the bright lamp, the red did not turn off.

During the experiment, the following indicators were evaluated in minutes (during the first minute - every 30 s):

horizontal activity (according to the number of passed field segments); vertical activity (number of racks); racks in the center were separately taken into account; the number of radial movements with the intersection of the outer circle (waste from the arena wall);

- 7 028069 the number of radial movements with the intersection of the inner circle (exits to the center of the arena); the number of radial movements with the intersection of both circles (transitions); grooming (washing);

the number of minks examined; minks in the center were separately taken into account; the number of holdings (rat fading in place for more than 5 s).

The latent period of exit from the center (when the animal reached the arena wall), the total time of hiding and the number of bowel movements during the experiment were also noted.

Test elevated cruciform labyrinth (PCL).

PCL is a horizontally lying equilateral cross made of plastic (beam length 50 cm, width 15 cm), two beams of which, located opposite each other, have side and end walls 30 cm high, and the other two are open. Open rays are labeled: one segment separates the light beam from the central square (inner border), the second divides the length of the beam in half (outer border). The whole structure is located on a stand at a height of 55 cm from the floor. Open rays are illuminated by bright lamps (60 W), located at a distance of 25-30 cm from the end of the beam. The animal at the beginning of the experiment was placed in the center of the PCL with his nose in the direction of light. Each rat spent 3 min in PCL. The following parameters were noted (for each minute and in total for the entire testing time):

the number of racks in the light and in the dark; the number of washings; number of harboring;

the number of peeps from the dark compartment;

the number of intersections of the inner border of the illuminated beam (exits to the 1st compartment); the number of intersections of the outer border of the illuminated beam (exits to the 2nd compartment); the number of hangings from the edge of the illuminated beam in the first and second compartments; the number of transitions through the center of light-light;

the number of transitions through the center of darkness-darkness.

The total time spent in the world, the first latent period of entry into the dark compartment, the total time of hiding, the number of bowel movements during the experiment were also noted.

The combination of testing in OP and PCL allows you to assess the balance of research activity and the level of anxiety of animals.

Test Forced swimming.

A transparent plastic cylinder 39 cm high and 20 cm in diameter was used, into which water was poured at a temperature of 23 ° C to a level of about 28 cm. The rat was placed in the water with its nose up. Each animal was in water for 5 minutes, the total time of active swimming, passive swimming and immobilization, as well as the latent period and the number of these reactions were recorded.

This test allows you to assess the severity of the behavioral manifestations of depression.

Test hot plate.

In the Hot Plate test (Idobacie, Italy), the animal was placed on a metal surface heated to 53 ° C. The latent period of the behavioral reaction to the pain stimulus was recorded - the time from the moment of landing to the first licking of the hind paw. 4 measurements of pain sensitivity were carried out with an interval of 15 min; in the processing of data, LP _ts , LP _max and ^{LP of} media were determined.

This test allows you to evaluate the pain sensitivity of animals.

Development of a conditioned reaction of passive avoidance of a pain stimulus (passive avoidance reaction). The installation is a wooden camera 50x25x40 cm, divided into two unequal parts by a partition with a door that can be raised (the rat can go to another compartment) or lowered to the floor. One half of the chamber (length 22 cm) does not have a top cover, the second (large, length 28 cm) is covered with a dark red transparent cover. The floor in the chamber is a metal grill and is connected to an ESL-1 brand electric stimulator. A bright lamp (60 W) is installed above the half of the chamber open from above. The animal was planted in the light compartment of the camera with its nose in the corner farthest from the dark half. The partition was raised. The behavior of the rat was recorded for 3 min: the latent period of entry into the dark compartment, time in the light, the number of peeps into the dark, entries into the dark and exits to the light, the number of grooming acts, stances and bowel movements. Since rats, as a rule, prefer not to be in the light, by the end of the first 3 minutes almost all animals were in a dark compartment. After making sure of this, the experimenter lowered the door and turned on the current (50 Hz, the duration of a single pulse of 10 ms) with a voltage of 100 V for 5-7 s, after which the animal was removed from the chamber. If the rat sat calmly for 5 s, the stimulus voltage was increased until vocalization appeared.

48 hours after electrical stimulation, the rat was re-placed in the chamber for 3 min and the behavior was recorded according to the scheme described above. The change in the recorded parameters relative to the first landing was judged on the learning ability of rats. In this case, electrical stimulation was not performed.

Passive avoidance reaction is a basic model for assessing the effect of substances on the formation and reproduction of a memorable trace in normal and cerebral dysfunction.

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Results.

The open field test results with interchangeable illumination are shown in FIG. 2. A significant difference between rats receiving human BCM-7 and rats receiving water was recorded in the number of mink peeks for 4 min, in the dark (p <0.05 according to Student ΐ criterion). This difference indicates a shift in the conflict of anxiety and orientation-research activity towards the latter.

The results of the elevated cruciform labyrinth test are shown in Fig. 3. Among the rats receiving human BCM-7 during the first two weeks, 6 rats made more than one exit from the dark compartment, while in the control group there were only 1 rats (p <0.05 by criterion hikvadrat). This difference also indicates a decrease in anxiety.

Thus, we can conclude that the introduction of human SCM-7 had a positive effect on the balance of tentatively research response and anxiety. Pups under the influence of the drug become less anxious, their research motivation increases.

During the forced swimming test, it was revealed that the time of the 1st episode of active swimming in 4 rats from the control group (receiving water) was less than 40 s, whereas in the group with the introduction of human BCM-7 there were no such rats (p <0.05 according to the chi-square criterion). This parameter is very important, it indicates a decrease in depression and activation of the dopaminergic system of the brain under the influence of the peptide. Also, in rats treated with human HSR-7, the number of immobilization acts was reduced (see Figs. 4 and 5). The data obtained indicate a weakening of the behavioral manifestations of depression under the influence of beta-casomorphine.

The hot plate test was used to measure the pain sensitivity of animals. It was found that under the influence of human VSM-7 in animals there is a normalization of pain sensitivity. It was previously shown that in rat pups with fetal valproate syndrome, this parameter is impaired, pain sensitivity is reduced, latent periods of the reaction are slowed down. In this test, the average latent period of paw licking in the VPK + HSR group was 8.3 ± 0.74 s, in the VPK + H ₂ O group - 12.2 ± 1.68 s (p <0.05 according to the Student ΐ criterion ) The results are shown in FIG. 6.

As you know, neurological disorders lead to a complex deterioration in the performance of the central nervous system. According to the test results, an open field, an elevated cruciform labyrinth, forced swimming and a hot plate can be concluded that the introduction of a heptapeptide normalizes the state of the central nervous system, has a restoring effect on those parameters that are impaired in the fetal valproate syndrome, counteracts the damaging factor and increases the resistance of the brain to aggressive exposure. These examples illustrate the neuroprotective component of the nootropic action of the peptide.

In FIG. 7 and 8 show the results of the development of a conditioned passive avoidance reaction in rats treated with water or human SCM-7. The latent periods on the day of training did not differ, whereas on the day of the test, the latent period in the experimental group with human SCM-7 was 133.6 ± 2.34, and in the control 72.8 ± 2.34; the difference (delta) between the latent period during training and testing is also significantly higher: in the experimental group - 111.1 ± 18.73, in the control group - 50.5 ± 18.72. It follows that the introduction of human SCM-7 improves the production of passive avoidance reaction and training with negative reinforcement. Since the production of passive avoidance reaction is a classic method for testing the nootropic function of a substance, it can be concluded that the neuropeptide beta-casomorphin-7 is a true nootropic and positively affects learning processes, memory and mental activity under conditions of aggressive influence of damaging factors on the central nervous system.

In the second part of the work, an MRI scan was performed.

Methodology

A study of the structures of the brain was carried out using magnetic resonance imaging (MRI), which was carried out in a small animal tomograph Vgikeg Vyu8res 70/30 (Vgikeg, Germany) with software RataMzyup 5.0. The study was conducted on rats at the age of 2 months. after the end of behavioral experiments. Two groups of animals were used: the group VPK + H ₂ O (n = 7) - negative control, rats with fetal valproate syndrome; VPK + BCM group (n = 7) - an experimental group, rats, which, against the background of fetal valproate syndrome, were administered the human BCM-7 peptide at a dose of 1 mg / kg intraperitoneally at 1-14 days of life.

Characteristics of the tomograph: constant magnetic field B = 7.05 T; hole diameter 310 mm; free access to generate 154 mm images; frequency of an alternating magnetic field of 50 GHz; maximum resolution 200 microns.

For MRI studies, rats were anesthetized with an intraperitoneal injection of 300 mg / kg chloral hydrate (Sigma) in a solution of No. C1 0.9% based on 100 mg / ml. Anesthetized animals were placed in a crib, maintaining a constant body temperature during the experiment, the head was fixed using dental and ear holders. Then the crib with the rat was placed in the coil of the device and the receiving coil was fixed on the head of the rat. An MRI scan was performed in the Τ2-ΚΆΚΕ mode to obtain T2-weighted images (T2VI) with high resolution.

- 9 028069

In this mode, the KAKE pulse sequence is used, which allows you to quickly obtain images with good resolution and contrast. The time of the study in this mode was 4 minutes. The image was built in a matrix of 156x156 pixels with a field capture of 26x26 mm. The image resolution was 167 μm / pixel.

As a result of the study, a series of tomographic sections of the rat brain were obtained in the frontal projection, starting from the cerebellum and ending with the olfactory bulbs. The distance between the planes of the slices was 1 mm, the thickness of the slices was 0.5 mm.

Processing of the obtained T2VI was carried out in program 1shade1. The volume of the left and right sides of the hippocampus was calculated by measuring the area of the zones on T2VI, comparing them with the corresponding images from the stereotactic atlas of the rat brain (Rakhtok, \ Ya150p. 1998). For the study, the hippocampal zones CA1 and CA3 were selected.

Results.

The MRI technique allows you to evaluate the formation of rat brain structures and identify the neurotrophic and neuroplastic components of the nootropic effects of human BCM-7 against the background of the development of fetal valproate syndrome.

The early data obtained indicate that prenatal administration of VPA in rats causes a reduction in the hippocampus and an increase in the level of the T2 signal in the hippocampus, which may be associated with neuronal death and flooding of these brain regions.

Hippocampal volume measurement.

We obtained data confirming the corrective effect of human SCM-7 on the pathology of the central nervous system. In rats of the VPK + SCM group, an increase in the volume of the left hemisphere of the hippocampus by 10.5% and in the right hemisphere of the hippocampus by 9% relative to the negative control, under the influence of the peptide, the volume of the hippocampus returns to the normal level (level of healthy animals). The results are presented in FIG. nine.

Studies were also conducted in the T2-tar mode (T2-map) for quantitative MRI studies in the following brain areas: hippocampal areas CA1 and CA3, thalamus, striatum, lateral part of the pale ball, tonsil.

Thus, the data obtained in the MRI study confirm the corrective and preventive effect of the human BCM-7 heptapeptide on the central nervous system dysfunction. Restoring the volume of the hippocampus is a very positive result, as this area of the head is connected with working memory, an objective and subjective idea of the world through the processing of external signals, verbal memory, and social interaction. Probably one of the components of the nootropic action of the heptapeptide is its protective effect directly on the hippocampal region.

Conducting experiments on a similar model of brain dysfunction using human betacazomorphine was conducted for the first time. It has been shown that human beta-casomorphine has a significant positive effect on the neurological state of animals. According to the results of the work, the effect can be unambiguously described as nootropic - human beta-casomorphine normalizes the development and maturation of the central nervous system, has a restoring effect on cognitive, higher mental functions, has a comprehensive neurotrophic, neuroprotective, neuroplastic effect, protects against damaging external influences, improves learning, memory and Attention. It can be assumed that brain regions such as the midbrain, cerebellum, hindbrain, thalamus, and limbic system are involved. It can also be assumed that the heptapeptide increases the level of expression of neurotrophic factors. Modulation of microelement homeostasis can be one of the essential components of the nootropic action of the peptide. It can be concluded that the peptide combines a neurotransmitter and metabolic effect. An MRI study showed that against the background of neurotoxic effects under the influence of human beta-casomorphine, the volume of the hippocampus is normalized. A similar effect can be extrapolated to healthy animals and even to humans - a nootropic effect can be expected with a high degree of probability. Human beta-casomorphine-7 has a multifactorial complex nootropic effect in conditions of brain dysfunction and increases the resistance of the central nervous system to damaging external influences. It should be noted that higher brain functions (memory, thinking, attention, perception) depend on many factors: energy supply, biosynthesis of information molecules, the state of the vascular system of the central nervous system, lipid and vitamins metabolism. Therefore, substances that can optimize these functions and, as a result, increase the stability of the central nervous system to damaging effects, should, first of all, have a beneficial effect on the energy, plastic and mediator metabolism in the central nervous system.

Example 3. Assessment of the nootropic activity of the heptapeptide Tug-Prgo-Pb-Va1-O1i-Prgo-11e in healthy laboratory animals.

This example is devoted to assessing the effect of a neuropeptide on normal laboratory rats that were not exposed to any damaging effect in order to reveal its nootropic effect.

- 10 028069

2 broods of pups were used, each brood was divided into 2 halves:

group H ₂ O + SCM (experimental): the calves were subjected to intraperitoneal administration of human beta-casomorphine-7 at a dose of 1 mg / kg (peptide dilution -1 mg / ml) from the 1st to the 14th day of life, 17 rats of both sexes ;

group H ₂ O + H ₂ O (control): received an equivalent volume of solvent, 16 rats of both sexes.

Tests were used to assess the development of the nervous system, as well as the development of passive avoidance reaction.

The following tests were used as tests for the psychomotor development and maturation of the central nervous system: Ktdyyid Geyekh test (reflex of a coup from the back to the legs);

the rat was turned over on its back, the time was noted when it would stand on all 4 paws; Oai Geyeh test (children's motor activity);

the rat was placed in the center of the circle (ά = 15 cm), the time was noted when it would cross the circle line with all 4 paws.

The passive avoidance reaction was developed using exactly the same methodology as in Example 2.

In the Ktdyyid geyekh test, the average coup time in rats of the experimental group was 1.7 s, in the control group - 2.5 s (p <0.05 according to the Student's t-test). The result is shown in FIG. 10.

In the Oai Geyekh test, there were 5 rats in the control group whose reaction time was more than 80 s; in the experimental group there were no such rats (p <0.05 according to the chi-square test). The result is shown in FIG.

eleven.

The results of these tests indicate that human beta-casomorphine has a nootropic effect on the developing brain. The obtained data characterize the presence of the neurotrophic component of the nootropic action of the peptide, because a similar effect is most likely realized through an increase in the number of neurotrophic factors, for example, ΒΌΝΡ.

In FIG. 12 and 13 show the results of the development of a conditioned passive avoidance reaction in rats treated with water or BCM-7. The latent periods on the day of training did not differ, whereas on the day of testing, the latent period in 8 rats in the experimental group with SCM-7 was more than 80 s, and in the control group only in 1 rat (p <0.05 according to the chi-square criterion ); the difference (delta) between the latent period during training and verification is also significantly higher: in the experimental group - 56.5 ± 15.77, in the control group - 13.2 ± 11.97. It follows that the introduction of human SCM-7 improves the production of passive avoidance reaction and training with negative reinforcement.

The experimental results showed that neurotransmitter, neurotrophic, neuroprotective mechanisms of the nootropic action of casomorphine are involved. The experiments presented in example 3 also confirm the presence of nootropic properties in human beta-casomorphine-7.

Example 4. Monitoring the health of laboratory animals in order to assess the possible toxic effects of human SCM-7 during experimental procedures.

The characterization of the possible damaging effect of the substance and the assessment of side effects were carried out at a qualitative level in parallel with the series of experiments described in Example 3. Despite the fact that the study was not preclinical, in assessing the negative effects, we were guided by the recommendations from the book Guide to experimental (preclinical) study new pharmacological substances edited by R.U. Khabrieva.

The similar nature of the study allows us to characterize the integral and physiological indicators of toxicity. As a result of the experiments, no intolerance of the substance in animals was revealed. In the group of animals receiving injections of human SCM-7, the absence of pathological changes in behavioral activity was recorded; normal intensity and nature of motor activity; lack of seizures and normal skeletal muscle tone; normal coordination of movements;

normal response to tactile and pain stimuli;

normal condition of the hair and skin, healthy color of the mucous membranes; the absence of any allergic reactions to the substance; lack of deformations of the tail;

normal urination and defecation; lack of changes in feed and water consumption; normal body weight.

Claims (12)

CLAIM 1. The use of the heptapeptide Tug-Rgo-Rye-Wa1-O1i-Rgo-11e as a means of having nootropic activity. 2. The use according to claim 1 in children aged 0 months to 3 years. 3. The use according to claim 2 in children who are breast-fed. 4. The use according to claim 2 in children who are breastfed. 5. The use according to claim 2 in children who are breastfed mixed type. 6. The use according to claim 1 in children aged 3 to 7 years. 7. The use according to any one of claims 1 to 6 for the prevention and / or treatment of hyperactivity, including attention deficit, neurosis-like, autistic and affective disorders. 8. The use according to claim 1 in patients aged 7 years. 9. The use of the heptapeptide Tug-Rgo-Rie-Ya1-O1i-Rgo-11e for the manufacture of a composition having nootropic activity. 10. The use according to claim 9, in which the composition is a pharmaceutical composition. 11. The use according to claim 9, in which the composition is a biologically active food supplement, a functional food product or a specialized food product for children or adults. 12. The use according to claim 11, in which the specialized food product for children is a nutritious mixture for infants or porridge for infants.