RU2364404C2 - Biotreatment blood serum, methods of production and application - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions concerns biology and medicine. There is disclosed method of biotreatment blood serum production involving electrostimulation of an animal, other than a human, blood drawing of specified animal, serum separation from specified blood and gamma irradiation in a dose 10 to 40 kGy. There is offered blood serum product and pharmaceutical composition containing said blood serum product, and also their application for treatment of various diseases.

EFFECT: invention provides production of biotreatment animal's blood serum applied for treatment of wide range of diseases, including epileptic attacks and apoplexy.

24 cl, 4 ex, 7 tbl, 11 dwg

Description

The present invention relates to a method for producing a blood serum product, a blood serum product and a pharmaceutical composition containing said blood serum product, as well as their use for treating various diseases and conditions, including epileptic seizures and apoplexy.

State of the art

Methods for preparing active substances from blood serum are known in the art. One of them is based on blood sampling from humans or animals, subsequent incubation, as well as separation of the active substance and, finally, preservation of the substance (see, for example, JP 2123287, EP 0542303, RU 2096041, RU 2120301). The prior art method relates to the production of blood serum, which increases the body's resistance to exogenous and endogenous factors, such as air pressure, air temperature, gravity, light, etc., as well as hunger, thirst, sleep and sexual desire, etc. . Blood serum is taken from a donor who has previously been brought into a certain functional state and depending on the duration of use, on the functional state and type of functional state, for example, insomnia, alcohol dependence, dependence on nicotine, etc., blood serum can be obtained with various biological activity, which exhibits mitogenic, dysfunctional, ophthalmic, audio-activating, thermo-activating, affecting diet, affecting sexual activity, antihypoxia sical, anti-alcohol and anti-nicotine action.

An excellent method is disclosed in EP 1283047 and relates to the treatment of animal blood serum with gamma radiation in order to increase the biological activity of the blood serum product.

Currently, a large number of studies focused on the mechanisms that regulate cell proliferation of various human tissues. In this regard, both stimulants and inhibitors of proliferation of normal and pathologically altered somatic cells, including nerve cells, were studied (see, for example, Aschmarin, 1. P. "Neurochemistry", Moskwa, Publishers of the Biomedical Chemical Institute of the Russian Academy of Science ", 1996).

It was noted that peptide growth factors, in addition to general activating functions, for example, stimulating mitosis, cell differentiation and cell growth of various types of normal tissue, increasing wound healing, can cause tumor formation and growth (see, for example, Bouneres, P. (1993) , Horm. Res, 40: 31; Robinson C., (1993) Ann. Med 25: 535; Dignez, C and Casanueva F. (1995) Trends Endocrin. Metab. 6:55, Menster D. et al. (1995 ) Clin. Exp. Metastasis 13: 67).

Peptides such as, for example, peptides of the thyroid gland, gastrin or bombesin contribute to the development of tumor cells, as well as the development of breast cancer, bone cancer and colon cancer (see, for example, Kitazawa S. and Maeda S. (1995) Clin. Orthop 312: 45-50 and Kaji et al. (1995) Endocrinology 136: 842).

Despite the fact that some peptides promote the division of normal cells and are stimulants for humans and animals, there is a danger that their use will lead to the development of tumor cells and, ultimately, to the development of cancer.

Earlier experiments showed that stimulation of animals with electricity leads to an increase in the level of β-endorphin in the blood (see, for example, Litvinova S.V. et al. (1990) Biomed. Sci. 5: 471). In reference work Udovitschenko W.L. Numerous data are presented regarding the results of stimulation or shock due to various reasons. It has been shown that, for example, electroshock leads to a marked increase in the concentration of β-endorphins, meta- and leyencephalins in the blood (see Udowitschenko W.I. (1989) "Xenogenic Opioid System in Shock" Pathiological Physiology and Experimental Therapy "6: 72-77).

SUMMARY OF THE INVENTION

The aim of the present invention is to develop a new method for producing biologically active blood serum from animal blood. It was unexpectedly discovered that biologically active blood serum obtained in accordance with the method of the present invention, showed new therapeutic properties.

Therefore, one aspect of the present invention is a method for producing biologically active blood serum, comprising the steps of:

a) electrical stimulation of a non-human animal,

b) taking blood from the specified animal,

c) isolating serum from said blood, and

d) gamma radiation of said serum.

In a preferred embodiment, the non-human animal is selected from the group comprising mammals and birds, preferably poultry, for example, chicken, duck, goose, ostrich and quail.

Although electrical stimulation can be applied to any part of the body, it is preferred that step a) of the method of the present invention is applied to the head, neck, body and / or one or more limbs of the animal. Of these, it is preferable to electrostimulate the animal’s head. In the context of the present invention, the terms electrical stimulation and electric shock are used interchangeably.

In a preferred embodiment of the method of the present invention, electrical stimulation is performed for a time interval between 1 and 60 seconds, preferably between 1 and 30 seconds, and more preferably between 2 and 10 seconds. It is also preferable that electrical stimulation is performed with a voltage in the range between 50 V and 150 V, preferably in the range between 80 V and 120 V, and more preferably in the range between 110 V and 120 V. When performing electrical stimulation, certain current values are preferred, and preferably, electrical stimulation is performed with a current strength in the range between 0.01 A and 0.4 A, preferably in the range between 0.02 A and 0.1 A, and more preferably in the range between 0.04 A and 0.06 A.

In a preferred embodiment of the method of the present invention, electrical stimulation is performed at a frequency in the range between 10 and 200 Hz, preferably in the range between 20-100 Hz and, more preferably, in the range between 45-65 Hz.

In yet another preferred embodiment of the method of the present invention, gamma radiation is introduced with an absorbed radiation dose of between 10 and 40 kGy, preferably 15 and 35 kGy, and more preferably between 20 and 30 kGy. The gamma radiation source can be any source, however, the preferred gamma radiation source is selected from the group consisting of ⁶⁰ Co, ¹³⁷ Cs, ⁶⁷ Cu, ⁶⁷ Ga, ¹¹¹ In, ¹⁹² Ir, ^99m Tc and ^l70 Tm.

In a more preferred embodiment of the method of the present invention, the method further comprises the step of incubating said blood before step c).

In a more preferred embodiment of the method of the present invention, the method further comprises the step of lyophilizing said serum before step d).

In a preferred embodiment of the method of the present invention, the blood is arterial and / or venous blood.

Another aspect of the present invention is biologically active blood serum produced in accordance with the method of the present invention.

An additional aspect of the present invention is a pharmaceutical composition comprising the blood serum of the present invention and one or more pharmaceutically acceptable diluents; carriers; excipients, including fillers, binders, lubricants, glidants, disintegrants, adsorbents and / or preservative.

In a preferred embodiment of the pharmaceutical composition of the present invention, the composition is formulated as a syrup, solution for infusion or injection, a tablet, capsule, solid tablet in the form of a capsule, troches, liposomes, suppositories, patch, bandage, sustained release capsules, powder or sustained release preparation . Preferably, the diluent is water, a buffer, a buffered saline or saline solution, and the carrier is preferably selected from the group consisting of cocoa butter and vitebesole.

An additional aspect of the present invention is the use of the blood serum of the present invention or the pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of a disease or condition that may be caused by an increase in the content of cyclic adenosine monophosphoric acid in the patient's brain.

Another aspect of the present invention is the use of the blood serum of the present invention or the pharmaceutical composition of the present invention for the manufacture of a medicament for improving cognitive and / or educational skills, in particular for improving long-term memory.

Another aspect of the present invention is the use of the blood serum of the present invention or the pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of seizures, in particular epileptic seizures.

An additional aspect of the present invention is the use of the blood serum of the present invention or the pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of proliferative diseases and apoplexy.

In a preferred embodiment of the application of the present invention, the proliferative disease is selected from the group consisting of malignant gastrointestinal or colorectal tract, liver, pancreas, kidney, bladder, thyroid gland, prostate, uterine mucosa, cervix, ovary, uterus, testicles, skin, oral cavity; melanomas; dysplastic oral mucosa; invasive cancers of the oral cavity; small cell and non-small cell lung carcinomas; breast tumors, in particular hormone-dependent breast cancers and hormone-independent breast cancers; transitional cell and squamous cell carcinomas; neurological malignancies, including neuroblastomas, gliomas, astrocytomas, osteosarcomas, meningiomas; soft tissue sarcomas; hemangiomas and tumors of endocrine glands, in particular, pituitary adenomas, pheochromocytes, paragangliomas, hematological malignancies, in particular, lymphomas and leukemia.

In addition, in another preferred embodiment of the present invention, the proliferative disease comprises cells similar to the human T cell lymphoma cell line Jurkat, the human B cell lymphoma cell Raji cell line, the human melanoma cell line Bro, the human cervical cancer cell line HeLa, MCF-7 human adenocarcinoma cell line, osteosarcoma Mg63 cell line, fibrosarcoma HT1080 cell line, neuroblastoma cell line IMR-32, and hepatocarcinoma cell HepG2 line.

In a further preferred embodiment of the use of the present invention, the drug is administered to a patient in an amount varying from 50 to 150 mg / kg body weight, preferably varying from 90 to 100 mg / kg body weight.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention unexpectedly found that stimulation of animals, in particular chickens, by electric current and additional processing of blood serum obtained from blood, γ-radiation leads to a significant increase in the biological activity of the blood serum product. The resulting products can have a positive effect on various body functions, conditions and diseases of the patient.

The beneficial effects of blood serum treated with electroshock and gamma radiation are unexpected for two reasons. Firstly, it is known in the prior art that electric shocks cause severe disturbances in all vital functions and systems in the body, in particular the central nervous system, blood and circulatory system and respiratory system (see, for example, Orlow, AN et al. (1977) Medicine). Secondly, it was also found that blood and serum obtained from blood are sensitive to radiation and are unstable, and are inactivated by ionizing radiation (see, for example, Radiomedicine - M Atomisdat (1972) 123-125 and Gergely, 3. et al. (1967) Radiosterilization of Medical Products 115-124).

These studies did not lead to the results of the present invention, and it could be assumed that it is impossible to obtain biologically active blood serum by a combination of electroshow and γ-radiation treatment. Thus, it was a surprise that biologically active blood serum can be obtained from arterial and / or venous blood taken from an animal, in particular from chicken treated with level II-III electroshock and gamma radiation. The specific activity of the thus obtained blood serum is described in more detail below.

Accordingly, a first aspect of the present invention is a method for producing biologically active blood serum, comprising the steps of:

a) electrical stimulation of a non-human animal,

b) taking blood from the specified animal,

c) isolating serum from said blood, and

d) gamma radiation of said serum.

Various animals may be used in the method of the present invention, however, preferably, the non-human animal is selected from the group comprising mammals and birds. Due to availability, in particular, it is preferable to use farm animals such as poultry, for example, chicken, duck, goose, ostrich and quail. A particularly preferred animal that can be used in the method of the present invention is chicken. The type of mammal that can be used in the method of the present invention is not particularly limited and includes, without limitation, rodents, for example, a mouse, a hamster and a rat, cats, dogs, horses, donkeys, sheep, cows and goats.

It is believed that electrical stimulation results in the release of certain compounds in the animal’s body that cause and / or contribute to the unexpected therapeutic effect of the biologically active blood serum of the present invention. A non-human animal can be stimulated in various areas of the body. Preferably, electrical stimulation is performed on the head, neck, body and / or on one or more limbs. You can stimulate the body in only one place or in several places at once. A particularly preferred body part for electrical stimulation is the head of the corresponding animal. When stimulating birds, in particular chicken, it is preferable to electrostimulate the head.

Electrical stimulation can be performed by methods known from the prior art, preferably using metal electrodes or bathtubs with water, which, for example, are used for culling livestock or killing poultry with electric current. Preferably, electrical stimulation is performed over a period of time between 1 and 60 seconds, preferably between 1 and 30 seconds, more preferably between 2 and 10 seconds and, most preferably, between 3 and 4 seconds. The duration of the time interval is longer if a large animal is subjected to electrical stimulation, and may be shorter if small animals are subjected to electrical stimulation. For example, to stimulate the heads of chickens, in particular, the preferred time interval for electrical stimulation is between 2 and 10 seconds, and more preferably between 3 and 4 seconds. Other parameters that can be changed during the electrical stimulation of the animal are voltage, current strength and current frequency, and the present inventors have identified some preferred ranges for these parameters. The selected current parameter partially depends on the size of the animal, as well as on the area of the animal's body that is stimulated. Essentially, larger animals and larger areas require higher voltage and amperage. Thus, electrical stimulation is preferably performed with a voltage in the range between 50 volts and 150 volts, preferably from 80 volts to 120 volts, and more preferably between 110 volts and 120 volts. The ranges for the current that can be applied are between 0.01 A and 0.4 A, preferably between 0.02 A and 0.1 A, more preferably between 0.04 A and 0.06 A and, most preferably about 0.05 A. The voltage, current, and application time are preferably selected to transfer energy in the range between 1 and 1000 watts · s, preferably in the range from 10 to 200 watts · s and, even more preferably, in the range from 15 to 100 watts · s.

To stimulate preferred animals, i.e. chicken, it is preferable to perform electrical stimulation with a voltage in the range between 80 volts and 120 volts, and more preferably between 110 volts and 120 volts. In addition, a current in the range between 0.04 A and 0.06 A, in particular 0.05 A, is preferable in relation to electrical stimulation of birds, in particular chicken.

In a particularly preferred embodiment of the method of the present invention, electrical stimulation of the bird, in particular chicken, is performed for 3-4 seconds at a voltage between 80 V and 120 V, in particular 110 V and 120 V. In this preferred embodiment, the current strength is preferably is between 0.04 A and 0.06 A and, most preferably, about 0.05 A.

Apparently, the frequency of electrical stimulation is not particularly important, but is preferably in the range between 10 and 200 Hz, more preferably in the range between 45 and 65 Hz and, most preferably, is about 50 Hz.

Gamma irradiation of serum during step d) of the method of the present invention can be performed using any gamma source, including x-ray sources and radionuclides. Preferably, the gamma radiation source is radionuclides with a specific gamma radiation pattern. Preferred gamma radiation sources are selected from the group consisting of ⁶⁰ Co, ¹³⁷ Cs, ⁶⁷ Cu, ⁶⁷ Ca, ¹¹¹ In, ¹⁹² Ir, ^99m Tc and ¹⁷⁰ Tm. Of these, for the inventors of the method of the present invention, ⁶⁰ Co, ¹³⁷ Cs, ¹⁹² Ir and ¹⁷⁰ Tm are particularly preferred sources of gamma radiation, and ⁶⁰ Co. is most preferred. The radiation dose absorbed by the serum is in the range between 10 and 40 kGy, preferably in the range between 15 and 35 kGy and, more preferably, in the range between 20 and 30 kGy, i.e. 25 ± 5 kGy.

Blood sampling from an animal can be carried out by any method known from the prior art and includes taking with a syringe, as well as piercing arteries or veins, or decapitation, in particular in relation to blood sampling from birds. You can take only part of the blood or all the blood of the animal. The latter is preferably used if a lethal dose of electricity has been applied to the animal. The collected blood may be arterial and / or venous.

Serum can be isolated from blood by any known method, including filtration, precipitation and centrifugation. However, it is preferable to incubate the blood for 4-72 hours at a low temperature, for example between 2 and 10 ° C, preferably between 4 and 8 ° C, to ensure blood coagulation, which leads to the release of additional factors into the blood. Thus, in a preferred embodiment of the method of the present invention, the method further comprises the step of incubating the blood after taking blood from the animal and before serum is isolated from the blood, for example, for 4-72 hours at a low temperature, for example between 2 and 10 ° C, preferably between 4 and 8 ° C.

In a further preferred embodiment of the method of the present invention, the method comprises an additional step of lyophilizing serum prior to the irradiation step d). Lyophilization provides easier treatment of serum during irradiation and optimizes the absorption of radiation by serum components.

An additional aspect of the present invention is directly biologically active blood serum, which is obtained by the method of the present invention. It differs from the blood serum of the prior art, upon receipt of which the steps of the method of the present invention are not used, a specific therapeutic effect is proved for it, which the blood serum products of the prior art do not exhibit.

Since it was unexpectedly discovered that the biologically active blood serum of the present invention provides a specific therapeutic effect, for example, antiproliferative activity or antiepileptic activity, an additional aspect of the present invention is a pharmaceutical composition containing biologically active blood serum obtained in accordance with the method of the present invention. Such a pharmaceutical composition may further comprise one or more pharmaceutically acceptable diluents; carriers; excipients, including fillers, binders, lubricants, glidants, disintegrants and adsorbents; and / or preservatives.

The pharmaceutical composition of the present invention can be administered by various well-known routes, including oral and parenteral administration, for example, intravenous, intramuscular, intranasal, intradermal, subcutaneous and similar routes of administration. Parenteral administration and certain intravenous administration are preferred. Depending on the route of administration, various pharmaceutical preparations are required, and for some of them it may be necessary to apply protective coatings to the drug to prevent the breakdown of biologically active serum, for example, in the digestive tract.

Thus, preferably, the pharmaceutical composition of the present invention is obtained in the form of a syrup, infusion solution or injection, tablet, capsule, capsule-shaped solid tablet, capsule-shaped solid tablets, troches, liposomes, suppositories, plasters, bandages, capsules prolonged action, powder or sustained release drug.

Particularly preferred pharmaceutical forms are those suitable for injection use and include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for an individual prescription. In all cases, the final form of the solution or dispersion must be sterile and liquid. Typically, such a solution or dispersion includes a solvent or dispersion medium containing, for example, aqueous buffer solutions, for example, biocompatible buffers, ethanol, polyhydric alcohol, such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils. The biologically active blood serum of the present invention can also be obtained in liposomes, in particular for parenteral administration. The advantage of a liposome is an increase in the half-life from the bloodstream compared with the free drug and an even longer release of the drug contained in it.

Sterilization of solutions for infusion or injection can be accomplished by any technique accepted in the art, including, but not limited to, the addition of preservatives, such as antibacterial or fungicidal agents, for example paraben, chlorobutanol, phenol, sorbic acid or thimersal. Additionally, isotonic agents such as sugars or salts, in particular sodium chloride, may be included in infusion or injection solutions.

Sterile injectable solutions containing biologically active blood serum are prepared by combining the biologically active serum in a predetermined amount in a suitable solvent, if necessary, with the various ingredients listed above, followed by sterilization. If necessary, to obtain a sterile powder, the above solutions are subjected to vacuum drying or freeze-drying. Preferred diluents of the present invention are water, physiologically acceptable buffers, physiologically acceptable buffered saline or saline. Preferred carriers of the present invention are cocoa butter and vitebesole. Excipients that can be used in various pharmaceutical forms of biologically active blood serum can be selected from the following non-limiting list:

a) binders such as lactose, mannitol, crystalline sorbitol, dibasic phosphates, calcium phosphates, sugars, coarse crystalline cellulose, carboxyl methyl cellulose, hydroxyethyl cellulose, polyvinyl pyrrolidone and the like;

b) lubricants such as magnesium stearate, talc, calcium stearate, zinc stearate, stearic acid, hydrogenated vegetable oil, leucine, glycerides and sodium stearyl fumarates,

c) disintegrating agents such as starches, croscaramellose, sodium methyl cellulose, agar, bentonite, alginic acid, carboxyl methyl cellulose, polyvinyl pyrrolidone and the like.

Other suitable excipients can be found in the Handbook of Pharmaceutical Excipients published by the American Pharmaceutical Association, which is incorporated herein by reference.

An additional aspect of the present invention is the use of blood serum or a pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of a disease or condition that may be caused by an increase in the content of cyclic adenosine monophosphoric acid in the patient's brain.

Another aspect of the present invention is the use of a biologically active blood serum or pharmaceutical composition of the present invention for the manufacture of a medicament for improving the patient's nootropic, cognitive and / or educational skills and, in particular, for improving long-term memory. The applicability for this indication is based on the discovery that the blood serum or pharmaceutical composition of the present invention enhances the learning abilities of patients.

In addition, blood serum or a pharmaceutical composition can be used to treat seizures of any type, in particular for the treatment of epileptic seizures. In particular, in severe forms of epilepsy and in large epileptic seizures, administration of the biologically active blood serum or pharmaceutical composition of the present invention can prevent death, which in some cases is associated with severe seizures. In connection with the observation that the present invention can be used to treat epileptic seizures, it has also been found that the blood, serum or pharmaceutical composition of this invention can be used to treat nervous diseases, including, but not limited to, bipolar disorder, depression, anxiety disorders , epilepsy, Alzheimer's disease, Parkinson's disease, peripheral neuropathy, amyloid angiopathy of the brain, neurodegenerative disorders and spinal cord damage.

An additional aspect of the present invention is the use of blood serum or a pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of proliferative diseases and apoplexy. It was especially unexpected that the biologically active blood serum or pharmaceutical composition of the present invention did indeed exhibit a marked antiproliferative effect when tested on various tumor cell lines in vitro. In this regard, it seems that the biologically active blood serum or pharmaceutical composition of the present invention, in particular, is effective for the treatment of proliferative diseases, and the preferred proliferative diseases that can be treated using the present invention are selected from the group comprising gastrointestinal malignomas or colorectal tract, liver, pancreas, kidney, bladder, thyroid gland, prostate, uterine mucosa, cervix, ovary, m heel, testes, skin, oral cavity; melanomas; dysplastic oral mucosa; invasive cancers of the oral cavity; small cell and non-small cell lung carcinoma; breast tumors, in particular hormone-dependent breast cancers and hormone-independent breast cancers; transitional cell and squamous cell carcinoma; neurological malignancies, including neuroblastomas, gliomas, astrocytomas, osteosarcomas, meningiomas; soft tissue sarcomas; hemangiomas and tumors of endocrine glands, in particular pituitary adenomas, pheochromocytomas, paragangliomas, hematological malignancies, in particular lymphomas and leukemias.

Since the antiproliferative effect of the biologically active blood serum of the present invention or the pharmaceutical composition of the present invention was primarily created for various tumor cell lines, it is particularly suitable for the treatment of proliferative diseases that contain cells and / or tumor tissue containing cells similar to the lines tumor cells used in these experiments. Accordingly, preferred proliferative diseases that are treated with a biologically active blood serum or a pharmaceutical composition containing biologically active serum contain cells similar to the human T cell lymphoma cell line Jurkat, human B cell lymphoma cell Raji cell line, human melanoma cell line Bro , human cervical cancer cell line HeLa, human adenocarcinoma cell line MCF-7, osteosarcoma cell line Mg63, fibrosarcoma cell line HT1080, neuroblastoma cell line IMR-32, and HepG2 cell line HCC approx. In this context, the term "similar cells" refers to cells that have the same origin, for example a T cell, a B cell or a sequence of generations of nerve cells, as the corresponding cell line, and which carry a mutation in the same or functionally equivalent gene, and where this mutation contributes to the proliferative activity of the cell, for example, a mutation in p53, pRb, cdc 2, cdk 4, cyclin A, cyclin B, p21 ^ras , c-fos, c-jun, p107, p130, etc .; which carry the same or functionally equivalent exogenous gene, for example, human papillomavirus (HPV), E 6 or E 7, insertion of hepatitis B virus in the cyclin B promoter, etc .; or which contain the same chromosomal rearrangement or violation, that is, a deletion, multiplicity of chromosomes, etc.

Based on the results obtained in animals and cell culture, certain amounts of biologically active blood serum are preferred for the treatment of diseases and conditions for which blood serum and a pharmaceutical composition can be used. However, it should be understood that in order to identify a therapeutic effect depending on the corresponding condition, as well as on the corresponding patient, that is, depending on the severity of the disease or condition, the general health of the patient, etc., various doses of biologically active blood serum or pharmaceutical composition. Determining the appropriate dose is at the discretion of the attending physician. It is expected that the dosage of biologically active blood serum in the therapeutic method of the invention should be in the range from about 0.1 mg to about 200 mg of serum per kg of body weight. However, in a preferred application of the present invention, biologically active blood serum is administered to an individual who needs it in the range of 50 to 150 mg / kg body weight, preferably in the range of 90 to 100 mg / kg body weight. The duration of biologically active blood serum therapy varies depending on the severity of the disease, and the condition, and idiosyncrasy of each individual patient.

The following examples are provided to demonstrate preferred embodiments of the invention. Specialists in this field should take into account that the methods disclosed in the following examples are those for which the inventors have found that they function well in the practice of the invention and, therefore, can be considered preferred variants of this practice. However, in the light of the present description, specialists in this field should take into account that in the specific disclosed embodiments, many changes can be made without departing from the meaning and scope of the invention, as set forth in the attached claims. All references cited herein are incorporated by reference.

Brief Description of the Figures

Figure 1 - cytotoxic effect of two pharmaceutical preparations on Jurkat cells.

The cytotoxic effect on the Jurkat cell line of human T-cell lymphoma is displayed for two different pharmaceutical preparations containing different concentrations of biologically active serum in accordance with the invention. Jurkat cell viability is displayed on the Y axis as a percentage, while the amount of biologically active serum is indicated on the X axis in mg / ml.

Figure 2 - cytotoxic effect of two pharmaceutical preparations on Raji cells.

The cytotoxic effect on the Raji cell line of human B-cell lymphoma is displayed for two different pharmaceutical preparations containing different concentrations of biologically active serum in accordance with the invention. Raji cell viability is displayed on the Y axis as a percentage, while the amount of biologically active serum is indicated on the X axis in mg / ml.

Figure 3 - cytotoxic effect of two pharmaceuticals on Bro B-19 cells.

The cytotoxic effect on the Bro B-19 cell line of human T-cell lymphoma is displayed for two different pharmaceutical preparations containing different concentrations of biologically active serum in accordance with the invention. Cell viability of Bro B-19 is displayed on the Y axis as a percentage, while the amount of biologically active serum is indicated on the X axis in mg / ml.

Figure 4 - cytotoxic effect of two pharmaceutical preparations on HeLa cells.

The cytotoxic effect on the HeLa cell line of human T-cell lymphoma is displayed for two different pharmaceutical preparations containing different concentrations of biologically active serum in accordance with the invention. The viability of HeLa cells is displayed on the Y axis as a percentage, while the amount of biologically active serum is indicated on the X axis in mg / ml.

5 is a cytotoxic effect of two pharmaceutical preparations on MCF-7 cells.

The cytotoxic effect on the MCF-7 cell line of human T-cell lymphoma is displayed for two different pharmaceutical preparations containing different concentrations of biologically active serum in accordance with the invention. MCF-7 cell viability is displayed on the Y axis as a percentage, while the amount of biologically active serum is indicated on the X axis in mg / ml.

6 is a cytotoxic effect of two pharmaceutical preparations on IMR-32 cells.

The cytotoxic effect on the IMR-32 cell line of human T-cell lymphoma is displayed for two different pharmaceutical preparations containing different concentrations of biologically active serum in accordance with the invention. Cell viability of IMR-32 is displayed on the Y axis as a percentage, while the amount of biologically active serum is indicated on the X axis in mg / ml.

7 is a cytotoxic effect of two pharmaceuticals on HT1080 cells.

The cytotoxic effect on the HT1080 cell line of human T-cell lymphoma is displayed for two different pharmaceutical preparations containing different concentrations of biologically active serum in accordance with the invention. HT1080 cell viability is displayed on the Y axis as a percentage, while the amount of biologically active serum is indicated on the X axis in mg / ml.

Fig. 8 shows the cytotoxic effect of two pharmaceutical preparations on HepG2 cells.

The cytotoxic effect on the HepG2 cell line of human T-cell lymphoma is displayed for two different pharmaceutical preparations containing different concentrations of biologically active serum in accordance with the invention. HepG2 cell viability is displayed on the Y axis as a percentage, while the amount of biologically active serum is indicated on the X axis in mg / ml.

Fig.9 is a "bell-shaped" curve of the proliferative activity of cells treated with mitogens.

The lymphocyte proliferative activity curve was analyzed based on DNA biosynthesis activity with respect to mitogen concentration. DNA biosynthesis activity is measured by the number of pulses per minute of radioactivity and insoluble acid on.

Figure 10 - mitogenic activity of the pharmacological composition.

The mitogenic effect of the pharmaceutical composition on the DNA biosynthesis activity is shown in the range of substance concentrations from 0.1 to 100.0 mg / ml. DNA synthesis activity is estimated based on the amount of radioactivity included in the DNA.

11 - the effect of the pharmacological composition on MCF-7 cells.

The activity of DNA synthesis in the MCF-7 line of human breast cancer cells is shown in relation to the amount of biologically active serum that is part of the pharmacological composition.

Examples

Example 1

The method of obtaining blood chicken subjected to electroshock

To obtain chicken serum, chicken was treated with level II-III electric shock (electrical voltage 80-120 V, current 0.05 A, frequency 50 Hz, application time 3-4 s, on the head). Blood was taken from the carotid artery and further incubated at a temperature of 4-8 ° C for 18-24 hours in plastic flasks. After complete removal of blood clots, the flasks were centrifuged at 3000 rpm for 20-30 minutes. Serum was separated from blood clots and lyophilized under conditions known in the art. Lyophilized serum flasks were treated with an RZ-100-M instrument with 20-30 kGy, preferably within 25 kGy, using ⁶⁰ Co. as a gamma source. Treated serum was stored at a temperature between 4-8 ° C for later use.

Evidence of Stimulating Effect of Electroshock Chicken Serum

The experiments described below were performed using male Wistar rats with an average body weight of 280-300 g. Animals were randomly assigned to 4 groups of 10 rats each. The first group was injected with 1.0 ml of physiological saline. The second group was administered a dose of blood serum of 100 ± 5.0 mg / kg of body weight of the present invention in 1.0 ml of solution. After 30 minutes after the injection, the rats were beheaded. Rats of the third group were injected with 1 ml of physiological saline, and rats of the fourth group received biological active serum (100 ± 5 mg / kg body weight) in a solution in a volume of 1.0 ml. After 30 minutes after the injection, animals that attached the load to the tail (10% of the body weight of the rat) were placed in a basin with water (25 ° C). After the first symptoms of torment, the animals were taken out of the water and beheaded.

As samples for further analysis, we took the brain, heart, liver (organs subjected to extensive energy stress in the processes of extreme adaptation), as well as skeletal muscles (as an organ subjected to the most powerful effect). Tissue samples of each rat were weighed, cooled with isotonic NaCl and quickly frozen in liquid nitrogen. The total time elapsed between the application of "stress" and the final processing of the samples was at most in the range between 5-6 minutes.

The amount of adenosine triphosphoric acid, adenosine diphosphoric acid, and adenosine monophosphoric acid was determined in rat skeletal muscle. Nucleotides were separated by ion exchange column chromatography using Anionit Dowex 1. The determination of adenosine triphosphoric acid, adenosine diphosphoric acid and adenosine monophosphoric acid was carried out spectrophotometrically in the range of 256 nm (Hitachi-557 spectrophotometer). The energy potential was determined according to the following formula

(ATP-0.5ADP) / (ATP + ADP + AMP)

The determination of the amount of cyclic adenosine monophosphoric acid in the brain, heart, and liver was performed using a well-known radioimmunoassay with an Amersham detection device (UK).

The determination of labeled adenosine monophosphoric acid was carried out using methods known in the art, using a GS-8 scintillation counter.

results

The difference in the amounts of adenosine triphosphoric acid, adenosine diphosphoric acid and adenosine monophosphoric acid and an increase in energy potential are obvious, compared with the third group or the control first group, or the second group (p <0.05), are obvious, as when comparing the fourth group or with the control of the first group, or with the second group (p <0.05) and, in addition, when comparing the third and fourth groups with each other (p <0.05) (see also table 1).

Table 1
The amount of adenosine triphosphoric acid, adenosine diphosphoric acid and adenosine monophosphoric acid and the value of the energy potential in skeletal muscle tissue with and without biologically active serum Rat group The number of nucleotides (μmol / l / g tissue) Energy potential (conditional coefficient) ATPh M ± m ADPh M ± m AMPh M ± m I (saline control) n = 10 7.55 ± 0.19 (7.28-7.85) 0.95 ± 0.12 (0.79-1.18) 0.25 ± 0.08 (0.14-0.37) 0.917 ± 0.080 II (serum) n = 10 7.89 ± 0.12 (7.65-8.02) 1.12 + 0.11 (0.93-1.27) 0.14 ± 0.05 (0.08-0.25) 0.923 ± 0.125 III (saline + dipping) n = 10 1.34 ± 0.08 (1.19-1.49) 3.56 ± 0.17 (3.21-3.81) 0.78 ± 0.07 (0.65-0.93) 0.549 ± 0.014 IV (serum + dipping) n = 10 4.78 ± 0.17 (4.35-4.95) 2.55 ± 0.11 (2.36-2.72) 0.33 ± 0.09 (0.19-0.48) 0.790 ± 0.095

The amount of adenosine phosphoric acid determined during the active period and 30 minutes after moderate stress associated with the injection (control group I) confirms that under the influence of serum in the muscle tissue, the amount of adenosine triphosphoric acid and adenosine diphosphoric acid is mixed at the upper norm, while as the amount of adenosine monophosphoric acid decreases.

These results can be interpreted so that biologically active serum leads to an increase in energy potential in muscle tissue. Calculation of energy potential confirms this trend.

Under the influence of extreme dipping stress in the third group (saline + dipping), a decrease in the content of adenosine triphosphoric acid and an increase in the content of adenosine diphosphoric and adenosine monophosphoric acid in relation to the control group were determined.

Under extreme stress of rats of the fourth group (serum + dipping), the general trends in the amounts of adenosine triphosphoric, diphosphoric and monophosphoric acids observed with the third group remained similar, however, the amount of adenosine triphosphoric acid remained significantly large, and in calculating the energy potential, an increase in the energy potential by 43 % (p <0.05) compared with the third group.

A comparative analysis of cyclic adenosine monophosphoric acid in the tissue of the heart and liver, as well as in the brain tissue of rats of the first and second groups showed that, under the influence of serum, an increase in the amount of cyclic monophosphoric acid can be detected in the brain tissue (p <0.01), while in the tissue of the heart and liver, a significant difference compared with the control group was not determined (see table 2).

table 2
Amount of cyclic adenosine monophosphoric acid AMP in the tissue of the brain, heart and liver after administration of biologically active serum Rat group The amount of cyclic adenosine monophosphoric acid (pmol / l / g untreated tissue) brain M ± m heart M ± m liver M ± m I (saline control) n = 10 3.95 ± 0.58 (3.32-4.55) 3.07 ± 0.11
(2.98-3.32) 2.75 ± 0.18
(2.47-2.99) II (serum) n = 10 4.58 ± 0.23 (4.26-4.85) 3.26 + 0.16
(3.02-3.47) 2.98 ± 0.99
(2.95-3.12) III (saline + dipping) n = 10 0.82 ± 0.13 (0.66-1.05) 0.49 ± 0.17
(0.32-0.78) 1.01 ± 0.07
(0.86-1.14) IV (serum + dip) n = 10 1.58 ± 0.13 (1.38-1.76) 0.83 ± 0.14 (0.56-0.96) 1.27 ± 0.08
(1.12-1.41)

Under the influence of extreme stress, in all tissues of rats of the third group (saline + dipping) and the fourth group (serum + dipping), a significant decrease (p <0.05) in the amount of cyclic adenosine monophosphoric acid was observed. However, this decrease decreased under the influence of biologically active serum. Accordingly, the amount of adenosine monophosphoric acid in the brain tissue was 92% larger than the corresponding amount in the third group (p <0.05), 96% large in the heart tissue and 25.7% large in the liver tissue.

Therefore, the serum in accordance with the present invention, treated with electric shock and γ-radiation, increases the energy potential in the skeletal muscle of rats, increases the amount of cyclic adenosine monophosphoric acid in the brain tissue both during the resting phases and during extreme physical stress, and contributes to an increase in the content of cyclic adenosine monophosphoric acid in the tissue of the heart and liver after physical stress in rats.

Example 2

Long-term memory

150 male Wistar rats were used in this experiment. All rats were pre-tested using the classic "open field" test and were divided according to activity into three groups: active, with medium activity and passive.

To create a reflex of the situation, rats were placed in a cylinder, which was located in a pool regulated by a thermostat. The cylinder was placed on three supports, and it allowed animals to dive under the lower edge of the cylinder to get to a platform located outside the cylinder.

The latent period for the first experiment was determined, as well as the latent period of the final solution of the “release” problem from a closed room and from water.

When the reflex was established, the rats were divided into three groups:

- “quickly emerging” - those rats that could find a way out within the first minute;

- “slowly emerging” - those rats that have just begun to look for a way out after 2-3 minutes;

- rats that did not want to solve the problem within 10 minutes.

Biologically active serum was injected into the abdominal cavity 30 minutes before the start of the experiment at a dose of 100 mg / kg body weight. Control animals were injected with 1 ml of physiological saline. The next day, the behavior of the reflex of the situation was tested and subsequently the rats were given a break of 40 days.

In accordance with this method, 120 rats were trained - 60 control rats and 60 rats receiving biologically active serum. Rats were preliminarily divided into three groups, as indicated above, that is, active, with medium activity and passive (20 animals in each group), in addition, for these analyzes they were divided into “fast” and “slowly” emerging.

results

Some rats did not exhibit a “diving reflex” at all (Table 3). In the control group, six of the active rats and rats with medium activity and 12 animals of the passive group did not exhibit a “diving reflex” at all. On the second day, this amount remained the same for active rats and for rats with medium activity, but decreased for passive animals to 7. After 42 days, the same 11 passive group rats refused to dive under the edge of the cylinder (1 rat died during the experiment).

Among the active rats receiving biologically active serum, there was not a single rat that refused to solve the problem on the second day. Of all the rats with medium activity, three refused to solve the problem on the first day, but, nevertheless, they solved it on the second day and retained this ability even after 42 days. Among the passive rats receiving serum, 4 rats showed no desire to solve the problem, but on the second day this number decreased to 2 and remained constant even after 42 days.

Table 3
The number of rats that did not show a "diving reflex" Test animal 1 day (24 hours) one 2 42 Active rats and rats with medium activity The control 6 6 6 Serum 3 0 0 Passive rats The control 12 7 11 ∗ Serum four 2 2 ∗ one rat died during the experiment

In the fast-emerging group, a single injection of serum in the 40-day interval of the experiment did not show a significant change in maintaining the reflex of the situation.

For the “slowly diving in” there was a significant, from the point of view of statistics, ability to maintain the ability to quickly find a way to avoid an extreme situation (Table 4). Thus, the reflex formation time on the second day decreased compared with the first day for both the control group and the test group. In the control group, these reflexes completely disappeared after 42 days, while in animals that received an injection of biologically active serum, the reflex was completely preserved. In addition, there was a difference in the time required for the reflex, which was 2-3 times greater (p <0.01) in control rats.

Table 4
The effect of blood serum on the formation and maintenance of the reflex of the situation in "slowly emerging" (c) Test animal 1 day (24 hours) one 2 42 LP-1 (latent period) LP-2 LP-1 LP-2 LP-1 LP-2 Active group The control 125.7 + 25 176.7 + 19.6 45.5 + 9.9 59.0 + 8.8 37.5 + 30.0 181.5 + 15.5 Serum 111.3 + 39.7 164.8 + 33.5 55.3 + 15.6 68.4 + 18.8 3.8 + 29.3 55.7 + 37.28 Medium activity group The control 144.8 + 21.8 191.2 + 16.3 49.6 + 11.2 62 + 14.3 3.8 + 24.0 19.2 + 9.8 Serum 118.2 + 71.6 178.6 + 76.8 3.8 + 50.3 77 + 54.1 9.2 + 28.8 54.6 + 39.7 Passive group The control 53.3 + 25.7 189.6 + 12.6 53 + 15.0 66.6 + 8.0 149.3 + 24.7 196.0 + 11.1 serum 18.2 + 71.6 178.6 + 76.8 63.8 + 50.3 77 + 54.1 39.2 + 28.8 54.6 + 39.7

In the formation and long-term maintenance of information, a significant role is attributed to H-cholinergic mechanisms. 30 animals, which were trained to dive under the "bell" for five days, were divided into three groups.

Physiological solution of cytisine (H-cholinergic antagonist of the brain) was administered at a dose of 1 mg / kg of body weight into the abdominal cavity 30 minutes before testing the animals of the first group (ten rats).

The animals of the second group (ten rats) were injected with biologically active serum at a dose of 100 mg / kg of body weight and after 30 minutes a solution of cytisine at a dose of 1 mg / kg of body weight was administered.

The third group of animals (control — ten rats) received 1 ml of saline injection.

The complex behavior of the animals was tested in accordance with the above method after 48 hours after the injection of the indicated substances. After 48 hours after the use of cytisine, biological tests showed a marked slowdown of the “release reaction” from the closed room, namely a 3-fold slowdown compared to control animals.

The administration of serum prior to cytisine injection not only reduced the effect of this antagonist, but also led to an increase in the “release” reaction by 20% compared with the control group, and the full effect of biologically active serum exceeded the antagonist effect by 5 times.

Table 5
Latent periods of the rats reaction to the situation in connection with the introduction of cytisine and biologically active serum (s) Time parameter Control (third group) The introduction of cytisine (first group) The introduction of serum + cytisine (second group) After 48 hours 13.33 ± 0.58 40.0 ± 14 8.3 ± 5.5 <0.05 > 0.05 <0.05

Based on the results, it was determined that brain N-choline receptors play a role in restoring the “release reaction” in connection with modeling complex animal behavior and that biologically active serum prevents learning impairment.

Thus, biologically active blood serum stimulates the formation of long-term memory and especially exhibits this effect in animals with a delayed release reaction from a closed room and from water. In addition, it appears that the H-cholinergic mechanisms of the brain play an important role in maintaining long-term memory, and the biologically active serum of the present invention can resist the action of substances negatively affecting the H-cholinergic mechanisms of the brain.

Example 3

Epilepsy

It is known in the prior art that camphor in a toxic dosage leads to hyperactivation of the motor region of the central nervous system, which in turn causes the development of tonic spasms. In this regard, camphor is used along with corazole on model animals to cause spasms. Depending on the doses of camphor administered, it is possible to cause all varieties of small and large epileptic seizures, including large epileptic seizures.

The experiments investigated the effect of biologically active serum on epileptic activity, which was induced by injection of camphor into the abdominal cavity of rats. In this experiment, 40 male Wistar rats weighing 190-210 were used. A solution of camphor oil (20%) was injected into the abdominal cavity in the amount of 0.25 and 0.5 ml (using 20 rats). Serum was administered at a dose of 100 ± 5.0 mg / kg body weight (using 20 rats).

Experts observed and evaluated seizures. The following parameters were evaluated. The latent period of the reaction, the type of spasm reaction (tonic-clonic, major and minor seizures), the duration of the epileptic seizure and the time intervals between them, loss of normal mobility and the final result (death of the animal or recovery from a pathological condition). The results are summarized in table 6.

Table 6
The effect of biologically active serum on experimentally induced epilepsy Doses of camphor Epileptic seizures Control group (camphor) Test group (camphor + serum at a dosage of 100 mg / kg) 0.25 The occurrence of tonic spasms 7'20 '' ± 10 '' 5'30 '' ± 15 '' The occurrence of clonic spasms in the form of large epileptic seizures after 12'30 '' ± 32 '' 8'45 '' '' ± 28 '' The average number of attacks One in 6'-7 ' One in 8'-9 ' Seizure duration 70'-80 ' 15 '' - 20 '' The state of the animal after 48 hours All survived All survived 0.5 ml The occurrence of tonic spasms 5'40 '' ± 30 '' 5'00 '' - 32 '' The occurrence of clonic spasms in the form of large epileptic seizures after 8'30 '' ± 27 '' 8'40 '' The average number of attacks One in 5 ' One in 7 ' Seizure duration 90 '' - 120 '' 25 '' - 30 '' The state of the animal after 48 hours 60% died All survived

Biologically active serum, administered at a dose of 100 mg / kg during the latent period of epileptic seizures caused by camphor injection, reduced all symptoms of seizure activity; the latent period of seizure activity increased, less severe clonic spasms were observed and normal mobility was maintained, and additionally, serum treated with electroshock prevented the death of animals in the model of severe epilepsy (all animals survived with the introduction of 0.5 ml of 20% camphor solution).

Example 4

The effect of blood serum of the present invention on the proliferation of human cells

The blood serum of the present invention is lyophilized chicken blood treated with level II-III electroshock and γ-radiation. Biologically active serum was used in two types of preparations: (i) serum resuspended in water at a concentration of 100 mg / ml (test fraction 1), and (ii) the supernatant of a suspension of 100 mg / ml biologically active serum in water after three minutes of centrifugation of the suspension at 10000 × g (test fraction 2).

Cell culture

Jurkat and Raji cells and human peripheral blood lymphocytes were cultured in plastic tissue culture plates (Nunc or Falcon) in 1640-RPMI medium (Sigma) containing 10% fetal calf serum (FCS, Gibco), 100 units / ml penicillin and 100 μg / ml streptomycin at a temperature of 37 ° C, at 5% CO ₂ and 95% humidity. Cell lines Bro, HeLa, MCF-7, Mg63, HT1080, IMR-32, as well as HepG2 were cultured as described above, but using DMEM (Sigma) medium instead of 1640-RPMI medium.

Isolation of mononuclear leukocytes (ML) in accordance with the Boyum method

Mononuclear leukocytes were isolated according to the method described by Boyum A. (Isolation of mononuclear cells and granulocytes from human blood, 1968, Cand. J. Lab. Clin, Invest, 120 (97): 9-18).

15 ml of ficoll-pack solution was placed in each of two conical tubes (Falcon), and then 25 ml of blood diluted twice in phosphate-buffered saline (PBS) was added to ficoll. Then the tubes were centrifuged at 400 × g and 20 ° C for 30 minutes in a bucket-rotor. The upper phase containing plasma was not used.

Mononuclear leukocytes (ML), concentrating at the interface between the plasma and the separation medium, were carefully aspirated with a pipette and collected in a centrifuge tube. After this, the cells were washed twice with PBS by centrifugation of the cells at 250 × g for 10 minutes and, ultimately, suspending them in the culture medium. This fraction contained 10-30% of monocytes and 80-90% of lymphocytes, which are called “lymphocytes” below. One part of the lymphocytes was used to study the mitogenic activity of the biologically active serum of the present invention and the other to study proliferation. For this purpose, phytohemagglutinin at a concentration of 20 mg / ml was added to the cell suspension.

Assessment of the activity of synthesis of deoxyribonucleic acid (DNA) in cells

To assess DNA synthesis, ³ H-thymidine was added to the cells, and the number of pulses of the incorporated radiation was estimated in the insoluble acid fraction. Briefly, lymphocytes were incubated in 96-well plates with 200 μl of medium containing 200-800 × 10 ³ cells. Different concentrations of biologically active serum of the present invention were added to each well. Two hours prior to completion of incubation, ³ H-thymidine (1 μCi / well, 40 μCi / mmol / L) was added. For radiometric analysis, cells were collected on filters using an automatic cell collection device. Soluble acid products were washed with 5% trifluoroacetic acid (H ₂ O) and the radioactivity of the detained substances was measured using a scintillation counter. DNA biosynthesis activity was measured in pulses / min.

Determination of cell survival after incubation with various substances using the MTT test

The MTT test was performed as described by Mosmann T. (Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays (1983) J. Immunol Meth. 65: 55-63).

To perform the test, cells were harvested in a logarithmic phase (adhesive cells were harvested when they filled approximately half of the tissue culture plate). They were placed in the growth medium in the Goryaev chamber, counted and then resuspended in the medium at a concentration of 50-100 × 10 ⁶ / ml. Cell solutions were placed in 96-well plates after various concentrations of test fractions were added in a total volume of 100 μl. To count viable cells, 50 μl of a solution of 3-4,5-dimethylthiazol-2-yl) -2.5 diphenyltetrazole (MTT) bromide in culture medium was added to each of the various 96-well plates after completion of incubation. To prepare the MTT solution, 1 ml of mother liquor MTT was mixed with 4 ml of culture medium. MTT stock solution was prepared by dissolving MTT in PBS (PBS contains 0.01 mmol / L sodium phosphate buffer, pH 7.4, with 0.15 mmol / L NaCl) with a concentration of 5 mg MTT / ml, followed by filtration through a filter with a pore size of 0.45 microns. The mother liquor was stored at + 4 ° C for up to one month. After adding the MTT solution, the tissue culture plates were further incubated for 4 hours in an incubator under identical conditions. After removing the nutrient medium of the next stage by suction using a pump, 150 μl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the blue formazan crystals formed and the optical density of the solution in each well was recorded with a multichannel spectrophotometer with a microplate reader at a wavelength of 540 nm (Labsystem). Cell viability with respect to the concentration of the added test fraction 1 and 2, respectively, is indicated as a percentage of the survival of the control cells. Data was processed using Origin software.

Results and discussion

The results are shown in figures 1-8. From these results, we can conclude the following: a high concentration of biologically active serum of the present invention (2.5-20 mg / ml) had an inhibitory effect on all cell lines, however, the response of cells from different types of tissue to test fractions 1 and 2, respectively, varies. The dose of IC ₅₀ (the concentration of the substance, which leads to 50% inhibition of cells) varies significantly between cell lines from 2.2 (Jurkat) to> 20 mg / ml (Mg63) for the test substance 1 and from 3.6 (peripheral blood lymphocytes) up to> 20 mg / ml (Mg63 and HeLa) for the soluble blood serum fraction of the present invention (test fraction 2). For all cell lines, the toxicity of the starting material (test fraction 1) was higher than the toxicity of the soluble substance (test fraction 2). However, it should be noted that the toxicity observed for individual cell lines was almost identical for both test fractions (Mg63, Rajii, peripheral blood lymphocytes) and also for others (Jurkat, MCG-7, IMR-32), moreover, there was a difference IC ₅₀ 2-3 times. The sensitivity of the cells to the test fractions compared to the conventional cancer treatment, doxorubicin, is shown in Table 7.

Table 7
Sensitivity of cell lines tested with doxorubicin Cell line IC ₅₀ , mmol / ml Jurkat one hundred Raji twenty Bro b-19 - ∗∗ Hela 400 Mcf-7 150 Mg63 - ∗∗ HT1080 - ∗∗ IMR-32 four Hepg2 one hundred Peripheral Lymphocytes 40 Comments:
∗ - mmol / l = 0.58 mg / ml
∗∗ - sensitivity to doxorubicin not tested

In addition, it was observed that in the concentration range from 0.3 to 3 mg / ml, depending on the cell line and type of substance (i.e., test fraction 1 or test fraction 2), this agent had a stimulating effect on some cells. The stimulating effect of the serum of the present invention was observed for Jurkat, Raji, Bro B-19, Mg63, HT1080 and HepG2 cells. The stimulating effect was negligible (10, 20, 40%), but was present in both types of test substance. Stimulation was not observed in HeLa, MCF-7, JMR-32 cell line cells and in peripheral blood lymphocytes. The stimulating effect of the test substance in the first form was observed at a much lower concentration than in the soluble fraction (test fraction 2). Perhaps the reason for this stimulation is not stimulation of proliferation per se, but rather an increase in the respiration rate of the cells, which could also be recorded using the MTT method used to assess cell viability. The question of whether the observed stimulatory effect was due to stimulation of proliferation or due to an increase in respiratory rate required some further study.

When studying the interaction between various substances with competent immune cells, essentially, it is necessary to discuss at least two issues:

1. Does the test substance have mitogenic activity, that is, does it have the ability to stimulate the proliferation of lymphocytes (in this case, an increase in the amount of the substance usually leads to an increase in the biosynthesis of lymphocyte DNA, which can be assessed by incorporating ³ H-thymidine)?

2. Does the test substance have a toxic effect (this question is usually solved by inhibiting the proliferation of lymphocytes, analyzed by the amount of ³ H-thymidine included, or by using vital MTT-type dyes on lymphocytes that were previously stimulated with mitogens)?

In addition, it should be noted that non-activated lymphocytes do not divide in the culture and that the degree of proliferation only increases with an increase in the number of mitogens added to the culture medium. This is expressed in an increase in radioactively labeled DNA.

The effect of mitogens on lymphocytes in relation to the administered doses can be described by the so-called "bell-shaped curve" (see Fig. 9). The first segment of the bell-shaped curve reflects the range of mitogen concentrations, where an increase in mitogen leads to an increase in proliferation (as measured by DNA biosynthesis) and where, in connection with this, there is a direct correlation between the mitogen concentration and the degree of proliferation. The second segment of curve (2) demonstrates the saturation effect, where a further increase in the concentration of mitogen does not lead to a further increase in the degree of proliferation, i.e., the mitogen has already exhibited the maximum effect. Cytotoxic activity has not yet been observed. Segment (3) shows the mitogen concentration range, where the mitogen exerts an increasing cytotoxic effect on lymphocytes.

The study of the mitogenic activity of the tested fractions 1 and 2 was performed in two experiments:

1. The subject of this experiment was the determination of the action of the tested fractions on inactive lymphocytes of human peripheral blood. For this purpose, the correlation of proliferative activity (i.e., action) with the administered doses was determined.

2. The subject of this experiment was to determine the effect of the test substance on the activity of DNA synthesis of lymphocytes activated by phytohemagglutinin (20 μg / ml FHA) in the second phase.

In the study of the substance, mitogenic activity was not observed, that is, in the range of substance concentrations of 0.1-100 mg / ml, stimulation of the biosynthesis of DNA of peripheral blood lymphocytes was not observed (Fig. 10).

With an increase in the dose of the tested fractions, the number of peripheral blood lymphocytes did not change significantly. When evaluating the effect of the test fractions on lymphocytes, which were subsequently activated with 20 mg / ml FHA, inhibition of activated lymphocytes by the test fractions was observed.

In experiments, it was determined that the test fractions inhibited the biosynthesis of DNA of stimulated lymphocytes at a concentration of 0.3 mg / ml (Fig. 11). However, the mechanism of action should still be determined. In this regard, additional experiments are important to explain the cytostatic and cytotoxic effects.

In order to determine how the results obtained in cell culture relate to human treatment, it is important to determine the concentration of the substance in various organs or tissues in animal experiments. In this context, it is important to investigate what differing concentrations of biologically active serum of the present invention can affect DNA synthesis in organs involved in lymphogenesis. Thus, it is important to determine the concentration of biologically active serum, in particular in the bone marrow, spleen and thymus of the mouse.

In addition to lymphocytes, the serum cytotoxic activity of the present invention was also investigated using cells from the MCF-7 cell line of human breast cancer in a monolayer. Due to contact inhibition, cell proliferation was not observed. In such a model, it is possible to determine the toxic effect of a substance, that is, it is possible to distinguish a cytotoxic effect from a cytostatic effect. Using this model, it was determined that the serum of the present invention exerted a cytotoxic effect at a concentration of 2.5 mg / ml, regardless of the fact whether the insoluble fraction of the serum of the present invention was removed or not, that is, regardless of whether the test fraction 1 was used or 2.

It is noteworthy that the toxic effect decreased markedly when the serum of the present invention was incubated with dividing cells, which is potentially due to the fact that the effect was measured after 24 hours after administration, and not after 72 hours after administration, in the presence of cell division (long incubation cells in a monolayer can lead to the death of control cells).

The above results demonstrate that the biologically active serum of the present invention has both a cytostatic effect and cytotoxic activity. However, cytotoxic activity was less pronounced, as the combination of cytotoxic and cytostatic activity, despite the fact that it was observed at the same concentration.

Combined blood serum from animals treated with level II-III electroshock and subsequently prepared biologically active substance increase the proliferation of Jurkat, Raji, Bro B-19, Mg63, HT1080 and HepG2 lines of human cells by 10-40% compared with the control group. In HeLa, MCF-7, JMR-32, and peripheral blood lymphocytes, the biologically active serum of the present invention showed significant inhibition of proliferation of all tested human cancer cell lines at higher dosages (2.5-20 mg / ml). The sensitivity of cells to the biological active serum of the present invention is much greater than the sensitivity to the widely used anti-cancer agent doxorubicin. In addition, when using DNA biosynthesis, as assessed by including ³ H-thymidine as a label, the substance of the present invention has both a cytostatic and cytotoxic effect.

Claims (24)

1. A method of obtaining a biologically active blood serum, comprising the steps of:
a) electrical stimulation of a non-human animal,
b) taking blood from the specified animal,
c) isolating serum from said blood; and
d) gamma irradiation of said serum, in which the absorbed radiation dose of said gamma irradiation is from 10 to 40 kGy. 2. The method according to claim 1, in which the non-human animal is selected from the group comprising mammals and birds. 3. The method according to claim 2, in which the bird is selected from the group comprising chicken, duck, goose, ostrich and quail. 4. The method according to any one of claims 1 to 3, in which, in step a), the head, neck, body and / or one or more limbs of the animal are subjected to (are) electrical stimulation, mainly the head. 5. The method according to claim 1, in which the electrical stimulation is performed during a time interval between 1 and 60 s, preferably between 1 and 30 s, and more preferably between 2 and 10 s. 6. The method according to claim 1, in which the electrical stimulation is performed with a voltage in the range between 50 V and 150 V, preferably from 80 V to 120 V, and more preferably between 110 V and 120 V. 7. The method according to claim 1, in which the electrical stimulation is performed with a current strength in the range between 0.01 A and 0.4 A, preferably between 0.02 A and 0.1 A, and more preferably between 0.04 A and 0, 06 A. 8. The method according to claim 1, in which the electrical stimulation is performed with a frequency in the range between 10 and 200 Hz, preferably in the range between 20-100 Hz and more preferably in the range between 45-55 Hz. 9. The method according to claim 1, in which the absorbed dose of gamma radiation is from 15 to 35 kGy, preferably from 20 to 30 kGy. 10. The method according to claim 1, wherein the gamma radiation source is selected from the group consisting of ⁶⁰ Co, ¹³⁷ Cs, ⁶⁷ Cu, ⁶⁷ Ga, ¹¹¹ In, ¹⁹² Ir, ^99m Tc and ¹⁷⁰ Tm. 11. The method according to claim 1, in which the method further comprises the step of incubating said blood before step c). 12. The method according to claim 1, wherein the method further comprises the step of lyophilizing said serum before step d). 13. The method according to claim 1, wherein said blood is arterial and / or venous blood. 14. Blood serum obtained by the method according to any one of claims 1 to 13. 15. A pharmaceutical composition comprising the blood serum of claim 14 and one or more pharmaceutically acceptable diluents; carriers; excipients, including fillers, binders, lubricants, glidants, disintegrants, adsorbents and / or preservatives. 16. The pharmaceutical composition according to clause 15, in which the composition is prepared in the form of a syrup, solution for infusion or injection, tablets, capsules, hard tablets in the form of capsules, troches, liposomes, suppositories, plasters, bandages, capsules, prolonged action, powder or preparation slow release. 17. The pharmaceutical composition according to claim 15 or 16, wherein the diluent is water, a buffer, a buffered saline or saline. 18. The pharmaceutical composition according to clause 15, in which the carrier is selected from the group comprising cocoa butter and vitebesole. 19. The use of blood serum according to claim 14 or the pharmaceutical composition according to any one of claims 15-18 for the manufacture of a medicament for the treatment of a disease or condition that may be caused by an increase in the content of cyclic adenosine monophosphoric acid in the patient's brain. 20. The use of blood serum according to claim 14 or the pharmaceutical composition according to any one of claims 15-18 for the manufacture of a medicament for improving cognitive and / or educational skills, in particular for improving long-term memory. 21. The use of blood serum according to claim 14 or the pharmaceutical composition according to any one of claims 15-18 for the manufacture of a medicament for the treatment of epileptic seizures. 22. The use of blood serum according to claim 14 or a pharmaceutical composition according to any one of claims 15-18 for the manufacture of a medicament for the treatment of apoplexy and proliferative disease selected from the group consisting of liver malignoma, cervical malignoma, breast malignoma melanoma, in particular hormone-dependent breast cancers and hormone-independent breast cancers; neuroblastomas, sarcomas of soft tissues and hematological malignancies, in particular, lymphomas and leukemia. 23. The use of the blood serum of claim 14 or the pharmaceutical composition of any one of claims 15-18 for the manufacture of a medicament for the treatment of a proliferative disease, wherein the proliferative disease contains cells similar to the human T cell lymphoma cell line Jurkat, lymphoma cell Raji line Human B cells, Human melanoma cell line Bro, human cervical cancer cell line HeLa, human adenocarcinoma cell line MCF-7, osteosarcoma cell line Mg63, fibrosarcoma cell line HT1080, neuroblastoma cell line IMR-32, and HepG2 cell line K lettuce hepatocarcinoma. 24. The use according to claims 19-23, wherein the drug is administered to the patient in an amount varying from 50 to 150 mg / kg body weight, preferably varying from 90 to 100 mg / kg body weight.

Images