RU2726854C1 - Polypeptide composition having property of normalizing cerebral functions - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: group of inventions relates to biology and medicine and concerns a novel composition of polypeptides recovered from bovine cerebrum cortex extract by gel filtration, a pharmaceutical composition based on said polypeptides and use thereof. Composition of polypeptides has nootropic, neuroprotective, antioxidant, tissue-specific action and is characterized by the following parameters: molecular weight of polypeptides - about 2600–3000 Da and 7500–7900 Da; amount of bound amino acids: arginine - from 29 to 108 mcg/mg, glycine - from 29 to 71 mcg/mg, asparagine - from 23 to 88 mcg/mg, and glutamine - from 23 to 104 mcg/mg, alanine - from 22 to 56 mcg/mg, proline - 19 to 73 mcg/mg, lysine - 16 to 124 mcg/mg, leucine - 21 to 82 mcg/mg, tyrosine - 16 to 44 mcg/mg, phenylalanine - 19 to 47 mcg/mg, valine - from 10 to 54 mcg/mg and isoleucine - from 13 to 46 mcg/mg of the dry residue of the composition. Pharmaceutical composition for normalizing cerebral function comprises said polypeptide composition and a pharmaceutically acceptable carrier. Also disclosed is use of said compositions for relieving convulsive syndrome, recovering disturbed cerebral reflex activity, memory, coordination of movements, with stroke, cerebral effusions or neuroprotector.EFFECT: composition of polypeptides has the properties of normalizing cerebral function, and has less side effects compared to the prototype when used.11 cl, 9 ex, 1 tbl, 8 dwg

Description

The technical field to which the invention relates

The invention relates to the field of biology and medicine, in particular, to the field of chemical and pharmaceutical industry. A new composition of polypeptides has been isolated, which has the property of normalizing brain functions, nootropic, neuroptrotective, antioxidant and tissue-specific action.

Prior art

To date, there is a very wide range of studies devoted to the problems of normalization of brain functions. Also, these drugs are usually used as nootropic, neuroprotective drugs and drugs with antioxidant and tissue-specific effects. Among such drugs are known: Cortexin (registration certificate
R N003862 / 02), Cerebrolysin (registration certificate
P N013827 / 01), Semax (registration certificate LS-002553) and others.

Of all the presented peptide drugs, Cortexin is the most studied. The drug CORTEKSIN ^® is a polypeptide drug isolated from the cerebral cortex of cattle and pigs and has a tissue-specific effect on the cerebral cortex, has cerebroprotective, nootropic and anticonvulsant effects, reduces the toxic effects of neurotropic substances, improves learning and memory processes, and stimulates reparative processes in the brain, accelerates the recovery of brain functions after stressful influences ( Lebedev AA, et al., 2006, Psychopharmacology and biological narcology, vol. 6, issue 3, 1275-1283 ) .

The mechanism of action of CORTEKSIN ^® is associated with its metabolic activity: the drug regulates the ratio of inhibitory and excitatory amino acids, the level of serotonin and dopamine, has a GABA-ergic effect, has antioxidant activity and the ability to restore the bioelectric activity of the brain.

Cortexin is a pharmacological agent containing, as an active principle, an isolated target product in the form of a complex of biologically active polypeptides with a molecular weight of no more than 15,000 Da and an isoelectric point of 3.5-9.5, and a maximum absorption in the UV spectrum, at a wavelength 275 ± 6 nm. it is proposed to use it in medical practice to normalize the functions of the brain (RU 2275924). In this case, the drug itself is a lyophilized powder of white or white with a yellowish tinge. Due to the fact that Cortexin is obtained by isolation from the cerebral cortex of cattle, it has a complex composition, which is almost impossible to characterize through the amino acid sequence of their content. Thus, the amino acid composition of Cortexin contains a wide range of amino acids, incl. proteinogenic: aspartic acid and glycine, as well as threonine, serine, glutamic acid, proline, alanine, valine, isoleucine, tyrosine, phenylalanine, histidine, lysine, arginine and other amino acids.

However, it should be noted that Cortexin contains a set of polypeptide fractions of different molecular weight and unknown composition, while it may contain polypeptide fractions that can cause side effects: drug intolerance, allergies, nausea, dizziness, etc. Additionally, in the presence of so-called inert or ballast fractions in the preparation, its administered volume may increase, which is not always suitable for use in pediatric practice. A drug with such an unknown composition is rather difficult to obtain and identify, as well as to control its quality, since composition is not precisely defined.

Thus, there remains a need to develop a more effective drug for normalizing brain function with nootropic, neuroptrotective, antioxidant and tissue-specific effects, while having fewer side effects.

The main reason limiting the effectiveness and safety of all existing neuropeptide drugs derived from the mammalian brain is their xenogenic origin. When isolating neuropeptides from the brain tissues of animals to reduce their immunogenicity (antigenic properties), they resort to acid or enzymatic hydrolysis of proteins and high molecular weight polypeptides, thereby reducing the amount of neurotrophic growth factors of protein and polypeptide nature [Khabrieva. U., 2005]. The peptide preparation obtained in this way contains denatured products of acid hydrolysis of proteins and polypeptides with a disturbed quaternary and tertiary structure, which reduces its specificity and biological activity, increases its immunogenicity, causing allergic reactions when applied. An additional cause of side reactions is, as a rule, the complex composition of such organic preparations, which includes a variety of proteins in terms of amino acid composition and molecular weight. Among these proteins, there are often proteins that cause allergic and other side reactions, but it is rather difficult and time-consuming to separate or purify the drug itself from them due to the similarity in properties with other proteins in the composition that carry a useful function.

To solve this problem, the authors have obtained a protein-peptide composition with a pronounced nootropic, neuroptrotective, antioxidant and tissue-specific effect. This composition is characterized by the fact that it contains proteins and polypeptides with a molecular weight of about 2600-3000 Da and 7500-7900 Da, the amount of bound amino acids is: arginine - from 29 to 108 μg / mg dry residue of the composition, glycine - from 29 to 71 μg / mg, asparagine - from 23 to 88 μg / mg, and glutamine - from 23 to 104 μg / mg dry residue of the composition.

In an additional embodiment, the amount of additional linked amino acids is:

alanine - from 22 to 56 μg / mg, proline - from 19 to 73 μg / mg, lysine - from 16 to 124 μg / mg, leucine - from 21 to 82 μg / mg dry residue of the composition.

In another embodiment, the amount of associated additional amino acids is: tyrosine - from 16 to 44 μg / mg dry residue of the composition, phenylalanine - from 19 to 47 μg / mg dry residue of the composition, valine - from 10 to 54 μg / mg dry residue of the composition, isoleucine - from 13 to 46 μg / mg dry residue.

In another embodiment, the polypeptide composition of the invention does not contain cysteine as linked amino acids.

In addition, there is provided a pharmaceutical composition comprising a polypeptide composition according to the invention and a pharmaceutically acceptable carrier.

Additionally, there is proposed the use of a composition of polypeptides according to the invention or a pharmaceutical composition comprising a composition of polypeptides according to the invention for relief of convulsive syndrome, restoration of impaired reflex activity of the brain, memory, coordination of movements, in case of stroke, effusions in the brain, or as a neuroprotector. The use in this case can be carried out by intravenous injection, intramuscular administration or rectally. Also, a polypeptide composition according to the invention or a pharmaceutical composition comprising a polypeptide composition according to the invention can be used alone or in combination with another neuroprotective agent. Another neuroprotective agent may be cortexin, cerebrolysin, actovegin, piracetam, phenotropil, encephabol, gliatilin, pantocalcin, glycine, phenibut, citicoline, picamilon, semax, mexidol, cytoflavin, cavinton, oxybral, sermion, trentalizin.

Brief description of the figures

In FIG. 1 shows the chromatogram of the extract obtained by gel filtration chromatography. The arrows show the fractions that were isolated.

In FIG. 2 shows a chromatogram of a polypeptide composition obtained using reverse phase chromatography. The arrow shows the fraction that is highlighted.

In FIG. 3 shows the mass spectrum of the composition of the polypeptides according to the invention.

In FIG. 4 shows the mass spectrum of the polypeptide composition corresponding to a retention time of 50.0-51.7 minutes.

In FIG. 5 shows the mass spectrum of the polypeptide composition corresponding to a retention time of 53.4-54.7 minutes.

In FIG. 6 shows the mass spectrum of the polypeptide composition corresponding to a retention time of 56.3-59.0 minutes.

In FIG. 7 shows the results of the study of biological activity.

In FIG. 8 shows the results of the study of biological activity (histogram).

Detailed description of the invention

The present invention consists in the fact that a composition of polypeptides has been obtained, which has the property of normalizing brain functions, nootropic, neuroptrotective, antioxidant and tissue-specific action, characterized by the following indicators:

- the molecular weight of the polypeptides is approximately 2600-3000 Da and 7500-7900 Da;

- the number of linked amino acids is:

arginine - from 29 to 108 μg / mg dry residue of the composition, glycine - from 29 to 71 μg / mg, asparagine - from 23 to 88 μg / mg, and glutamine - from 23 to 104 μg / mg dry residue of the composition.

In an additional embodiment, the amount of additional linked amino acids is:

alanine - from 22 to 56 μg / mg, proline - from 19 to 73 μg / mg, lysine - from 16 to 124 μg / mg, leucine - from 21 to 82 μg / mg dry residue of the composition.

In another embodiment, the amount of associated additional amino acids is: tyrosine - from 16 to 44 μg / mg dry residue of the composition, phenylalanine - from 19 to 47 μg / mg dry residue of the composition, valine - from 10 to 54 μg / mg dry residue of the composition, isoleucine - from 13 to 46 μg / mg dry residue.

In another embodiment, the polypeptide composition of the invention does not contain cysteine as linked amino acids.

This composition is obtained from mammalian brain extract. The extract is obtained from the brain of cattle according to a technique known from the prior art. Further, from the obtained extract, the polypeptide composition according to the invention is isolated.

The resulting composition of polypeptides has nootropic, neuroptrotective, antioxidant and tissue-specific effects. Additionally, this composition is characterized by the following indicators:

- the molecular weight of the polypeptides is approximately 2600-3000 Da and 7500-7900 Da;

- the number of linked amino acids is:

arginine - from 29 to 108 μg / mg dry residue of the composition, glycine - from 29 to 71 μg / mg, asparagine - from 23 to 88 μg / mg, and glutamine - from 23 to 104 μg / mg dry residue of the composition.

In an additional embodiment, the amount of additional linked amino acids is:

alanine - from 22 to 56 μg / mg, proline - from 19 to 73 μg / mg, lysine - from 16 to 124 μg / mg, leucine - from 21 to 82 μg / mg dry residue of the composition.

In another embodiment, the amount of associated additional amino acids is: tyrosine - from 16 to 44 μg / mg dry residue of the composition, phenylalanine - from 19 to 47 μg / mg dry residue of the composition, valine - from 10 to 54 μg / mg dry residue of the composition, isoleucine - from 13 to 46 μg / mg dry residue. In yet another embodiment, the polypeptide composition does not contain the amino acid cysteine.

It has been shown that the composition of the polypeptides according to the invention has a neuroprotective effect. The neuroprotective effect suggests that in acute and chronic damage to neurons, there is a decrease in cytotoxic cerebral edema, normalization of reparative processes in the brain and remyelination processes of the peripheral nervous system, restoration of stable brain activity, reduction of neuronal damage in various endogenous neurotoxic factors and toxic effects of neurotropic substances and apoptosis neurons. The nootropic effect of the composition of the polypeptides according to the invention involves the preservation and enhancement of brain functions, improvement of learning and memory processes, concentration of attention, general tone of the nervous system and resistance to various stressful influences. The antioxidant effect involves the inhibition of lipid peroxidation in neurons and glial cells, maintaining the stable functioning of all parts of the nervous system, and activating the protection of neurons under conditions of oxidative stress. Tissue-specific action means the activation of the metabolism of neurons in the central and peripheral nervous system and the normalization and functions of the cerebral cortex.

In general, the composition of polypeptides by properties is a neuropeptide drug that affects the bioelectrical activity of the brain, regulates the ratio of inhibitory and excitatory amino acids, provides a GABAergic effect, reduces the toxicity of neurotropic substances and controls gene expression and protein synthesis in cells. All these processes are included in the cascade of pathogenetic correction of the postanoxic state and other types of damage, accompanied by impaired autoregulation of cerebral blood flow and neuronal damage.

Water-soluble polypeptides in the context of the present invention means polypeptides composed of amino acids that are soluble in water. Methods for determining the quantitative content of water-soluble polypeptides can be different, for example, using spectrometry, for example, by the method of spectrophotometric determination according to the Bradford method.

Molecular weight in the context of this invention means the molecular weight of a polypeptide, expressed in units of dalton (Da). The polypeptides included in the composition of the polypeptides of the invention have a molecular weight of from about 1800 Da to about 3510 Da. Methods for determining molecular weight are known from the prior art: chromatographic, physical, using spectrometry, for example, mass spectrometry.

Associated amino acids in the context of this invention means amino acids that are in an associated state (in connection with some nature) with proteins and other structures or substances in an organ or organism, or in the form of proteins or peptides themselves, forming them. Previously, it was found that the role of linked amino acids lies not only in their neurotransmitter functions, but also in the modification of proteins, as well as the regulation of metabolic processes that ensure brain adaptation.

Free amino acids in the context of this invention refers to both protein and non-protein amino acids. Protein (proteinogenic) amino acids can be unbound into proteins or peptides.

Below are the formulas of the amino acids that make up the composition:

Name Formula Arginine Arg (NH-C (NH ₂ ) NH (CH ₂ ) ₃ CH (NH ₂ ) -COOH) Glycine Gly NH ₂ -CH ₂ -COOH Asparagine Asp HOOC-CH (NH ₂ ) -CH ₂ -CONH ₂ Glutamine Glu O = C (NH ₂ ) -CH ₂ -CH ₂ -CH (NH ₂ ) -COOH Alanine Ala NH ₂ -CH (CH ₃ ) -COOH Proline Pro

Lysine Lys HOOC-CH (NH ₂ ) (CH ₂ ) ₄ NH ₂ Leucine Leu HOOC-CH (NH ₂ ) CH ₂ CH (CH ₃ ) ₂ Valine

Tyrosine

Phenylalanine

Cysteine

The term "about" is used to indicate that the value includes the inherent error of the device, the technique used to determine the value, and / or fluctuations that are inherent in the objects of interest. For example, but without limitation, if the term "about" is used, then the specified value may be more or less by 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%.

The composition of the polypeptides of the present invention may be in the form of a lyophilisate. Methods for preparing a lyophilisate are known in the art. Typically, the lyophilisate is prepared by freezing a solution containing the polypeptide composition and optionally auxiliary substances such as stabilizers, fillers, preservatives, and subjecting this solution to freeze-drying under reduced pressure.

Additionally, the composition of the polypeptides according to the invention can be presented as a pharmaceutical composition containing the composition of the polypeptides in an effective amount and a pharmaceutically acceptable carrier.

Thus, a pharmaceutically effective amount can be from 1 mg to 50 mg per gram of the pharmaceutical composition. In this case, the pharmaceutically acceptable carrier in the specified pharmaceutical composition can make up the rest, up to 1 g. The pharmaceutically acceptable carrier can be a carrier, solvent or diluent, auxiliary substances traditionally used for the preparation of finished dosage forms. So, in the case of injectable forms, for example, in the case of finished dosage forms in the form of an injection administered intravenously or intramuscularly, the carrier or solvent can be water for injection or any solution, incl. and water-based, intended for administration in this way, and the auxiliary substances can be stabilizers, substances that provide isotonicity, substances for adjusting the pH. viscosity, preservatives. In the case of rectal forms, the carrier can be a suppository base, which can be a hydrophilic, hydrophobic, emulsion or other type of base suitable for the preparation of these formulations. The rectal form may also contain excipients traditionally used to obtain rectal forms, for example, preservatives, emulsifiers, thickeners, plasticizers and other excipients.

In addition, the present invention also includes the use of the polypeptide composition for the relief of convulsive syndrome, restoration of impaired reflex activity of the brain, memory, coordination of movements, in stroke, effusions in the brain, or as a neuroprotector.

Convulsive syndrome is a condition that occurs suddenly and is characterized by involuntary muscle contraction, may be accompanied by a change in the person's consciousness. Treatment or relief of a seizure syndrome consists in reducing the number of relapses, alleviating symptoms, slowing or stopping the progression of this condition.

The reflex activity of the brain consists in a quick and adequate response of the body to external or internal stimulation. In the reflex mechanism, it is customary to distinguish three parts: sensory, central and motor. The irritant causes the process of excitation in the terminal branches of the sensory nerve. Excitation along the sensory nerve is transmitted to the center (brain), where it switches to the motor nerve and through it reaches the working organ, and a response to the stimulation arises. These three parts of the reflex mechanism are collectively called the reflex arc. In this case, in the brain, these mechanisms are carried out due to various receptors and signaling substances, with the participation of various departments. Thus, it is rather difficult to establish the nature and cause of the disturbance of the reflex activity of the brain. With violations of reflex activity, as a rule, deviations from the norm are diagnosed both in behavior and in the vegetative sphere. They belong to the category of neuroses and are expressed in a violation of the orientation-research reflex, in inadequate responses to signals from the external environment, in memory defects, in poor learning, unstable manifestation of skills, insufficient spatial orientation, emotional shifts, as well as in pathological changes in the cardiac -vascular, digestive, endocrine and other systems of the body.

Recovery with this disorder means an improvement in reflex activity compared to the state before treatment, a weakening of symptoms or a slowdown in their progression, restoration of orientation functions in space, an increase in the efficiency of reactions to external stimuli, an increase in the compensatory functions of the body, an improvement in cognitive functions, a more even behavior in everyday life, restoration of a normal psycho-emotional character.

Memory impairment means a condition in which there is a decrease or loss of the ability to remember, store, recognize and reproduce information. In various diseases, individual components of memory may suffer, such as memorization, retention, reproduction.

Recovery from memory impairment means stopping the progression of memory impairment, improving the condition or weakening the symptoms of memory impairment, while this can be expressed in a decrease in episodes of memory loss, an increase in the volume of memorized information, and an improvement in cognitive skills.

Impaired coordination of movements can be expressed in patient instability, swaying from side to side when standing or walking, loss of balance, falls when walking on flat surfaces. In severe cases, this condition may be accompanied by nausea and vomiting. Also, the condition can lead to dysarthria and nystagmus, tremors of the extremities, decreased muscle tone. The restoration of this function when taking funds can be expressed in the weakening of symptoms, alleviating the condition and slowing down its progression, increasing the general muscle tone and compensatory functions.

A stroke is an acute violation of the blood supply to the brain (acute cerebrovascular accident), characterized by a sudden (within a few minutes, hours) appearance of focal and / or cerebral neurological symptoms that persist for more than 24 hours or leads to the death of the patient in a shorter period of time due to cerebrovascular pathology and effusion into the brain. The consequences of a stroke and effusion to the brain can be expressed in paresis of the muscles of the face and body, impaired coordination and motor function, decreased muscle tone, confusion, impaired memory and reflex brain activity, and cognitive disorders. Treatment consists in restoring lost functions and increasing the compensatory functions of the body, reducing the consequences of this condition.

Neuroprotective agents are substances or pharmaceuticals that help protect brain cells from damaging influences, prevent their death or improve their functions, mainly by restoring connections between neurons, improving the quality of signal transmission, which ultimately can lead to an improvement or normalization mental activity, weakening or restoration of impaired functions, incl. functions of memory, motor and coordination functions, cognitive skills and reflex activity of the brain, increasing the resistance of the brain to hypoxia, general mental and physical performance.

In this case, the use of a polypeptide composition or a pharmaceutical composition containing a polypeptide composition according to the invention for the treatment or restoration of the above conditions is possible in the form of an intravenous injection, an intramuscular injection or a composition in the form of a rectal form.

However, the composition of the polypeptides can be administered alone or in combination with another neuroprotective agent. Another neuroprotective agent is a drug that is used to restore or normalize the activity of the brain and nervous system, increase mental and physical performance.

In particular, the other neuroprotective agent may be cortexin, cerebrolysin, actovegin, piracetam, phenotropil, encephabol, gliatilin, pantocalcin, glycine, phenibut, citicoline, picamilon, semax, mexidol, cytoflavin, cavinton, oxybralizin, sermion, tssentaln, tssentaln.

For relief of convulsive syndrome, restoration of impaired reflex activity of the brain, memory, coordination of movements, in stroke, effusions in the brain or as a neuroprotector, you can use not only the composition of the polypeptides according to the present invention, but also the pharmaceutical composition containing this composition of polypeptides. The pharmaceutical composition can be administered by intravenous injection, intramuscular administration or rectal administration. In this case, the pharmaceutical composition can be used alone or in combination with another neuroprotective agent. In particular, another neuroprotector is Cortexin, Cerebrolysin, Actovegin, Piracetam, Phenotropil, Encephabol, Gliatilin, Pantocalcin, Glycine, Phenibut, Citicoline, Picamilon, Semax, Mexidol, Cytoflavin, Cavinton, Oxybral, Sermion, Cinnamon.

The present invention is given below in the form of examples, which in no way limit the scope and are intended for illustrative purposes only.

Examples of

Example 1

Method for producing mammalian brain extract

The raw material for the isolation of the composition of polypeptides was obtained according to the method described in patent RU 2275924. The extract after receiving was lyophilized, obtaining a white or yellowish-white powder.

Example 2

Isolation of polypeptide composition using gel filtration chromatography and reversed phase chromatography

The extract obtained at the previous stage is a complex of water-soluble polypeptides with a molecular weight of up to
10,000 Yes. For separation into fractions, separation using gel filtration chromatography was used. The conditions are shown below:

Separation conditions on a HiLoad 16/60 Superdex 30 prep grade column

Device Liquid chromatograph Akta Pure150 Column HiLoad 16/60 Superdex 30 prep grade, Vcol = 120 ml Mobile phase Highly purified water (MilliQ) Flow rate, ml / min 1,0 Column temperature, ° C 20-24 Sample temperature, ° C 20-24 UV detector parameters Wavelength 220 nm Injected sample volume, ml 5.0 Separation time, min 125.0

The results obtained from the analysis of the obtained extract by gel filtration chromatography are shown in FIG. 1.

A fraction was isolated at retention times of about 88 to about 109 minutes. Further, the resulting fraction was separated by reverse phase chromatography under the following conditions:

Separation conditions on reverse-phase sorbent

Daisogel SP-100-10-C8-PK

Device Liquid chromatograph Akta Pure150 Column Daisogel SP-100-10-C8-PK, Vcol = 1.66 ml Mobile phase (PF) A: 0.05% TFA in highly purified water
B: 0.05% TFA, 50% acetonitrile, 50% highly purified water.
Step elution: Flow rate, ml / min 0,4 Column temperature, ° C 20-24 Sample temperature, ° C 20-24 UV detector parameters Wavelength 220 nm Injected sample volume, ml 10.0 Separation time, min 125.0

A fraction of polypeptides with a retention time of 28-35 minutes was isolated. The volume was about 6.7 ml, the weight of the product was about 4.5 mg. The resulting fraction was dried to constant weight.

Example 3

Analysis of the resulting composition of polypeptides

The analysis of polypeptides included in the previously obtained fraction was carried out by spectrophotometry, HPLC-UV analysis, and HPLC-MS analysis.

The amount of water-soluble peptides was determined by the Bradford method using spectrometry. We used standard solutions and reagents. The optical density of the test solution was measured on a spectrophotometer at a wavelength of 595 nm. A standard Cortexin sample (without artificial glycine) in an amount of 50 mg was used as a calibration sample. The amount of water-soluble peptides ranged from about 12.0 to about 49.8 mg per mg dry matter.

The total amount of amino acids was determined by acid hydrolysis followed by derivatization using HPLC-UV analysis.

To determine the amount of amino acids that make up the peptides, it was necessary to determine the amount of free amino acids in a sample and the total amount of amino acids in the same sample. Derivatization was performed to determine free amino acids. To determine the total amount of amino acids, the sample was derivatized after preliminary acid hydrolysis followed by neutralization. For this, 10 μL of the sample was placed into a glass vial of the autoinjector of the chromatograph, 70 μL of AccQ • Tag Ultra Borate buffer (borate buffer) and 20 μL of AccQ • Tag Ultra reagent (commercial reagent) were added. The contents of the vial with a tightly closed lid were immediately vortexed and incubated first at room temperature for 1 minute and then at 55 ° C for 10 minutes. The derivatives were stable at room temperature for 1 week.

The chromatographic conditions are shown below:

Device Waters Acquity UPLC Liquid Chromatograph equipped with Spectrophotometric Diode Array Detector Column AccQ • Tag Ultra Colum, 2.1 x 100 mm, sorbent diameter 1.7 μm (Waters, USA), cat. No. 186003837 Mobile phase А: diluted solution AccQ • Tag Ultra Eluent A Concentrate;
B: AccQ • Tag Ultra Eluent C
Gradient Mode Flow rate 0.7 ml / min Column temperature 55 ° C Sample temperature 20 ° C Diode Array Detector Parameters Wavelength λ = 260 nm
Speed 10 points / sec
Time constant 0.200 sec Injected sample volume 1 μl Analysis time 11 minutes

The amount of free amino acids was determined by the method of derivatization without hydrolysis, using HPLC-UV analysis. To calculate the number of amino acids contained in peptides, that is, bound amino acids, the amount of free amino acids was subtracted from the total amount.

The number of bound amino acids was:

arginine - from 29 to 108 μg / mg dry residue of the composition,

glycine - from 29 to 71 μg / mg,

asparagine - from 23 to 88 μg / mg, and

glutamine - from 23 to 104 μg / mg dry residue of the composition.

The amount of other amino acids was:

alanine - from 22 to 56 μg / mg,

proline - from 19 to 73 μg / mg,

lysine - from 16 to 124 μg / mg,

leucine - from 21 to 82 μg / mg dry residue of the composition.

It should be noted that the following amino acids were also found in the composition from significant amounts of bound amino acids in the composition:

tyrosine - from 16 to 44 μg / mg dry residue of the composition, phenylalanine - from 19 to 47 μg / mg dry residue of the composition, valine - from 10 to 54 μg / mg dry residue of the composition, isoleucine - from 13 to 46 μg / mg dry residue ... However, no cysteine was observed as a bound amino acid.

The molecular weights of the peptides were determined by HPLC-MS, followed by the calculation of the molecular weight of the characteristic peptides based on the obtained chromatograms.

Analysis conditions are presented below:

Column ACCQ-TAG ULTRA C18, 1.7um, 100mm x 2.1mm (Waters)
S / N 02953704525122 Flow rate 0.3 ml / min Eluent PP A: 0.1% formic acid in water
PF B: 0.1% formic acid in acetonitrile
Gradient Mode Column temperature 60 ° C Injected sample volume 5 μl

Mass detector parameters:

Ionization mode positive ES + Scan duration, sec 0.25 Detection mode: scanning, in the range from 300 to 3000 m / z Ion Source Settings Capillary voltage, kV 4.0 Cone voltage, V 44 Extractor voltage, V 3.00 Lens voltage (RF lens), V 2.50 Source temperature, ° C 150 Desolvation gas temperature, ° C 350 Desolvation gas flow, L / h 600

The results of mass chromatography of the resulting polypeptide composition are shown in FIG. 2-4.

The deconvolution method is used to determine molecular weight from low-resolution mass spectra.

The result of determining the molecular weight of the main peptides of the composition of the polypeptides according to the invention after separation in the gel filtration chromatography mode

Peak retention time, min. The magnitude of the ion charge Molecular weight value, Yes 50.0-51.7 6+ 2600-2700 5+ 4+ 53.4-54.7 15+ 7500 - 7900 14+ 13+ 12+ 11+ 10+ 9+ 56.3-59.0 6+ 2700-3000 5+ 4+ 3+

The chromatogram of this composition of polypeptides clearly shows the main peaks with a retention time of 50.0-51.7 min, 53.4-54.7 min, 56.3-59.0 min, which include peptides with a molecular weight of 2600-2700 Yes, 7500-7900 Yes, 2700-3000 Yes, respectively.

In addition, the resulting composition was diluted for complete dissolution in water and the pH of the resulting solution was determined. The pH was from 5.0 to 6.6.

Example 4

Determination of the biological activity of the resulting composition of polypeptides in chicken embryos

The biological activity of the composition of polypeptides was assessed by the effect on the growth of nerve elements or outgoing cells in the growth zone in the culture of the brain tissue of chicken embryos when compared with the control.

The brain of chicken embryos removed from eggs after 9-12 days of incubation was used as a biological test system.

A quantitative assessment of the effect of the drug on the growth of the brain tissue of embryos was performed by the morphometric method followed by the calculation of the cell growth area index (hereinafter PI) and the calculated PI% value, which expresses the difference in the area of the control and experimental samples, expressed as a percentage.

As a reference was adopted activity minimum allowable SP% brain tissue after cultivation with PL Cortexin ^® in concentration of 20 ng / ml = 20%.

As a result of the studies, it was shown that the composition of polypeptides has biological activity: it stimulates an increase in the area of growth of nerve tissue by at least 20% relative to the control.

An egg with a chicken embryo 9-12 days old was taken for the analysis, the egg was washed with soap and water. The chicken embryo was removed. Surgical instruments treated in a 96 ° solution of ethyl alcohol followed by filling, cut the film covering the brain, isolated the brain tissue and placed it in a sterile Petri dish with several drops of Hanks solution. Attached to the surface of six Petri dishes, 12-14 fragments of neural tissue with a size of about 0.5 mm, then the dishes were closed and kept for 2-4 minutes in a laminar flow cabinet at room temperature. Then, 3 ml of culture medium was added without adding test objects, after which test objects were added to the test dishes with a concentration of 20 ng / ml in a volume of 3 ml.

Experimental and control Petri dishes were covered with lids and placed in a CO ₂ incubator with 5% carbon dioxide content and a temperature of (37 ± 1) ° C for 50 hours.

The biological activity of the composition of polypeptides was determined by the effect on the growth of nerve elements or outgoing cells in the growth zone in the culture of the brain tissue of chicken embryos when compared with the control. The test was carried out using a light microscope.

The initial area of a nerve tissue fragment was measured by tracing it using the Draw or Erase Feature tool of the TotalLab Colony Counter module. The value of the "area" parameter was entered into the results registration table in the "min" column. The area of the entire fragment of the nervous tissue of the brain, including the peripheral growth zone, was measured in the same way and the result obtained was entered into the table of registration of results in the column "max".

PI was calculated as the ratio of the area of the entire fragment of the nervous tissue of the brain, including the peripheral growth zone, to the initial area of the fragment of the nervous tissue (max / min).

To predict the biological activity of the test objects, the PI% was calculated using formula 1.

IP% = ((IPo-IPk) / IPk) × 100% (1)

Where: PI% - index of the area of cell growth for the test object; IPo is the index of the cell growth area of the experimental sample; IPc - index of the cell growth area of the control sample.

The test object was considered biologically active if the PI% of brain tissue after cultivation with the test objects at a concentration of 20 ng / ml was at least 20% more than the PI in the control.

Statistical processing of the PI calculation results was performed using the t-test for independent samples using the Prism 6 software (GraphPad Software, USA). Differences were considered statistically significant at p <0.05. In the report, the results are presented in the form of histograms, reflecting the means and standard deviation and statistical differences, the value of n (number of measurements in each group) was equal to 14.

In FIG. 7 shows photographs of cell growth in a chicken embryo in the case of control and polypeptide composition. It is seen that in the case of the composition of the polypeptides according to the invention, the growth area of the nerve cells is higher.

Thus, the tissue-specific and neuroprotective activity of the polypeptide composition was demonstrated.

Example 5

Determination of the chronic toxicity of the composition

A mouse model was used to assess chronic toxicity, with a period of 3 months selected.

In experiments on rodents (rats of both sexes), the polypeptide composition obtained in Example 2 was injected intramuscularly for 90 days in 3 doses: 10 times greater than the therapeutic for humans - 1.5 mg / kg; maximum 150 mg / kg (1000 times more therapeutic for humans, does not cause death in rats after a single administration) and intermediate - 15 mg / kg.

The studies were carried out in accordance with the experimental program:

Animals - female rats, male rats.

The route of administration is intramuscular.

Doses: 1 dose - 1.5 mg / kg; 2nd dose - 15 mg / kg; 3 dose - 150 mg / kg.

The frequency of administration is within 90 days.

The solvent is water for injection.

Number of animals - 1 dose - 20 males, 20 females;

2nd dose - 20 males, 20 females;

3rd dose - 20 males, 20 females.

The total number is 160, of which 40 are the control group.

During the experiment, the following indicators were assessed: general condition, weighing, water consumption, hematological, biochemical studies, physiological studies, macroscopic examination and weighing of internal organs, histological studies.

According to the results of the study, it was found that the composition of the polypeptides according to the invention in rats of both sexes intramuscularly once a day for 90 days at doses of 1.5; 15; 150 mg / kg does not affect the appearance, general condition and behavior of animals, does not adversely affect the biochemical parameters of blood and basic physiological functions of the body, does not cause pathomorphological changes. Thus, it should be concluded that the composition is well tolerated, harmless, does not have acute and chronic toxicity.

Example 6

Study of biological activity

To confirm the biological activity of the claimed pharmaceutical agent and its ability to normalize the functions of the brain, studies were carried out to study the pharmacological activity on the model of acute traumatic brain injury (TBI).

To simulate traumatic brain injury, a special installation was used, consisting of a metal tube 0.5 m long, 2 cm in diameter and a steel ball weighing 0.03 kg. To fix the head, the rats were placed in standard plexiglass houses, in the front of which there was a hole corresponding to the diameter of the tube. Trauma was inflicted when a ball fell on the parietal region of the skull of animals. After injury, rats showed muscle relaxation, short-term loss of consciousness, soporosis, and short-term convulsions. It was noted that ataxia persisted in rats for 30-40 minutes, and reflex reactions were disturbed for 1.5-3 hours.

The composition of polypeptides was started to be injected intramuscularly from the next day after acute trauma at doses of 1.5 mg / kg once a day for 10 days.

The effectiveness of treatment was judged by the clinical picture, dynamics of body weight, relative weight of the brain, functional state of the central nervous system, indicators of tissue respiration, activity of metabolic processes in the brain and histological picture of the brain.

Body mass and relative brain mass of experimental animals after acute trauma and treatment with the target product Indicators Experimental groups Intact (healthy) Without treatment (after 10 days) Composition treatment (after 10 days) Body weight, g 185 ± 6 160 ± 5 182 ± 7 Relates. brain weight, mg / 100 g body weight 9.5 ± 0.5 12.4 ± 1.1 10.3 ± 0.9

The table shows the state with TBI of the functions of the central nervous system (disorders of learning, memory, excitation and inhibition) without treatment and after using the composition according to the invention.

Indicators of the functional state of the central nervous system (SPP, CPAR, open field test) of experimental animals with TBI and treatment with a composition of polypeptides Indicators Experimental groups Intact (healthy) Without treatment (after 10 days) Treatment with a composition of polypeptides (after 10 days) SPP, V 12.1 ± 1.2 15.1 ± 1.3 13.4 ± 0.7 URPI Latent period of the transition reaction, sec 75 ± 4 136 ± 5 72 ± 4 Time spent in the light chamber, sec 84 ± 2 41 ± 4 80 ± 3 Time spent in a dark chamber, sec 4.8 ± 0.5 14.7 ± 1.2 4.2 ± 0.6 Number of trained animals (% of the whole group) 10 (100) 2 (20) 10 (100) Open field, registration within 5 minutes Horizontal activity 38 ± 3 9 ± 1 33 ± 3 Vertical activity 23 ± 2 7 ± 1 18 ± 2 Mink reflex 15 ± 3 4 ± 1 13 ± 2 Boluses 10 ± 1 5 ± 1 8 ± 1 Integral activity 67 ± 4 13 ± 2 63 ± 3 Notes: SPP - summation-subthreshold indicator;
Passive avoidance reflex is a conditioned reflex of passive avoidance.

From the data in the table it can be seen that the composition of the polypeptides according to the invention normalizes the functions of the central nervous system.

The data in the table show that as a result of TBI, muscle tone increased, and the magnitude of the reaction to mechanical stimulation decreased markedly, which indicates violations of the conjugation of reflex processes in the central and peripheral nervous systems. The use of the polypeptide composition contributes to the normalization of the peripheral nervous system and muscles.

The value of physical endurance of rats by the duration of hovering (min) in the test of static power load with their own weight Indicators Experimental groups Intact (healthy) Without treatment (after 10 days) Treatment with a composition of polypeptides (after 10 days) Hovering time, min 28.1 ± 1.3 9.2 ± 0.8 24.9 ± 1.1

The presented results indicate the effectiveness of the composition of the polypeptides in the treatment of the consequences of acute traumatic brain injury. Also, based on the results obtained, we can talk about an effective nootropic, neuroptrotective, antioxidant and tissue-specific action of the polypeptide composition according to the invention. The composition according to the invention can be used for relief of convulsive syndrome, restoration of impaired reflex activity of the brain, memory, coordination of movements, in case of stroke, effusions in the brain, or as a neuroprotector.

Example 7

Research of allergic reactions

As a study of possible side effects, a model was chosen on 2-month-old rats of the Norwegian brown breed, weighing about 150 g. The rats were divided into 3 groups, 8 in each (control (intact animals); with the introduction of albumin from egg yolk 0.1 mg; with the introduction of the composition of the polypeptides 1.5 μg / kg). Allergic reactions were induced by oral administration of egg yolk albumin for 10 days. The polypeptide composition was also administered orally for 10 days.

The rats were kept in a clean room, on a non-allergenic diet, and observed for 30 days. When a skin allergy was identified, symptoms were recorded and measurements taken (if applicable).

The results included the following description:

- No allergic reactions were noted in intact animals, incl. in the form of skin.

- In 6 animals from the group that received albumin from egg yolk (Group 1), on the 3rd day from the start of administration, active skin reactions developed: the formation of skin irregularities and irritation, scratching of irritated areas, itching and anxiety of the animal, dermatitis on the paws. On the 11th day (1 day after the completion of albumin intake), skin reactions intensified, lethargy and drowsiness of the animals appeared. On day 15 and further, 4 of the 6 animals described showed skin reactions, but with a reduced effect.

- In 3 animals from the group receiving the composition of polypeptides (Group 2), itching and skin irritation, scratching of the irritated areas were diagnosed. By the 20th day, these phenomena were not noted.

In parallel with an external examination, blood was taken and a clinical blood test was performed for the presence of basophils (B), eusinophils (E) and neutrophils (N) (indicators of an allergic reaction). The average of 8 animals is taken, the volume content is given in%.

Indicators, day Day 10 Day 15 Day 20 Day 25 Day 30 Intact animals B -0.3
E - 0.1
H - 10 B -0.3
E - 0.15
H - 10 B -0.3
E - 0.15
H - 10 B -0.3
E - 0.15
H - 10 B -0.3
E - 0.15
H - 10 Group 1 B - 1.0
E - 2.1
H - 18 B - 1.3
E - 1.9
H - 18 B - 1.3
E - 1.7
H - 17 B - 1.3
E - 1.5
H - 15 B - 1.3
E - 1.5
H - 15 Group 2 B - 0.4
E - 1.5
H - 14 B - 0.4
E - 1.5
H - 14 B - 0.4
E - 1.2
H - 14 B - 0.3
E - 1.2
H - 12 B - 0.3
E - 1.0
H - 10

As can be seen from the results, the polypeptide composition practically does not cause allergy in the rat model.

Example 8

Preparation of pharmaceutical compositions comprising a polypeptide composition by injection

The polypeptide composition obtained in Example 2 was lyophilized, dissolved in various amounts in a pharmaceutical solvent (water, novocaine) and analyzed for stability.

Composition 1 2 3 4 five 6 Composition of polypeptides 5 mg 10 mg 15 mg 5 mg 10 mg 15 mg Water or 0.9% sodium chloride solution (saline) up to 1 ml. up to 1 ml. up to 1 ml. 0.5% novocaine up to 1 ml. up to 1 ml. up to 1 ml.

All of these solutions were stable and active against the growth of nerve cells (analysis similar to Example 4).

These solutions were intended for intramuscular or intravenous administration.

Example 9

Preparation of pharmaceutical compositions comprising a polypeptide composition for rectal administration

The polypeptide composition in the form of a lyophilisate from Example 2 was dissolved in a small amount of water and introduced into the "Witepsol W" suppository mass. WITEPSOL W is a commercially available suppository base containing 20-50 hard fats. This base contains triglycerides (65-80%), diglycerides (10-35%) and monoglycerides (1-5%). The incomplete glycerides included in the composition slow down the deposition of solid particles, contribute to the wetting of mucous membranes and improve the absorption of active components.

Received a pharmaceutical composition in the form of suppositories for rectal administration with the following compositions:

Composition 1 2 3 Composition of polypeptides 5 mg 10 mg 15 mg Witepsol up to 2 g. up to 2 g. up to 2 g.

All compositions were stable, melted at 35-37 ° C and allowed the active substance to be released within 45 minutes.

Claims (25)

1. Composition of polypeptides obtained from the extract of the brain of cattle using gel filtration, which has nootropic, neuroprotective, antioxidant and tissue-specific action, characterized by the following indicators: - the molecular weight of the polypeptides is approximately 2600-3000 Da and 7500-7900 Da; - the number of linked amino acids is: arginine - from 29 to 108 μg / mg, glycine - from 29 to 71 μg / mg, asparagine - from 23 to 88 μg / mg, and glutamine - from 23 to 104 μg / mg dry residue of the composition; - the number of additional linked amino acids is: alanine - from 22 to 56 μg / mg, proline - from 19 to 73 μg / mg, lysine - from 16 to 124 μg / mg, leucine - from 21 to 82 μg / mg, tyrosine - from 16 to 44 μg / mg, phenylalanine - from 19 to 47 μg / mg, valine - from 10 to 54 μg / mg and isoleucine - from 13 to 46 μg / mg dry residue of the composition. 2. The composition of the polypeptides according to claim 1 in the form of a lyophilisate. 3. A pharmaceutical composition for normalizing brain function, containing a composition of polypeptides according to claims. 1, 2, and a pharmaceutically acceptable carrier. 4. The use of the composition of the polypeptides according to PP. 1, 2 for the relief of convulsive syndrome, restoration of impaired reflex activity of the brain, memory, coordination of movements, in case of stroke, effusions in the brain, or as a neuroprotector. 5. Use according to claim 4, characterized in that the application is carried out by intravenous injection, intramuscular injection or rectally. 6. Application according to PP. 4, 5, characterized in that the polypeptide composition is administered alone or in combination with another neuroprotective agent. 7. Use according to claim 6, characterized in that the other neuroprotector is cortexin, cerebrolysin, actovegin, piracetam, phenotropil, encephabol, gliatilin, pantocalcin, glycine, phenibut, citicoline, picamilon, Semax, mexidol, cytoflavinral, cavinton, sermion, cinnarizine, trental. 8. The use of a pharmaceutical composition according to claim 3 for the relief of convulsive syndrome, restoration of impaired reflex activity of the brain, memory, coordination of movements, in case of stroke, effusions in the brain, or as a neuroprotector. 9. Use according to claim 8, characterized in that the application is carried out by intravenous injection, intramuscular injection or rectally. 10. Application according to PP. 8, 9, characterized in that the composition according to claim 3 is administered alone or in combination with another neuroprotective agent. 11. The use according to claim 10, characterized in that the other neuroprotector is Cortexin, Cerebrolysin, Actovegin, Piracetam, Phenotropil, Encephabol, Gliatilin, Pantocalcin, Glycine, Phenibut, Citicoline, Picamilon, Semax, Mexidol, Cytoflavibral, Cavinton, Oxidon sermion, cinnarizine, trental.

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