RU2238756C1 - Agent stimulating reparation of damages and eliciting tissue-, organ- and stage-specificity and antiviral activity - Google Patents

Abstract

FIELD: medicine, biotechnology.

SUBSTANCE: invention relates to agents stimulating reparation of cell damages and can be used in therapy and cosmetology. Agent is prepared by isolation of fraction of low-molecular ribonucleopeptides with molecular mass 3.0-6.5 S (Swedberg's units)from nuclei of mammal cells. Reparative effect of agent is confirmed in biomodels and shown that agent elicits tissue-, organ- and stage-specificity, antiviral activity and changes an average half-time life of cells. Invention allows expanding region of using low-molecular ribonucleopeptides, to enhance biological effects of their effect and to obtain substances functioning in body for a long time without loss of ability to carry out the regulation of cell functions and to correct metabolism in different pathological disorders.

EFFECT: valuable biological and medicinal properties of agent.

11 tbl, 13 dwg, 14 ex

Description

The invention relates to medical and biological chemistry, cytology, molecular biology and genetics and can be used in studies of tissue differentiation and dedifferentiation, biosynthesis, replication and transcription with different functional effects on the body as a way of isolating the active RNA fraction in genetic engineering and as a method of regulating metabolism and biosynthesis, cell proliferation of certain organs in order to stimulate the repair of cell damage and restore damaged cell functions and organs, regulating the level of hormones and other metabolic regulators, activating adaptive responses and protective reactions of the body to allergens, viruses, microorganisms, harmful physical and chemical influences, normalizing impaired structures and functions caused by aging, and is intended to produce biological drugs that stimulate specific organ function, which is achieved by fractionation of cellular or nuclear nucleoproteins from the organs of cattle, selection of active organ-specific rib onucleopeptides-regulators (RNP), sterilization, lyophilization and packaging in ampoules, followed by use in experimental and medical veterinary medicine and cosmetics and for targeted developments in the medical field.

Most currently used drugs are certain natural metabolites or their synthetic analogues, i.e. links of the lower level of multifaceted structures and systems of metabolism, and act on one specific link of metabolism - protein or enzyme. To date, pharmacologists have learned to adjust the work of approximately 500 different proteins from more than 30,000 functioning in the body. However, it has been proven that many difficult to cure or even incurable diseases (diabetes, sclerosis, depression, obesity, etc.) are violations of 10 or more jointly functioning metabolic units, and for some of these units modern therapeutic drugs are ineffective. Naturally, it is almost impossible to adjust the operation of each link, and then adjust their joint functioning with the help of a limited set of highly specialized drugs. On the other hand, with the help of well-known systemic regulators of the whole organism (e.g. hormones), it is possible to stimulate, inhibit or cause systemic changes in the work of a huge number of different reactions, most of which are not related to the pathology that they are trying to cure in this way. The disadvantage of this approach is also that the excess of the hormone or metabolites regulated by it is perceived by the body as a signal to inhibit the synthesis of its own hormone and its products.

It is known that in normally functioning organs there are systems of regulators specific for each type of cell that synchronize the work of cells of a given organ and exchange information and interact with other organs and systems of cells. The evidence of the functioning of the organo-and stage-specific regulatory systems in the body is the known facts of humoral information exchange between cells, according to which the cells of some tissues in response to external influences, forcing them to adaptive rearrangements, are able to send other cells chemical signals about their physiological state and the commands necessary to correct functions. According to some authors, the most likely carriers in such cases are proteins and low molecular weight peptides, and according to others, nucleic acid molecules.

Obviously, at present, it is necessary to search for such regulatory substances that would ensure maximum preservation and, if necessary, enhancement, for example, of the endocrine capabilities or immune responses of certain cells, enhancement of reparative processes in damaged cells, shift of metabolic processes to values characteristic of normal healthy organism, that is, they would enhance the production of regulators in cases when it is weakened, and would reduce to normal when it is excessively enhanced. With the help of modern pharmaceuticals and medical preparations, it is not yet possible to obtain such effects.

In order to restore normal regulatory processes at the level of individual organs and systems of cells, applicants and some other researchers have been trying for many years to use procedures for transplanting pieces of tissue and cells into certain organs: transplantation of embryonic nerve tissue into the brain / Alexandrova M.A. et al. Transplantation of brain tissue in biology and medicine. M .: Nauka, 1993, p. 173. Vitvitsky V.N. Doct. diss. (1988) /, tissue of islets of Langerhans, adrenal glands and other tissues / Finke E. et. al. Transplat. Proc. V.23 (1), p.772 (1991) /, tissue of the parathyroid glands / Smith M.A. et. al. Am. Surg. V. 56, No. 7, p. 404 (1990) /.

The processes of normalization of organ functions and stimulation of reparations observed in these experiments can be explained by the action of compounds possessing the properties of inducers or depressors of certain regions of the recipient's cell genome. Our studies have shown that the most likely candidates for the role of such regulators are low molecular weight nucleopeptides (NNPs), mainly ribonucleopeptides (RNPs), which are found in the nucleus and in the cytoplasm of cells and sometimes make up a significant part (in some cases up to 7-10% nuclear RNA).

The ability of various drugs containing DNA, RNA, and NK-protein complexes to actively influence the functions of cells and cellular systems of the body as a whole has been repeatedly noted by different authors. Various methods are described for isolating such preparations (patent WO 97/28171/1996) and their use in cosmetics and medicine (patent US 04885157, 12/05/1989, patent US 05849517, 12/15/1998, patent WO 02/076490 A1, 2002). The positive effects of DNA preparations (300-12000 KD) containing up to 6% DNA are described in the treatment of burns and wound healing (patent RU 2099094, 6 A 61 L 13/32, A 61 K 9/70, 04/06/94). Antiviral and immunotropic effects of preparations of DNA and DNA-RNA hybrids of mol. masses of 300-500 KD isolated from animal raw materials (patent RU 2108797, 6 A 61 K 35/60, C 07 H 21, 04/09/94, patent RU 9403643, 6 A 61 K 35/60, 09/29/94). It is also known that the addition of RNA preparations to azidothymidine and interferon significantly reduces toxicity and enhances the antiviral effects of these substances (patent RU 2016572, A 61 K 31/70, 37/66, 22.03.88). The possibility of using riboxin, the main component of which is RNA, has been declared as a means of changing biological age, in particular rejuvenating the body and accelerating cell renewal (patent RU 93043514, 6 A 61 K 31/00, 33/14, 09/01/93).

However, as our studies and studies by other authors have shown, an important property of RNAs that are part of the described preparations is tissue, organ specificity and specificity depending on the stage of ontogenesis of the cells from which this RNA is derived (stage specificity). These properties, apparently, are partly possessed by Regeneresen preparations produced by the laboratory to them. prof. Dyckerhoff (Germany) / Regeneresen nach Prof. Dr. H. Dyckerhoff. Lab. Prof. Dr. H. Dyckerhoff. Koln, 1992 /. These drugs can be considered as distant analogues of the funds proposed by this patent. They are coarse RNA extracts from the organs of farm animals, to which a significant amount of RNA nucleotides of bacterial origin are added. Injectable drugs are used to specifically stimulate the metabolism in tissues. The procedure for isolation and purification of the drug does not include special techniques that allow you to separate fractions of nucleoproteins, which are more active than just RNA, and more stable.

The predecessor of the substance protected by this patent and a special case of the use of organ-specific regulators for the specific regulation of a specific function is the patent for an agent that stimulates hair growth published earlier by the applicants and the method for its preparation / patent RU 2144366, A 61 K 35/36, 03.03.1998 /, which describes a method for isolating nuclear RNA from hair follicles of young animals. The target product of this study is an RNA kit with a sedimentation constant up to 8 S, the bulk of which were molecules with a length of 100-250 nucleotides. The drug contains an admixture of protein up to 5% and DNA.

This patent protects substances isolated from the brain, liver, endocrine glands and immunocompetent tissues using more advanced methods of isolation. These substances also have a stimulating effect on the functions of the organs from which they are derived, and are also able to stimulate the repair of cell damage to these organs caused by both adverse external influences and pathological processes. In addition, it was shown that preparations isolated from the tissues of young healthy animals, when introduced to animals of a more mature age, are able to shift the metabolic rate to the values characteristic of younger individuals.

The main task solved by this invention is to expand the scope of low molecular weight nucleopeptides, to enhance the biological effects of their action by obtaining end products of higher quality than described in the prototype, to obtain substances that function in the body for a long time without losing the ability to regulate cell functions and adjust metabolism in various pathological disorders.

The solution of this problem is achieved due to the fact that a natural substance that is released from the cells and is a fraction of RNP is determined.

Technologies have been developed to isolate sufficient amounts of RNP from different organs and glands for pharmacological and clinical use.

Using the obtained substances, methods have been developed to stimulate the functions of different organs and normalize the functioning of organs in pathological disorders of their functions.

Data on the mechanism of action of RNP are discussed. When RNP is introduced into the body of the recipient, they selectively bind to the cells from which they are isolated, enhancing or weakening the work of individual genes and forcing those genes that are selectively activated by these RNPs to work actively in the cells. Since it is known that RNPs have poor species specificity, it is quite possible and permissible to use in clinical practice RNP isolated from animal tissues and organs as pharmacological agents.

The essence of the applicant's proposal is as follows.

The substance is a set of RNP fractions with a molecular mass determined by ultracentrifugation from 3 to 6.5 S (Swedberg units), which strictly corresponds to the average molecular weight parameters of the class of substances described in the literature as low molecular weight nuclear RNPs obtained by different authors with using different isolation procedures (Jian Gu and Ram Reddy. Nucleic Acids Research, vol. 25, No. 1, p. 98, 1997. Mazin, A.L. Molecular Biology. Volume 17, issue 4, P.755, 1983).

The total content of NK in our preparations is 90% or more, protein 0.5-5.0 weight%, impurities of DNA and high molecular weight RNA up to 100 weight% are present. The average molecular weights of substances obtained using different chemicals and the conditions of extraction described below, as well as the composition of impurities, differed very slightly, which makes it possible to assume that all the procedures for the isolation of RNP that we used lead to the same substances, having the same physico-chemical properties. Comparison of the biological activity of drugs obtained in different ways makes it possible to assume that the same substances are the active principle in all drugs.

The biological activity of RNP is determined by studying the effect of RNP preparations when administered to normal adult animals on the incorporation of labeled DNA precursors in vitro into cells and tissues and in vivo.

RNP is isolated from the nuclei of mammalian cells, which involves a number of mandatory technological steps. When isolating RNP, it is necessary to preliminarily separate and discard all cytoplasmic RNA in the process of isolating cell nuclei by centrifugation, followed by phenol-chloroform deproteinization, in which different nuclear RNPs are sequentially isolated in several stages, then they are purified by reprecipitation, treated with proteases, deproteinized again, high molecular fractions are removed, precipitated with chilled alcohol, dissolved in pyrogen-free water, sterilized by filtration, lyophilized and re-sterilized with new radiation exposure.

The method of application is reduced to sequential, multiple or simultaneous subcutaneous or intramuscular injections of NSAIDs in saline or in a solution of anesthetics (in particular, in novocaine solution), or in the form of enemas or cutaneous applications.

In more detail, the essence of the method for obtaining RNP from cell nuclei and their characteristics is illustrated by the following examples.

Method 1

The tissue of the liver of cattle is separated from the connective tissue membranes, blood vessels, fatty layers and washed from blood cells in physiological saline. 100 g of the thus treated tissue is homogenized at low temperature for 5 minutes in 1000 ml of a solution containing 0.01 M Tris-Hcl buffer pH = 8.5, 0.001 M calcium chloride, 0.001 M EDTA and 0.6% Triton X- 100. The homogenate is centrifuged at 800 g for 10 minutes. The supernatant is used to isolate the cytoplasmic RNP fraction, and the precipitate is washed in the same solution, but without Triton X-100, and suspended in 250 ml of sodium acetate solution containing 0.02 M Tris-Hcl buffer pH = 5.0, 0.001 M EDTA and 0.5% sodium dodecyl sulfate, then 350 ml of a phenol-chloroform mixture (1: 1) are poured onto it and heated for 10 minutes in a water bath at a temperature of 40 ° C. After cooling, the system is centrifuged at 5000 rpm for 20 minutes and the upper phase containing RNA is taken, 350 ml of a phenol-chloroform mixture (1: 1) are again poured onto it, heated and centrifuged. An equal volume of the same acetate buffer and a phenol-chloroform mixture are added to the interphase, heated at a temperature of 50 ° C, cooled and centrifuged. This procedure is successively repeated at 60, 70 and 80 ° C. The obtained phases of centrifugates are again deproteinized, removing most of the proteins, DNA and high molecular weight RNA that are removed by centrifugation from solutions after adding NaCl up to 1-2 M. 2.5 volumes of chilled ethanol are added to the obtained phases of the centrifugate, the precipitate is separated, washed again with ethanol sterilized by filtration, poured into ampoules and freeze-dried. Ampoules are sealed under vacuum and stored in the refrigerator.

In the same way, using the same solutions and heating at the same temperatures, fractions of cytoplasmic RNP are isolated from the supernatant after the first centrifugation.

The yield of the drug is 350-450 μg from 1 g of tissue. The resulting product after additional deproteinization contains 2-5% protein and more than 94.0% of low molecular weight RNA. The bulk of the impurities are high molecular weight RNA and DNA. The sedimentation constants of individual fractions were in the range from 3.0 to 6.5 S, and the main amount of active fractions in the range of 4-6.5 S.

When isolating preparations from other tissues, higher or lower tissue: ratios were used: buffer, other X-100 triton concentrations, and other heating temperature ranges. For preparations isolated from different tissues, the maximum biological activity was observed in the fractions isolated at different heating temperatures.

Method 2

The tissue of the kidneys of cattle is processed according to the scheme described in Example 1, and the cytoplasmic fraction is isolated. To 50 ml of material, add 150 ml of boiling 0.01 M Tris-Hcl buffer pH = 6.8, containing 2% sodium dodecyl sulfate and 2% isoamyl alcohol. The mixture is homogenized at 8000 rpm. Within 1 minute, bring the temperature to 90 ° C and immediately cool to zero temperature, draining it into a cooled container. The mixtures thus obtained are centrifuged for 10 minutes at 1000 g at 0 ° C, the supernatant is collected and 2 volumes of chilled ethanol are added to it. The precipitates are resuspended in 50 ml of a 67% ethanol solution, 1 ml of a 2 M sodium chloride solution is added to the suspension and centrifuged again. The last procedure is repeated several times. The precipitates are dissolved in 50 ml of distilled water, adjusted to pH = 7.0 and the solution is clarified by centrifugation for 1 hour at 25000 g at zero temperature. Sodium chloride was added to the supernatant, left for 30 minutes at a temperature of about 0 ° C, and high molecular weight RNA was precipitated by centrifugation. Low molecular weight RNA is precipitated from the supernatant by adding sodium acetate to the solution, followed by 2.5 volumes of ethanol.

The yield of the substance is 1.0-1.2 mg from 1 g of tissue.

The resulting product contains 2.9-4.2% protein and 90-92% low molecular weight RNA. The bulk of the impurities are high molecular weight RNA and DNA. The average value of the sedimentation constant is 3.5-6.5 S. The sedimentation constants of individual fractions were in the range from 3.0 to 6.5 S, and the main number of active fractions was in the range of 4.0-6.5 S, which indicates on the identity of these drugs with drugs obtained from cell nuclei by method 1.

Method 3

At the first stage, brain tissue is processed according to method 1 to obtain a fraction of cell nuclei. To an aliquot of the obtained nuclear material, 5 volumes of a solution containing 6 M guanidine isothiocyanate, 5 mM sodium citrate with pH = 7.0, 0.1 M beta-mercaptoethanol and 0.5% sarcosyl are added. In this medium, the material is homogenized at 4000 rpm for 2 minutes and 1 g of cesium chloride is added for every 2.5 ml of homogenate. The homogenate is layered on a 1.2 M-5.7 M gradient of cesium chloride in 0.1 M EDTA at pH = 7.5 in centrifuge tubes. The system is centrifuged for 20 hours at room temperature at 30,000 rpm. The supernatant is discarded, and the RNA pellet is dissolved in sodium chloride solution, adjusted to pH 7.0, cooled to zero temperature, and high molecular weight RNA is precipitated by centrifugation. Low molecular weight RNA fractions are precipitated from the supernatant with 2.5 volumes of chilled ethanol and purified by reprecipitation from ethanol.

The yield of the substance is 100-210 μg from 1 g of tissue.

The resulting product contains 1.2-3.5% protein and 94-96.5% of low molecular weight RNA. The bulk of the impurities are high molecular weight RNA and DNA. The sedimentation constants of individual fractions were in the range from 3.0 to 6.58, and the main number of active fractions was in the range of 4.0-6.5 S, which indicates the identity of these drugs with drugs isolated by other methods.

The study of the properties of RNP as regulators of biosynthetic processes, cell proliferation, and correctors of the structure and functions of different organs and tissues was carried out in several main directions. At the first stage, using compensated RNPs described by these methods, the compensatory-restoration processes and damage repair processes were regulated. The experiments of this series are more convenient to carry out on tissues and cells with weak regeneration, for example, on fully differentiated neurons of the cerebral cortex, which, as a rule, are unable to divide and are completely degenerated in response to hypoxia, trauma and other influences.

Example 1. The effect of RNP on damaged neurons.

Damage to adult neurons in these experiments was achieved with the help of hypoxia, local injuries or the introduction of 10% NaOH, which, judging by the cytological data, caused the death of up to 36% of nerve cells in the cortex, hippocampus and other parts of the brain. The first series of experiments was conducted on 120 normal adult female Wistar rats. Hypoxia was used as a factor damaging neurons, and the blood-brain barrier (BBB) was opened with 10% NaCl, 35 days after hypoxia, when the BBB was completely restored. To open the BBB, rats were injected with a hypertonic 10% NaCl solution at a dose of 0.7 ml per 100 g of body weight for 3 minutes and RNP was injected intraperitoneally after 30 minutes. In the experiment, RNPs isolated from cattle brain tissue were used according to the method described above as Method 3. The preparations contained 94-96.5% of low molecular weight RNA, the average value of sedimentation constants was 3.0-6.5 S. NaCl injections and RNP was performed 3 more times at intervals of 1-2 days. After 42 and 162 days after hypoxia, the rats were injected with ³ N-leucine at a dose of 7 μCi / g body weight (W.A. 2460 TBq / mol) and the animals were decapitated an hour later. For biochemical analysis, 4-5 rats were examined in each group. Fractions enriched in neurons were isolated from the cerebral cortex of each rat according to the method of Freys / Freysz RBJNeurochem. 1968, v.15, No. 4, p.307). The amount of protein in the fractions was determined spectrophotometrically using an Ultrospec 4056 instrument (LKB, Sweden), and the amount of leucine included in neurons was calculated on a Magk-1 scintillation counter (USA).

The following series of animals were examined: norm (H) - intact rats, norm + NaCl (HNa), norm + NaCl + RNP (HNaP); hypoxia (G); hypoxia + NaCl (CNa); hypoxia + NaCl + RNP (GNaP).

The results of a radiometric study of the fractions enriched in neurons and glia are shown in FIGS. 1 and 2. The effect of 10% NaCl on hypoxia-affected neurons is an additional strong decrease in protein synthesis. Subsequent administration of RNP shifts the indicators characterizing protein synthesis towards the values characteristic of a normal organism. The tendency toward normalization of protein synthesis under the influence of RNP is apparently more characteristic of glia.

Our preliminary studies showed that RNPs isolated from the brain of younger animals are more active in stimulating the repair of neuron damage, in the next series of experiments we used RNPs isolated from the brain of cattle embryos by the method described above (Method 3). RNP preparations from the brain of embryos contained 94-96.5% of low molecular weight RNA, the values of sedimentation constants were determined as 3.0-6.5 S.

In this experiment, 100 Wistar female rats were used, 60 of which were subjected to a session of hypoxic hypoxia. 21 rats died while the rest tested cytologically dystrophy up to 36% of cortical neurons. Control and hypoxic animals were injected intramuscularly with RNP isolated from brain cells (125 μg in 1 ml of physiological saline, 1, 3, 6, and 7 days after hypoxia). 8 days after hypoxia, rats were injected with ³ N-leucine at a dose of 7 μCi / g body weight (U.A. 2460 TBq / mol) and the animals were decapitated an hour later. For biochemical analysis, 4-5 rats were examined in each group. Fractions enriched in neurons were isolated from the cerebral cortex of each rat according to the method of Freys / Freysz RB, J. Neurochem. 1968, v.l5, No. 4, p.307). The amount of protein in the fractions was determined spectrophotometrically using an Ultrospec 4056 instrument (LKB, Sweden), and the amount of leucine included in neurons was calculated on a Magk-1 scintillation counter (USA). The resulting radioactivity values are shown in figure 3. In the experiment there were the following series: norm (N) - intact rats; normal + RNP (HP); hypoxia (G); hypoxia + RNP (GR). A biochemical study showed that the effect of hypoxia leads to a decrease in protein synthesis in neurons in the early (8 days) terms of the experiment. Intramuscular injection of RNP isolated from embryonic brain cells into animals significantly increases protein synthesis in neurons of animals undergoing hypoxia.

In general, the results of experiments in this series indicate that the administration of RNP preparations extracted from brain cells to animals subjected to hypoxia and the action of hypertonic NaCl solutions significantly restore biosynthetic processes, shifting the corresponding indices to the values characteristic of a normal adult organism. The described changes in biosynthesis occur immediately after the introduction of RNP and persist for a long time - up to 4 months or more. Especially significant effects were observed when using in experiments RNP isolated from cells of the developing brain of embryos.

Example 2. The effect of RNP on damaged testis cells.

We studied the synthesis of DNA and proteins in the cells of the testes of animals exposed to non-lethal doses of gamma radiation, and the change in these processes under the influence of RNP.

In the experiment we used RNP isolated from cattle testicles according to the scheme described above (Method 2). The resulting product, regardless of the tissue from which it was isolated, contained 2.9-4.2% protein and 90-92% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs. Traces of DNA are identified. The sedimentation constants of individual fractions were in the range from 3.0 to 6.5 S, and the main amount of active fractions was in the range of 3.5-6.5 S.

The selected model made it possible to achieve much more than with senile changes in dysfunction and cell damage.

The experiment was carried out as follows: 40 adult Wistar male rats weighing 300-340 g were divided into 5 equal groups. The first 8 rats were left as a control, and all the rest were exposed to a single dose of gamma radiation at a dose of 700 rad. Irradiation was carried out on a GUPOS unit with a source of 137Cs (dose rate 4.7 Gy / min) at 20 ° C. 8 animals from the irradiated group made up the 2nd control group, and animals of the other three groups after irradiation were treated with RNP preparations from the testes. The course of treatment included 7 injections of RNP according to the scheme we developed, which is described in more detail below in experiments on the repair of the effects of gamma radiation using RNP. After 30 days, to establish a stable metabolic rate in the irradiated animals, all animals were decapitated and thin sections of tissue were prepared from testes for in vitro studies. 200 mg of tissue was placed in 2.0 ml of Hanks solution, to which up to 0.1 M HEPES and either 20 μCI ³ N-thymidine (W.A. 59 Ci / mM) or 20 μCi ³ H-leucine (W. A. 40 Ci / mM) and incubated for 120 min at 37 ° C with gentle stirring with shaking. Then tissue sections were washed by centrifugation from unlabelled labeled precursors and planted on nitrocellulose filters, adding an equal volume of chilled 10% TCA to the homogenates and washing the filters with 5% TCA and 80% ethanol. The radioactivity on the filters was calculated on a LS-6800 BECKMAN counter using a scintillator for heterogeneous counting. The results of this experiment are presented in Table 1 as the intensity of label inclusion with the entire mass of DNA or organ protein. This is due to the fact that 30 days after intensive gamma irradiation of animals, a decrease in protein and DNA synthesis in testis cells occurs against the background of a sharp decrease in organ weight (from 3.14 to 1.18 g) and the appearance of softness and sagging tissue.

Treatment of animals with RNP preparations from the testes of cattle increased the level of biosynthesis of DNA and proteins in the cells of the testes of irradiated animals. These changes occur against the background of some normalization of organ weight. The results of this experiment indicate the ability of RNP preparations isolated from the testes to normalize the biosynthetic processes in the tissues of the testes, similar to the experiments described previously (example 1). Note that the normalization of biosynthesis under the influence of RNP is quite effective in the case of damage caused by hypoxia, as well as damage caused by radiation.

The following experiments were performed to correct the functions of the immune system using NNP. We conducted this series of experiments on RNP preparations from immunocompetent organs: thymus, spleen, and bone marrow.

Example 3. The effect of RNP from immunocompetent tissues on the number of deaths in animals exposed to lethal doses of gamma radiation.

In this series of experiments, RNPs isolated from the small intestine, bone marrow, thymus and spleen of cattle were used according to the scheme described above (Method 1). Within the limits of the determination error, all preparations contained 2-5% protein and more than 94% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs; traces of DNA have been identified. The sedimentation constants of the individual fractions were in the range from 3.0 to 6.5 S. The preparations from which organs were used in each particular case, it was noted in the description of the results. We studied the effectiveness of correcting the functions of the immune system using RNP by reducing the number of deaths resulting from the introduction of RNP to animals exposed to high doses, and by a more specific criterion - changing the biosynthesis of DNA, RNA and protein, as well as the proliferation of cells of the immune system.

The work of the 1st stage of this series of studies was performed on adults weighing 150-200 g, Wistar rats. The animals were irradiated with a GUPOS apparatus and a 137 Cs radiation source with a power of 4.7 Gy / min. The following drawing (figure 4) demonstrates the ability to change the course of development of pathological syndromes of gamma radiation, acting with the help of organospecific RNP directly on different parts of the immune system. The drug obtained from the thymus, which positively affects the number of T-lymphocytes in the blood, can also significantly increase the percentage of animals surviving after irradiation. Adding RNP from the spleen to the thymus preparation significantly enhances the therapeutic effect. Thus, this experience demonstrates the advantages of using mixtures of RNP isolated from different organs for the treatment of radiation sickness. When animals were irradiated with doses higher than the average lethal, preparations of the thymus + spleen and thymus + spleen + bone marrow mixtures showed more significant therapeutic effects than preparations from individual organs.

As is known from the literature, at relatively low doses of gamma radiation, the body suffers from disorders of various parts of the hematopoietic system (bone marrow syndrome), and at higher doses, other disorders occur: pathology of the structure and functions of the small intestine epithelium - intestinal syndrome. With this in mind, the authors conducted studies to determine the most effective schemes for administering RNP to animals when exposed to doses of gamma radiation, in which some animals die from bone marrow, and some from intestinal syndromes. In the experiment we used RNP preparations isolated from the small intestine (Tk) and from the bone marrow (Km) (Fig. 5). In the control, 40% of the animals died by the fourth day after irradiation (intestinal syndrome), another 40% by the tenth day (bone marrow syndrome), and 20% remained alive 30 days after irradiation. The introduction of any of the studied drugs before irradiation stimulated the death of animals, moreover, the effect of RNP from the small intestine enhanced the effects of the small intestine syndrome, and from the bone marrow, the effects characteristic of bone marrow syndrome. These results can be considered as further additional evidence of the tissue specificity of the action of the substances protected by the patent.

The results of this experiment are consistent with the views of the authors of the patent on the mechanisms of action of RNP. Since one of the mechanisms of action of RNP, according to the authors, is associated with increased cell proliferation, the introduction of RNP into the body before irradiation, i.e. their use as radioprotectors should have a rather negative effect (actively dividing cells are less stable with respect to external influences). A positive effect is achieved only in cases of the introduction of RNP after irradiation, i.e. the use of RNP as therapeutic agents for the treatment of radiation sickness syndromes (Schemes II, III, and IV in FIG. 5). The effects were highly dependent on the dose of the drug, and even more on the pattern of introducing RNP into the body. Three intramuscular injections of RNP from the small intestine (Tk) or bone marrow (Km) led to a significant increase in the life span of irradiated animals and to a decrease in the number of deaths. Testing the drug administration scheme made it possible to achieve more significant effects. The 4-fold administration of NNP from the small intestine in the first 4 days after irradiation (Tk) and the 6-fold administration of RNP from the bone marrow on days I, 2, 3, 6, 8, and 10 were more effective. If in such experiments we use animals irradiated with a dose of LD50, then after 30 days all of them will remain alive. A more frequent administration of the drug (Km), as well as an increase in the dose of the drug by 2-3 times, is unfavorable. With a dose reduction of 5-10 times, the effects were significantly reduced.

To study the mechanisms of action of RNP at the cell level, the cytotoxic and cytogenetic effects of adverse physical (gamma radiation) and chemical (poisoning by heavy metal salts) effects and modifications of these effects using RNP were studied. This group of studies was carried out on adult sexually mature females of hybrids of the 1st generation of linear CBAxC57BL mice, which are an ideal and well-studied model for such studies.

In cytogenetic experiments performed on RNP preparations extracted from bone marrow, we studied the change in the ratio of polychromatophilic erythrocytes (reticulocytes) to normal mature R / E erythrocytes, which is normally approximately equal to I and significantly changes with different external influences, reflecting changes in the kinetics of cell maturation (cytotoxicity). The change under the influence of external influences of the number of micronuclei (MN) in reticulocytes was also determined. These particles are formed as a result of ruptures of 2 DNA strands in the chromosomes and the formation of an additional smaller nucleus during cell division, which remains in the red blood cell after removal of the main nucleus from it. In a normal body, 1-2 such particles per 1000 cells are formed. With various adverse effects, this value can vary by tens of times. Cytological preparations were prepared according to the method proposed by Schmidt, stained according to Giemsa and the number of different types of cells was counted on a microscope with an immersion lens with an increase of x900. Statistical processing of the results was carried out using Student's t-distribution and the corresponding criteria for the significance of differences.

In the 1st experiment of this series, the antimutagenic and antitoxic effects of RNP from bone marrow on bone marrow and spleen cells of animals irradiated at a dose of 4 Gy were studied (Table 2). For 1000 reticulocytes studied, 2.4-4.0 cells with micronuclei were registered in the control, and the R / E ratio was 0.7-0.8. After the action of gamma radiation, the number of reticulocytes with micronuclei in preparations from the bone marrow increased to 25.5, which indicates a sharp increase in the number of cells with DNA damage. The R / E ratio under the influence of irradiation decreased to 0.38, which indicates the death of a certain number of cells in the early stages of erythropoiesis or a change in the kinetics of erythrocyte maturation. At all studied concentrations, injections of RNP from bone marrow led to a decrease in the mutagenic effect. We can conclude that we are dealing with a rather active therapeutic drug of mild action. An insignificant change in the effect with a significant increase in the concentration of the drug is characteristic mainly for effective general strengthening and healing agents. It is noteworthy that only a few micrograms of RNP is sufficient to obtain a clear antimutagenic effect. In the case of studying the effect of RNP from bone marrow on spleen preparations, the antimutagenic effect is clearly weaker (a change in the number of MYs varies from 18.2 to 14.4). This is a clear confirmation of the specificity of the action of RNP on the cells of the same organ.

Table 3 presents the results of the effects of animal poisoning with hexavalent chromium ions (Cr VI) and the combined effects of gamma irradiation and Cr VI on bone marrow cells, and correction of the cytotoxic and cytogenetic effects of the combined effects on the body of these two adverse factors using RNP. The combination of the effects of radiation and injection into animals of a solution of K ₂ Cr ₂ O ₇ gives some summation of cytogenetic effects. The application of the compensating factor of RNP on these effects caused a significant decrease in the total mutagenic effect (from 22.6 to 10.8, i.e., more than 2 times).

The general conclusion from the experiments in this section is that RNP from the immunocompetent tissues studied by us have a clearly expressed ability to restore the functions of damaged cells of the same tissue, slightly affecting others, i.e. do not show pronounced side effects. The effect of RNP on damage caused by gamma radiation or salts of heavy metals, reveals only a slight specificity in the course of reparations. These results make it possible to characterize RNP as quite active substances of biological origin, which can be used for the selective treatment of certain types of cells, cell systems and organs, and the selective restoration of the most damaged functions.

The following series of experiments was carried out with the aim of proving the possibility of regulating the level of hormones using RNP and the effect of using RNP preparations from hormone-producing organs on the synthesis of the corresponding hormones and metabolism of the whole organism.

Example 4. The effect of RNP from the thyroid gland of cattle on the intensity of total metabolism and on the synthesis of thyroxine and triiodothyronine.

In the experiment we used RNP isolated from cattle thyroid gland according to the scheme described above (Method 1). The preparations contained 2-5% protein and more than 94% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs; traces of DNA have been identified. The sedimentation constants of individual fractions were in the range from 3.0 to 6.5 S.

In this experiment, thyroid preparations were administered to old female rats that showed impaired synthesis of thyroxine (T4) or triiodothyronine (T3) and significant excess weight.

36 rats, weighing from 400 to 475 g, were divided into 6 equal groups (the average weight of the animals in each group was approximately 440 g) and all animals, except the control and 5th groups, were injected every other day during the month of RNP thyroid gland in quantities: 0.1 mg (group 1), 0.3 mg (group 2), 0.6 mg (group 3), 1.2 mg (group 4), animals 5th group of RNP was administered 3 times 2.5 mg, and the animals of the control group were injected with saline according to the accepted scheme. The overall picture of the weight change of animals in the control and with the introduction of RNP is presented in Fig.6. The unit of scale on the abscissa corresponds to 10 g. During the experiment, control animals, which, like everyone else, on the usual vivar diet, increased their weight by about 2% (from 440 to 450 g, control curve of Fig.6).

Injection into animals of RNP in doses of 0.3 mg led to weight loss from 440 to 420-425 g. The decrease in weight became noticeable on the 10th day of the experiment. The maximum weight loss is observed on the 30th day, approximately 5%. The mass growth curve after stopping RNP injections at a dose of 0.3 mg is approximately parallel to the control, and thus, even on the 100th day of the experiment, the weight of the animals remains lower than the weight of the animals in the control group.

Animals that were given higher doses of RNP (0.6 and 1.2 mg, respectively), which were higher but completely harmless to the animal organism, showed a much more significant decrease in body weight (from 440 g to 390-405 g, i.e. by 10 or more percent). The decrease in weight becomes noticeable after 5 days after the start of the injection and continues for several days after the cessation of injection. There is a clear dependence of the effect of reducing the weight of animals on the administered dose of RNP from the thyroid gland. With increasing doses, the effect increases significantly. The slope of the mass growth curve (gain) after cessation of RNP injections lags behind the animal mass growth in the control. By the end of the observation (day 100), the injected animals remained with much lower mass than the control. Some of them do not gain the weight that they had at the beginning of the experiment, until the end of life, that is, for several months.

Since it is obvious that for the reconstruction of the genome using RNP from the thyroid gland there is no need for a long, multiple injection of RNP, experiments were carried out with short-term pulsed use of the drug. The upper curve in Fig. 6 shows the result of a total 3-fold administration of 2.5 mg of RNP to animals over 1 day at 8-hour intervals.

A 3-fold injection of animals with RNP leads to a significant (up to 5-7%) weight loss with a consequence of up to 2-3 months. Weight loss continues in some cases up to 20 days after the cessation of injection. Perhaps this phenomenon, which could not be observed so clearly with other schemes for the administration of RNP, can be explained by a larger percentage of thyroid cells that were affected by RNP, given their daily rhythm of mitoses. After the cessation of injection, the weight of the animals decreases for another 15 days. Then the mass gradually increases, but the rate (dynamics) of its increase remains lower than the growth rate of the mass of animals in the control, and at the end of the experiment, the mass of animals in the experiment remained significantly reduced compared to the control.

The following general conclusions can be drawn:

1) there is a clear tendency to reduce the weight of animals in the experiment at all doses of the administered drug;

2) the level of weight loss was strictly dependent on the dose of the drug, and the greatest effect was observed at the maximum dose (average weight of the animals decreased from 440 to 390 g);

3) a total of 3-fold administration of the drug in doses at which no side effects or adverse reactions from the body were observed causes a significant reduction in the weight of the animals.

The weight of the animals, when administered with sufficiently effective doses of the drug (0.6 mg and 1.2 mg), remained reduced for more than three months and, after the end of the injections, changed less than in control animals that did not receive RNP. Under the influence of RNP, the weight of animals decreased on average from 440 to 390 g, and 3 months after discontinuation of the drug, it changed from 390 to 410 g. Note that 3 months for rats is a significant part of life. In all cases, a decrease in overweight was accompanied by an increase in basal metabolism. Animals lost weight, being on the same as the control, vivar diet, due to increased mobility and overall activity. At the same time, old individuals acquired a smooth white coat and looked healthier.

At the end of the experiment, 1 ml of blood was taken from the tail vein in all animals and the levels of T3 and T4 were determined using radioimmunoassays. The amount of triiodothyronine (T3) and thyroxine (T4) in the blood of animals was determined the day after the cessation of RNP injections from the thyroid gland. The results of determining the level of hormones in the blood of the control and all experimental groups are shown to the right of the curves in Fig.6.

The introduction of already minimal doses of RNP (0.1-0.3 mg) significantly increases the amount of T3 compared with the control. With a further increase in the injected concentration of RNP, the amount of T3 increased, reaching a maximum at an RNP concentration of 0.6 mg. A further increase in the injected concentration of RNP does not lead to an increase in the amount of T3, despite the fact that the decrease in animal weight continues. A similar, but even more pronounced phenomenon is defined for T4. It should be noted that the absence of stimulation of hormone biosynthesis and, possibly, inhibition of further growth in the amount of hormones by an excess of regulator, is generally characteristic of regulators in which, for the manifestation of a positive effect, it becomes necessary to bind to certain limited centers on the surface of the cell or intracellular structures.

We also tested the effect of thyroid-medication from a thyroid gland on several volunteer patients with impaired lipid metabolism and overweight.

Example 5. Patient K. Age 58 years, height 185 cm, weight 104 kg, A / D 110/180. The diagnosis of hypertension, overweight, cardiosclerosis. Over 3 days, the patient was injected intramuscularly with 20 mg of RNP from the mammalian thyroid gland in a 0.5% novocaine solution. After completing the injection course, a daily examination of the injection sites, control weighing and measurement of blood pressure were carried out daily for the next 15 days. No reactions from the skin at the injection site were found. On the 5th day (one day after the last injection), the patient's weight decreased to 91 kg, A / D decreased to values 90/140. Over the next 15 days, the weight and blood pressure of the patient changed slightly within the usual daily norms.

Thus, as a result of a trial test of the effectiveness of using RNP from the mammalian thyroid gland in overweight patients, a persistent positive effect was shown to reduce body weight and normalize the biosynthesis of hormones (TK and T4) in the blood after 3-5-fold intramuscular injections. The above studies indicate the fundamental possibility of using RNP from the thyroid gland in clinical practice and the prospects of further studies with the aim of developing drugs that can adjust the level of metabolism, hormonal status, decrease and normalization of the body mass of mammals and humans.

The following experiments were conducted on preparations isolated from the testes.

Example 6. The effect of RNP from the testes of young bulls on cell division of spermatogenous epithelium (replicative DNA synthesis) of rats.

In the experiment we used RNP isolated from cattle testicles according to the scheme described above (Method 2). The resulting product contains 2.9-4.2% protein and 90-92% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs. Traces of DNA are identified. The average value of the sedimentation constant is 3.5-6.5 S. The sedimentation constants of individual fractions were in the range from 3.0 to 6.5 S, and the main number of active fractions in the region of 3.5-6.5 S.

The experiment was performed on male Wistar rats weighing 250-270 g. All animals were divided into 3 groups: 1 - control, instead of RNP, the animals were injected with saline solution, 2 - the animals were injected with RNP from the bull’s brain, 3 - the animals were injected with RNP from the testes of the bull.

On the 1st and 2nd days of the experiment, animals were injected intraperitoneally 3 times at 8-hour intervals (taking into account the daily rhythm of mitoses), either 700 μg of RNP from testicles in 1 ml of saline (3rd group), or 700 μg of RNP from brain in 1 ml of saline (2nd group), or 1 ml of saline (control).

8 hours after the last administration of NSAIDs, 14 C-thymidine (64 μCi per 1 kg of weight, specific activity 69 Ci / mmol) was started to be administered in 1 ml of saline 3 times at 8-hour intervals. 8 hours after the administration of thymidine, the animals of all three groups were decapitated, testicles were isolated, they were fixed in 10% formalin and weighed parenchyma samples of approximately 20 mg from them into separate tubes, 200 μl of solubilizer were added to them, kept in an incubator at 60 ° C until the tissue is completely dissolved, 5 ml of a liquid toluene scintillator (4 g of PPO + 50 mg of PPO + 1 l of toluene) was added and counted on a liquid scintillation counter, determining the values of dpm. The average values obtained from 3 determinations in individual animals are shown in Fig.7. A similar procedure three times with 8-hour intervals of 14C-T administration was also carried out 2, 3, 6, 9, 13 and 20 days after the administration of RNP. The results of all these definitions are presented as separate points.

Administration to rats of RNP from the testes on the 1st and 2nd day increases the incorporation of thymidine into the cells of the testes of the recipients. This is the so-called "non-specific" increase in DNA replication, which can be achieved by the introduction of any other nucleic acids, their precursors, and even a set of nucleotides. Starting from the 3rd to the 6th day, the effect of RNP on DNA synthesis is leveled with the help of cellular regulatory mechanisms, and on the following days until the 20th there is growth and the establishment of persistent specific stimulation of DNA synthesis in testis cells under the influence of RNP administration (in control experiments with oxyurea, it was shown that DNA synthesis in this case is associated with cell replication, and not with damage repair). Fluctuations in the level of DNA synthesis over time is a common occurrence with the feedback regulatory mechanism. The drug RNP from the brain in this experiment behaves completely differently (Fig. 8). It is characterized by only nonspecific stimulation of DNA synthesis in the testes up to 6 days. Then the level of cell proliferation returns to normal and remains so in the future.

Example 7. The study of the effects of RNP on the genetic effect of gamma radiation in mice and on genetic damage in germ cells.

In the experiment used RNP isolated from cattle testicles according to the scheme described above (Method 2). The resulting product contains 2.9-4.2% protein and 90-92% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs. Traces of DNA are identified. The average value of the sedimentation constant was 3.5–6.5 S. The sedimentation constants of individual fractions were in the range from 3.0 to 6.5 S, and the main amount of active fractions was in the range of 3.5–6.5 S. on mice, males and females, hybrids (CBAxC57BL) F1, male hybrids (BALBxC57) and mice of the BALB line, having reached at least 2 months of age by the beginning of the experiment. Irradiation was carried out on a gupa unit GUPOS 137Cs, dose rate 6.12 Gy / min. RNP was administered to animals in a saline solution of 0.2 ml in doses of 4, 10 and 20 mg / kg of weight once or repeatedly both before and after irradiation. The size of embryonic mortality before and after implantation in the offspring of irradiated males and the frequency of Abnormal Sperm Heads (AGS) were evaluated. In addition to genetic indicators, the fertility of male mice was also studied by the percentage of effective crosses and the change in the mass of the testes, reflecting the cytological effect of radiation.

To study the magnitude of embryonic mortality, 3 intact females were planted in male mice after exposure for a week, 3 females were changed after a week. Depending on the variant of the experiment, 3-4 consecutive transplantations were carried out. During this time, mature sperm were realized during fertilization, having experienced effects at different stages of spermatogenesis. 18 days after replanting, the sacrificed pregnant females were opened and the number of corpus luteum in the ovaries, implantation sites, and live embryos in the uterine horns was counted. The ratio of these indicators was determined mortality of embryos before and after implantation.

The frequency of occurrence of AGS was analyzed on air-dried smears prepared from the contents of the epididyms of irradiated animals. From each male, at least 300 sperm were counted. In terms of slaughter of males, i.e. 35 and 45 days after the end of the exposure, the body weight and the weight of the testes were determined.

7a. Single exposure.

Embryonic mortality.

Table 4 presents data on the effect of daily RNP administration for five days to irradiated hybrids (CBAxC57BL) F1 male mice on the percentage of effective crosses and embryonic mortality. There is a clear tendency for a decrease in the magnitude of induced embryonic mortality under the influence of NNP. In terms of effective crosses, there was an increase in the percentage of effective crosses for all three studied replants. When summarizing these data for all plantings, the differences were statistically significant.

It should be noted that RNP increased the fecundity of males in non-irradiated animals, especially if the males were more than 4 months old.

The frequency of abnormal sperm heads and the mass of the testes. From the data shown in Table 5, it is seen that with the introduction of RNP in doses of 4 and 10 mg / kg to male hybrid mice subjected to a single irradiation at a dose of 3 Gy, a tendency to a decrease in the frequency of AHS induced by irradiation was observed. Table 6 shows data on the effect of daily administration of RNP (10 mg / kg) for five days after irradiation with a dose of 3 Gy of BALB mice on the frequency of AGS and the weight of testes. Linear mice are more radiosensitive, the frequency of induced AGS is higher, and the degree of decrease in testis mass is stronger than in hybrid animals. An analysis of these indicators revealed that at 30 and 45 days after irradiation, the weight of the testes was slightly higher in the variants with the introduction of NNP. At both stages of the analysis, the frequency of AGS was lower in the case of administration of the drug. When summarizing the data of the analysis results at 30 and 45 days after irradiation, the degree of decrease in the frequency of AGS in the variants with RNP was statistically significant.

7b. Fractional exposure.

Embryonic mortality.

Table 7 presents the results of the analysis of embryonic mortality in the offspring of males irradiated with a total dose of 3 Gy, fractionated at 0.6 Gy for 5 days, which were administered after irradiation with a minimum dose of RNP of 4 mg / kg for 10 days. Within 4-6 weeks after irradiation, the fertility of males in the control significantly decreased until complete sterility. An analysis of the results already in the 3rd week of mating revealed a positive effect of RNP on the mortality rate of embryos before the implantation period. Summing up the study of the genetic effect of RNP, it should be noted that in all the experiments conducted, a tendency toward the therapeutic effect of RNP was observed. A statistically significant decrease in the degree of radiation damage was found by the percentage of effective crosses when NNP (10 mg / kg) was administered five times after irradiation with a dose of 3 Gy of gray CBAxC57BX F1 hybrids. In addition, a decrease in the frequency of AGS was detected in BALB mice in a similar experiment. Three similar experiment variants (3 Gy + 10 mg / kg RNP × 5) were carried out on mice of three different genotypes (gray hybrids, white hybrids and linear BALB mice). The therapeutic effect was most pronounced in BALB mice. A positive effect in hybrid mice was found only in experiments on old animals. This suggests that the beneficial effect of NNP is associated with the stimulation of the cell repair system, which in linear and old mice may be weakened. In general, the favorable effect is most pronounced with a relatively high dose of radiation and with the introduction of NNP after irradiation.

Example 8. The effect of RNP from the brain and RNP from the adrenal cortex on the severity of food anaphylaxis and on the values of the anaphylactic index.

In the experiment used RNP isolated from cattle testicles according to the scheme described above (Method 2). The resulting product contains 2.9-4.2% protein and 90-92% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs. Traces of DNA are identified. The average value of the sedimentation constant was 3.5–6.5 S. The sedimentation constants of individual fractions were in the range from 3.0 to 6.5 S, and the main amount of active fractions was in the range of 3.5–6.5 S. The experiments were carried out on male guinea pigs with an initial weight of 250-300 g. Chicken ovalbumin sensitization was carried out according to the method developed at the Institute of Nutrition AMS. The animals were administered three times daily oral solution of ovalbumin in a dose of 100 mg per animal. The introduction of a permissive dose of ovalbumin was performed on the 14th day. The severity of anaphylactic shock was assessed by the level of mortality, the number of convulsive manifestations and the magnitude of the anaphylactic index. In the first series of the experiment, the drug NNP from the adrenal glands of cattle, from a whole organ was used. The drug was administered to animals at a dose of 0.1 mg / kg, 1 mg / kg and 10 mg / kg daily for three days before the introduction of the authorizing dose. In the second series of the experiment, NNP preparations were used, isolated from adrenal cortex at a dose of 0.1 mg / kg and 1 mg / kg, adrenal medulla at a dose of 1 mg / kg and from the brain at a dose of 1 mg / kg. The drug NNP from the brain was taken as a control to identify the specificity of the effects.

The experimental results are presented in Table 8. It was shown that administration of an RNP preparation from whole adrenal glands at a dose of 1 mg / kg reduces the severity of anaphylactic shock in all studied parameters, whereas a preparation from whole adrenal glands at a dose of 10 mg / kg increased the number of convulsive manifestations. When studying NNP preparations isolated separately from the cortical and medulla, it was found that the drug from the brain layer when used at a dose of 1 mg / kg significantly enhances the severity of food anaphylaxis, while NNP preparations from the adrenal cortex and from the brain did not have a similar effect. Apparently, after further clarification regarding the applied doses and methods of administration, the studied NNP can be recommended as substances that can regulate the intensity of the development of certain allergic reactions of the body.

The general conclusion from all the experiments presented in this section is that RNP preparations isolated from different tissues and organs responsible for the synthesis of hormones are able to specifically normalize the functions of the corresponding organs and tissues in disorders caused by pathological processes and aging, and enhance the repair processes damage to the corresponding cells and tissues caused by various harmful effects.

The following experiments were carried out in order to determine the effect of RNP on the biological age of organs and tissues, to detect the “effects of rejuvenation” by the action of drugs from the tissues of physiologically young animals.

In the experiments described above, using the RNP bioregulators that we isolated, a stable effect of specific stimulation of cell proliferation was obtained. Is it good or bad for the body? How different are the newly appearing cells from the previous ones, how will they function in the body, are they “younger” or “older” of their predecessors in terms of physicochemical and functional characteristics?

To answer this question, we briefly consider one of the experiments carried out on the liver of rats of different ages and on the liver of ordinary adult animals subjected to partial resection of the liver, in which the kinetics of inclusion and subsequent excretion of ³ H-thymidine from animal organs, as well as the average half-life, were studied cells. As follows from the results shown in Fig.9, these indicators in animals of different age groups are significantly different. After adverse effects (in this case, hepatectomy), a month after the operation, when the compensatory and recovery processes are mostly completed, the organ is substantially aged (according to our calculations, the operation in this case is equivalent to 3-4 months of aging, which for a rat total life extension).

Given the results of this experiment, a scheme was constructed that, according to the authors, reflects modern ideas about cell division and its relation to the aging of organs and their cells (Fig. 10). An ordinary cell, the cycle length of which from division to division = 2 conventional units of time between divisions (lifetime), during this time is divided into two. As recognized by most authors, the cycle duration in daughter cells is somewhat different. For simplicity, we take the asymmetry coefficient = 2. Thus, as a result of the first division, we obtain cells with a lifetime of 2 times longer (4 conventional units) and 2 times less (1 conventional units). A cell with a cycle time = 1 dies and is lysed, and a cell with a lifetime = 4 during this time is divided into two, with a time of 8 and 2, etc.

The average life time of cells in an organ (Tav.) After a lapse of time equal to 2 cell cycles is (4 + 1) / 2 = 2.5, after another 4 cycles (8 + 2) / 2 = 5 - is constantly increasing with each new division.

As you know, starting from a certain stage of development (in our scheme after 8 complete divisions), each cell goes into a “rest state” and does not divide, but performs its functions. With age, the number of cells that have passed into this state increases, and fully differentiated cells that do not undergo division for a long time have characteristics similar to those of resting cells. After some time, the number of such cells reaches half and stops at this level, which is observed in organs, the volume of which remains constant.

Short-term intensive stimulation of the proliferation of all or part of the cells of our model at any stage, for example, partial resection of the liver (the lower part of the scheme), can only cause an increase in the growth of Tcp values compared to a normally developing organ (at the stage 12 after resection, Tcp = 6.6 instead of 6 , 0, and at stage 14 - 7.6 instead of 6.6). If at some stage labeled compounds, for example, thymidine, are introduced into the body, its incorporation into the cells of this organ will depend on the number of dividing cells at this stage of development, and its excretion will depend on the number of lysed cells. It is clear that the same parameters determine Tcp at this stage of development.

Using such experiments, we can quantify Tcr, i.e. characterize the biological age of a given organ or tissue (such a concept is used in modern gerantology). This age does not necessarily correspond to the passport, it depends on the conditions of development of the body and can vary significantly with different external influences. Thus, using this scheme, we can try to calculate how useful or harmful this effect, including the effect of RNP or other factors or conditions, on cell proliferation.

Example 9. Determination of the half-life of cells (T / 2) and changes in this indicator in old animals with the introduction of organ-specific RNP.

In the experiment used RNP isolated from cattle testicles according to the scheme described above (Method 2). The resulting product contains 2.9-4.2% protein and 90-92% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs. Traces of DNA are identified. The average value of the sedimentation constant was 3.5–6.5 S.

As in the previous experiment, animals (24 old Wistar males, aged 2 years or more, weighing 420-470 g) were injected with RNP from cattle testicles, 3 times 700 μg at intervals of 8 hours. The control group (also 24 rats) was injected with saline. After 20 days, upon the achievement of stable stimulation of proliferation of testis cells, the animals of the control and experimental groups were injected with 600 μCi ³ H-thymidine (W.A. 59 Ci / mm) in three doses at intervals of 8 hours. The animals were slaughtered one day, 20 days, 44 days and 56 days after the pulse label with thymidine.

The label removal curve 1, 20, 44, and 56 days after the pulse labeling of the pretreated RNP animals and the corresponding control curve are shown in FIG. 10A. As expected, RNP significantly stimulated the overall level of cell proliferation (inclusion rate of ³ H-T) on the 20th day after administration RNP, as evidenced by almost two-fold increase in the intensity of the labeling in one day. The kinetics of label removal from tissues treated and untreated RNP is completely different. In animals that were injected with RNP, the rate of label excretion was significantly higher and the average half-life of the cells was reduced from 21 to 16 days (total preparations including all cells of the organ were examined).

Thus, the results of this experiment are quite amenable to calculations, similar to those described above, to determine the degree of "aging" or "rejuvenation" obtained with this effect. It is shown that in this case we can talk about the "rejuvenation" of the testes cells of old animals and even determine the degree of "rejuvenation" quantitatively. The results of these experiments also suggest an increase in the productivity of testis cells under the influence of RNP by approximately 30%. In a parallel study on the same animals, we found stimulation of sperm motility under the influence of RNP. This indicator in control animals was 46%, and in animals treated with RNP - 52%.

Example 10. Determination of the effect of RNP isolated from the skin on the intensity of reproductive DNA synthesis, cell division and on the synthesis of the most functionally important proteins of different fractions of collagen and elastin in the dermis, as well as keragins in the epidermis of animals of different ages.

In the experiment used RNP isolated from cattle testicles according to the scheme described above (Method 2). The resulting product contains 2.9-4.2% protein and 90-92% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs. Traces of DNA are identified. The average value of the sedimentation constant is 3.5–6.5 S. The sedimentation constants of individual fractions were in the range from 3.0 to 6.5 S, and the main number of active fractions was in the range of 3.5–6.5 S.

Initially, in this experiment, an attempt was made to determine the intensity of DNA synthesis in the cells of the total tissue of the skin of animals (epidermis and dermis) of different age groups and to study the effect of RNP on DNA synthesis in cells of normal animals (middle and older ages). The animals of the experimental group were distinguished by the fact that daily, within 10 days before the start of the experiment, NNP in the gel was applied to the skin-cleansed areas of the skin (10 mg of RNP was dissolved in 1 ml of 0.5% Carbopol 940 Gel, pH 8.0. Gel dose 200 mg per 4 cm ^{2 of} skin), while the animals of the control group received a gel without RNP.

Figure 11 (Control) demonstrates the inclusion in the DNA of ³ N - thymidine per unit mass of the skin of animals of 3 studied age groups (2, 10 and 20 months). A characteristic decrease in DNA synthesis with the age of animals can be noted. These results are consistent with most studies of age-related parameters of DNA synthesis in skin tissues and cells of its individual layers. The impact on the skin of animals of NNP in the form of applications (Fig. 11, Experience) causes a significant active stimulation of DNA synthesis, while there is a shift in biosynthesis parameters to values characteristic of animals of a younger age.

Considering that changes in the intensity of DNA synthesis do not always unambiguously indicate “rejuvenation” of the cells of a given tissue, for a more correct study of the effect of “rejuvenation”, it seems necessary to use additional indicators of quantitative changes in the content and synthesis of the most important proteins for the normal structure and functioning of this protein tissue.

For this purpose, animals of the control group were injected with 3H amino acids from both the dermis and separate fractions of collagen and elastin were isolated. All these procedures were then duplicated in experiments in the in vitro system on skin preparations from the same animals, studying the incorporation of ¹⁴ C-amino acids. The animals of the experimental group differed in that, as in the previous experiment, RNP in the gel was applied to the skin-cleansed areas of the skin daily, 10 days before the start of the experiment (10 mg of RNP was dissolved in 1 ml of 0.5% Karbopol Gel 940 pH 8.0, Gel dose 200 mg per 4 cm ^{2 of} skin), while animals of the control group received a gel without RNP. The results of studying the inclusion of labels per unit mass of collagen fractions: K 1 (salt-soluble fraction), K 2 (fraction soluble in acidic acetate buffer), K 3 (insoluble collagen) and elastin are presented in Table 9, in vivo experiment (inclusion of ³ H- amino acids) and in vitro (inclusion of ¹⁴ C-amino acids) \. Noteworthy is the presence of certain changes in biosynthesis with age in all fractions except K2, which in all cases does not show clear significant changes. It should be noted that in the literature there are studies of individual authors, in which it is shown that this fraction of collagen varies with age and it is characterized by an increase in biosynthesis at certain certain stages of development. The effect of RNP drugs significantly changes the processes of incorporation of labeled amino acids into elastin and collagen. The superposition of the values obtained in these experiments on the scale of changes in the studied proteins in ontogenesis makes it possible to determine the age of the tissue site of the treated RNP (the so-called "biological" age of this tissue after the experiment) and its difference from the age of the "passport" control. The results of such comparative constructions show that after exposure to RNP in the form of applications, the level of synthesis of all the studied proteins shifts significantly towards “rejuvenation”, just as this was the case in experiments to determine the intensity of DNA synthesis. This regularity in the study of protein biosynthesis manifests itself quite clearly, despite the fact that in this case, unlike DNA synthesis, there is no reason to assert that each of the studied proteins (fractions K1, K2, K3 and E) uniformly changes the intensity of synthesis with age .

Data on determining the mass of individual fractions extracted from a dermis preparation with an area of 1 cm ² in the control and after exposure to RNP are presented in Table 10. Despite the fact that the degree of extractability of different proteins from the dermis can both increase or decrease with the age of the animals, this In this case, the procedure for determining the “biological” age of the dermis site treated with RNP is quite possible based on the experimental characteristics of protein fractions (collagen and elastin). Note that RNP more actively affects the dermis in the case of the old age group of animals.

Similar studies were carried out by us on separate fractions of keratin from the epidermis, Table 11. In this case, we can conclude that, after exposure to RNP, there is also a tendency to shift the studied parameters towards values characteristic of younger animals.

Thus, the results of this series of experiments indicate that in all cases, RNP uniquely activates DNA biosynthesis in the studied fractions of collagen, elastin, and keratins towards indicators that are characteristic of younger animals, which can be interpreted as a “rejuvenating” effect on condition of the skin, layer of the dermis and epidermis and individual protein fractions.

The next section of the study is devoted to the study of the antiviral effects of RNP drugs.

In this series of experiments, RNPs isolated from the liver, thyroid gland, skin, brain, testicles, thymus, and cattle lymph nodes were used according to the scheme described above (Method 1). Within the limits of the determination error, all preparations contained 2-5% protein and more than 94% low molecular weight RNA. The bulk of the impurities are high molecular weight RNAs; traces of DNA have been identified. The sedimentation constants of the individual fractions were in the range from 3.0 to 6.5 S. Which preparations were used in each particular case was noted in the description of the results.

The antiviral effect of RNP drugs was tested in a clinical setting and on an outpatient basis for volunteers infected with hepatitis A, B, and C herpes viruses, as well as papillomoviruses.

Example 11. Evaluation of the effectiveness of RNP in patients with herpes simplex virus infection caused by Herpes Simplex Virus (HSV).

176 patients of different age groups participated in clinical trials, including 12 people under 20 years old, 69 people from 21 to 30 years old, 32 people from 31 to 40 years old, 27 people from 41 to 50 years old and 36 - over 51 years old. 129 women and 47 men. Herpetic rashes in patients were localized in the area:

- red border of lips and surrounding smooth skin - in 47 patients,

- nasal mucosa - in 14 patients,

- smooth skin in various parts of the body - in 8 patients,

- transitional epithelium of the anus and surrounding smooth skin - in 23 patients,

- groin - at 31,

- genital mucosa - in 53 patients.

The main requirement for selection was a chronic recurrent course, with a relapse rate of at least 1 time in 6 months.

At the time of treatment of 176 HSV patients, 41 had periods when the relapse rate reached 1-2 times a month, in 79 - 1 and 2 times in 2-4 months and in 56 - 1-2 times in 4-6 months; 86 patients with relapses usually took Zovirax (Acyclovir) and noted a positive effect in resolving herpes infection, and 1 patient with genital herpes indicated the absence of any positive dynamics when taking Zovirax both to prevent relapses and to stop the acute condition .

In all patients, the disease was chronic, recurring in nature for several years.

For the treatment of HSV infection, RNPs isolated from various organs and tissues were used. The drug was applied on the skin, at a dose of 5-10 mg, for 1-2 days.

In 68 patients with HSV, NNP from the thyroid gland was used, in 41 patients from the cerebral cortex, in 38 patients from the skin (epidermis and dermis), in 21 patients from the thymus, and in 8 from the testes.

When applied to the affected areas of NNP from the thyroid gland, brain and skin, a similar picture of the dynamics of the resolution of the skin process was noted. If the drug was applied in the initial period of the development of the disease, i.e. at the time of appearance of parasthesia and the appearance of mild hyperemia of the skin in the fixed areas of the appearance of rashes, 30% of patients had a complete resolution of the skin process during the day, and 70% showed the appearance of tiny erosions against the disappearance of parasthesia, while the stage of formation of the vesicles was not visually determined . Within 1-2 days, erosion was epithelized, and the skin process was resolved in 6-7 days. If RNP was applied to the lesions during the period of the developed clinical picture of the disease, i.e. on vesicular or zrozirovanny areas of the skin, then soreness and various tactile sensations disappeared in 10-12 hours, and epithelization in the foci lasted no more than 1 day. The skin process was completely resolved in 7-10 days. The patient, who, as prescribed by the doctor, repeatedly and unsuccessfully used Acyclovir, also had a quick resolution of the skin process.

All patients of these groups (147 people), after treatment with RNP, were observed for 1 year, in 28 patients the first relapse occurred after 8-10 months, and this group of patients consisted of those patients who used RNP in the initial period of the disease. The remaining 119 patients with HSV after 12 months of recurrence were not noted.

In 21 patients with HSV who used the drug from the thymus, there was a more diverse dynamics and clinical picture of the skin process after application of the drug. In 16 patients, the same dynamics in resolving the skin process was observed as in the main group (147 patients), with no relapse within 1 year. However, the nature of the contents of herpetic vesicles instead of serous had purulent contents. In 4 patients, the skin process after applying RNP proceeded, as usual, without obvious acceleration, with the appearance of relapses with the same regularity as before treatment. New relapses were milder, with mild burning sensations, tingling, soreness. In 1 patient, the skin process took a protracted nature: after applying NNP, hyperemia, mild edema, parasthesia persisted for 3 weeks. During this period, isolated very small erosions appeared, covered with dotted serous crusts. The process was resolved by the end of 4 weeks of observation. New relapses appeared with the same regularity as before treatment, but, according to the patient, they were milder.

In 8 patients receiving RNP from the testes, a positive effect on the course of herpetic infection was also noted, however, due to the lack of a further drug, studies were discontinued.

Example 12. The effectiveness of drugs NNP in relation to the normalization of transaminases in the blood of patients with hepatitis B and C.

Clinical studies of the effect of RNP preparations from the liver and thyroid gland on the level of transaminases: alanine aminotransferase, aspartate aminotransferase and total bilirubin in the blood of patients with acute viral hepatitis B (2 patients, drug administration by i / m injection of 1 injection of 10 mg of NNP daily for 5 days or 5 enemas of 20 mg) and acute viral hepatitis B + C (1 patient, the introduction of the total drug containing 10 mg of NSAIDs from the liver and from the thyroid gland by 5 i / m injections).

A marked decrease in the level of enzymes in the blood in all cases began from 10-14 days. after the start of treatment, and by the end of the third week the level of enzymes was normal in all patients.

Example 13. Clinical evaluation of the effectiveness of RNP in patients of Herpes virus varicellae (Varicella-Zoster Virus - VZV).

Under observation were 2 patients with VZV, a woman 38 years old and a man 53 years old. The disease was acute, with high fever and general malaise, a strictly left-sided lesion of the chest skin along the intercostal nerves in both patients. On the day of treatment, the woman had a disease duration of 12 days, the picture of the rashes was polymorphic: there were large sizes (up to 1 cm) of blisters filled with hemorrhagic contents, and many small, fresh vesicles that were located around massive crusts. The pain syndrome is expressed significantly - the patient could not breathe freely, and the movements of the left arm were limited. From day 4 of the disease, she received intensive therapy, including high doses of vitamins, antibiotics, interferon of 40,000 units once a day (No. 7), Imunofan 1.0-1 times a day (No. 5), but any improvement was noted against the background of the treatment did not have. The man turned on 4 days from the moment the rashes appeared, and the disease progressed in a milder form. I did not receive treatment.

To stop the disease, a drug from the thyroid gland was used in a dose of 40-50 mg, which was dissolved in 5 ml of physiological saline and was injected into the rectum with an enema at night, for a total of 80-100 mg. On the 2nd day from the moment of drug administration, a sharp improvement in the general condition of patients was noted, and by 3 days the patient stopped having pain. In patients on the 3rd day, a significant decrease in edema and hyperemia in the lesions was noted, the rash dried up, crusted. On the 7th day, the lesions had a stagnant-cyanotic color with persistent crusts in places of erosion, the general condition of patients is satisfactory. On the 30th day, the foci retain a cyanotic color, scar formation is noted at the site of large erosion in the patient. Thus, both a single cutaneous application of RNP at a dose of 5 mg and a double application at a dose of 10 mg are quite effective (in cases of herpes infection).

With prolonged use of drugs to achieve a therapeutic effect, as a rule, 5-7 applications are sufficient for 2 weeks.

Example 14. The effectiveness of drugs NNP in the treatment of papillomavirus infections.

An outpatient study of the effectiveness of drugs isolated from the thymus and lymph nodes of cattle in the treatment of human papillomavirus infection in patients with genital warts and intrarectal localization of genital warts was also conducted. Previously, all patients did not receive treatment for papillomavirus infection. Patients in this group underwent one course of treatment for RNP. 10 mg of the drug was diluted in 2 ml of saline. Injections were performed intramuscularly every other day. Only 5 injections.

In patient M. (phimosis, condylomas were localized in the foreskin), after the third injection, the condylomas began to become covered with hyperkeratic layers and soon decreased by half, however, in parallel with this, the patient began to notice the appearance of multiple boils on the skin of the body, which resolved independently after 2-3 days from the moment of appearance. Within four months, the rash regressed by 2/3.

In patient N. (condylomas were localized in the region of the labia minora, labia minora, vagina, cervical canal), against the background of the therapy, after the fourth injection, 2/3 of all formations disappeared. Complete clinical remission was noted 1.5 months after the start of treatment. When re-examining the immunological status, normalization of all indicators was noted.

In patient A. (the formation was localized in the scaphoid fossa and was clinically morphological of the papillary genital warts type) after injections, the patient independently removed the formation using the Solcoderm drug. As a result, when assessing the clinical condition after 2 months, there was a lack of education, as well as relapses throughout the observation period (up to today); in addition, when conducting a PCR study, a previously detected type 33 papillomavirus was not detected in the re-study.

In all the cases described, there was a lack of side effects during treatment with NNP, good tolerance, pain in the first minutes after the administration of the drug in / to or / m.

Conclusion

The above experimental material suggests that the ability to adjust the effect of regulatory systems of cells and systems of information exchange between cells, including regulatory signals of the genome, is quite achievable at the modern level of science, and new opportunities that open RNP in the treatment of various diseases surpass the wildest assumptions. The exceptional ability of RNP isolated from normally functioning organs of a healthy organism to selectively correct metabolic disorders of various etiologies and repair a wide variety of damage to precisely those cells whose functions are impaired was even somewhat unexpected for the authors and causes some difficulties in interpreting the possible mechanisms of such phenomena.

Apparently, the set of normal cellular RNP contains a sufficient number of regulators - signals that can include portions of genes that are repressed or vice versa, that are excessively active as a result of various adverse effects on the cell. Entering this body from the outside in excess, RNP stimulate the work of all normally functioning links and adjust the work of those links whose activity is impaired.

Such correction occurs regardless of the cause of this violation, and leads to the correction of all parts of the broken chain from the lowest to the highest. It is very important that it uses compounds that work in the same cells normally, are completely harmless to the body, and there is no need for synthetic compounds, complex extracts from plants and other similar substances. All this gives grounds to consider the use of exogenous regulators of the physiological state of cells, tissues and organs as a new, very promising approach to the correction of metabolism and treatment of pathologies, including at the genome level, and consider RNP as one of the new, effective classes of biologically active substances and regulators used in genetic therapy.

The authors have developed several models to quantify the effect of RNP on cells and organs from the point of view of their rejuvenation or aging, and to determine the biological age of an individual organ, tissue, organ system, organism as a whole. This approach can be recommended for solving a number of problems in biology and medicine, in particular, for quantifying the benefits or harms of various external influences, testing the degree of damage and pathological changes in different organs and tissues, and developing individual patterns of using RNP, their mixtures and other drugs in order to obtain the maximum therapeutic effect.

Claims (1)

An agent that stimulates the repair of cell damage, including low molecular weight ribonucleopeptides isolated from mammalian cell nuclei with a molecular weight of 3.0-6.5 S (Swedberg units), containing more than 90 wt.% Low molecular weight RNA, 0.5-5.0 wt. % protein and impurities of high molecular weight DNA and RNA up to 100 wt.%, with the following properties: tissue-, organo- and stage-specificity, antiviral activity, which changes the average half-life of cells.

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