RU2240810C2 - Method for producing and applying embryonic antitumor modulator agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves vaccinating patients with embryonic antitumor modulator agent containing wide range of oncofetal antigens. Vaccination is carried out in subcutaneous application once a year at a dose of 0.02-0.04 mg/kg. The preferential dose is equal to 0.03 mg/kg of body weight.

EFFECT: high stability of the agent; stimulated antitumor resistance.

4 cl, 2 dwg, 4 tbl

Description

FIELD OF THE INVENTION

The invention relates to medicine, in particular to oncology. The embryonic antitumor modulator disclosed in the invention, as well as the method for its preparation, can be used in the medical industry to obtain a highly effective agent for the treatment of precancerous conditions and the prevention of malignant neoplasms.

State of the art

Compared with pathogenic stimulants of antimicrobial, viral and parasitic origin, the immunogenicity of tumor-associated antigens is rather weak. This is the main obstacle to the formation of oncotoxic T cells, which requires the connection of tumor antigens with the main histocompatibility complex of class I-II. In order to solve this issue, a number of vaccines have been obtained for the treatment of certain types of malignant neoplasms and the prevention of relapse.

Known vaccines against melanoma, which are allogeneic tumor cells and their lysates, autologous tumor cells, various antigens (MAGE-1, MAGE-3, MART-1, gr 100, etc.). These vaccines increase the activity of cytotoxic lymphocytes in relation to malignant cells, activate the proliferation of T-lymphocytes, tumor infiltration of CD8 +.

Vaccines are also known for the treatment of cancer of the breast, prostate, kidney and colon. Moreover, vaccines of varying degrees of purity are used with the use of thermal effects, radiation, etc. The preparation of vaccines is based on genetic engineering techniques, DNA recombination, introduction of the interleukin-2 gene, tumor necrosis factor into tumor cells, etc. They are used mainly for the purpose of anti-relapse immunotherapy and are not a preventive measure in the sense of increasing the antitumor resistance of people. Obviously, they are not a universal prophylactic and are used against one specific nosological form of cancer. These vaccines contain tumor cells, although neutralized.

By Tsang K.Y. (et.al., 1995) discloses a method that describes the immunogenicity of a tumor embryonic antigen (OEA) for humans. It contains the OEA gene of the recombinant virus and causes a special T-cell response in humans. This vaccine can only be used in the treatment of such human tumors that cause this antigen.

The temporary pharmacopoeial article (VFS 013-52-98), approved by the Agency for Medicines and Medical Technologies of the Ministry of Health of the Republic of Armenia on May 29, 1998, discloses an embryonic antitumor modulator (EPOM), which is the first among the authors of this invention obtained. This modulator has a number of advantages. However, a temporary pharmacopoeial article does not suggest or disclose these benefits. The present invention is the result of lengthy and strictly controlled studies that led to the unexpected discovery of a source of antimutagenic, immunomodulating properties, antiviral and other activities that explain not only the pronounced preventive antitumor effect, but also the sanitizing effect in a number of other pathological conditions.

SUMMARY OF THE INVENTION

Thus, the objective of the present invention is to improve the effectiveness of the treatment of a number of precancerous conditions and the prevention of malignant neoplasms. This problem is solved by increasing the natural antitumor resistance of the individual organism.

One of the aspects of this task is to increase the specific antitumor resistance in high cancer risk groups. Within this aspect, vaccine prophylaxis is proposed by an embryonic antitumor modulator, which is administered subcutaneously once a year in an effective amount, preferably at a dose of 0.02 to 0.04 mg / kg body weight, most preferably at a dose of 0.03 mg / kg.

Compared with known methods and drugs, the proposed method and drug are intended for patients with precancerous pathology and people over 35-40 years old who are at increased risk of developing cancer.

A feature of the proposed method is filling out a questionnaire (test) and determining the level of predisposition to tumor pathology. Another feature is that the group of high oncological risk includes, for example, long-term smoking individuals, persons with a family predisposition to cancer, as well as those patients in whom the amount of fibrinogen in the blood is 1.5-2 times higher normal level.

Hyperfibrinogenemia is also considered a risk factor due to the fact that, as it was established by the studies of the author of this application, if it is present, the cancer cells are covered with a “fibrin shield”, imitate a common general pathological process and thereby escape from immune surveillance.

To achieve the solution of this problem allows the use in the proposed method of a new embryonic antitumor modulator, which contains a wide pool of normal fetal proteoglycans (see below in the section “Qualitative and quantitative composition of the target product (EPOM)”).

The new embryonic antitumor modulator Mkrtchyan proposed in this invention has a preventive antitumor effect, antimutagenic and anticlastogenic effect, interferonogenic and immunomodulating action, as well as antiviral activity.

Without claiming to be an exhaustive and final scientific justification of the positive effect of the proposed modulator on the body, it is believed (based on existing knowledge in this area) that the specific sensitization of effector immunity cells with respect to oncofetal antigens plays a key role in the mechanism of realization of the antitumor effect of EPOM. increased killing of malignant cells.

The reason for this conclusion is the data obtained by the author of the suppression reaction of adhesion of leukocytes (RPPL), the reaction of passive hemagglutination (RPHA), the complement binding reaction (CSC) and the delayed-type hypersensitivity reaction (RGT). Targeted stimulation of antitumor protection is accompanied by an increase in the production of γ-interferon, IL-6 (seven times in the spleen in relation to the control) and prolactin — the level of the latter in the central organ of immunogenesis — the thymus, was 6 times higher than the initial background. Below (in tables 1-3) are some of the above observations.

A positive immunomodulatory effect was also detected in patients with EPOM vaccinated, suffering from pre-tumor pathology, smoking for a long time and having a family predisposition to cancer.

For a number of parameters - a preventive effect against the occurrence of histogenetically different forms of tumors, the use of small doses in the form of subcutaneous injections in the inner surface of the forearm with the determination of the severity of the local reaction, as well as the "vaccination" once a year - the embryonic antitumor modulator is likened to a polyvalent anticancer vaccine . During the observation of patients, it also turned out that EPOM has a tonic and anti-aging effect on the body.

The next aspect of the invention is the development of a method for producing an embryonic antitumor modulator, which provides the target product having the properties and qualities described above.

The features of the method according to this invention are: extraction of embryonic proteoglycans in a state as close to native as possible, obtaining a wide pool of oncofetal antigens from normal embryonic tissues of various origins, lyophilization (see further in the section “Method for producing EPOM”).

An additional aspect of the invention is the use of an embryonic antitumor modulator according to this invention for the manufacture of a preparation (in particular an anti-cancer vaccine) for the treatment of precancerous conditions and cancer prevention.

The invention is further illustrated by non-exhaustive examples of embodiments and drawings, which are intended only to demonstrate the feasibility of the invention, but not to limit.

Brief Description of the Figures

Figure 1 presents the results of gel filtration computer chromatography performed on a device PEV-2 (Pharmacy).

Figure 2 presents the results of liquid chromatography under pressure (HPLC SP8000 PEV-2) (USA).

Detailed Disclosure of Invention

The method of obtaining EPOM

Embryonic substances are thoroughly washed for 4-5 hours in running water and placed for 1.5 hours in 70 ° alcohol (final concentration). Then the tissue is cut into pieces, frozen with liquid nitrogen and homogenized in a specially prepared homogenizer with high impact force to a powder state. The homogenate is diluted with distilled water in a ratio of 1: 4, agitated and the suspension is placed in a ball mill for 20 minutes, the bowls and balls of which are prepared from metabolically inert materials. Previously, the latter are cooled to a temperature of 0 ° C. The number of revolutions is brought up to 400 per minute. The quality control of the disintegration of embryonic tissue is a cytological study to identify rectified cell nuclei (more than 80%), the smallest cytoplasmic breaks and fragments of fibrous structures. In a flat glass dish, the homogenate is exposed to ultraviolet radiation for 25 minutes, after which it is centrifuged in a PC-6 refrigerator centrifuge for 15 minutes at 6000 rpm.

96% wine alcohol is added to the clear supernatant in an amount sufficient to bring the alcohol concentration in the solution to 84 °, which ensures the precipitation of glycoproteins. The latter are separated by centrifugation at 16-20 thousand rpm (g - 58400) for 20 minutes in a zone rotary ultracentrifuge type UP-65. The precipitate is dissolved from a volume ratio of 1: 2 in distilled water, processed in a ball mill for 10 minutes and centrifuged again at 20 thousand rpm (acceleration - 58400) for 15 minutes in a UP-65 centrifuge. Protein (42-57%) is determined in the supernatant, viscosity is established (in different series 13-18), and after passing through sterilizing membrane filters (millipores - 0.8), the supernatant is lyophilized.

Parietal freezing is carried out in a special sterile rotating chamber at a temperature of 25-30 ° C. Quenching is carried out for 24 hours in a low-temperature cabinet. A gradual increase in temperature over 28-30 hours from -40 to + 37 ° C provides complete sublimation.

Qualitative and quantitative composition of the target product (EPOM)

The drug contains a pool of proteins - glycoproteins, extracted by the method of alcohol deposition. According to the results of analytical electrophoresis in 10% polyacrylamide gel according to Davis, the preparation contains 5 fractions of protein. In the lyophilized series of the drug, the concentration of total protein (as determined by Lowry, followed by spectrophotometric analysis) varies in different series, from about 42 to 57 wt.%. In addition to proteins, EPOM contains about 1.5% free and about 8-10% bound carbohydrates. Of the enzymes, alanine aminotransferase (ALT) is present - approximately 1.4 U / L, aspartate aminotransferase (ACT) - approximately 71.9 mmol / L, lactate dehydrogenase (LDH) - approximately 3.0 u / L, alkaline phosphatase (AL) - approximately 24 , 7 u / l, also contains trace elements: Mg - approximately 1.7 mmol / L, K - approximately 0.96 mmol / L, Ca - approximately 0.65 mmol / L, P - approximately 0.52 mmol / L, (according to Hitachi's biochemical analyzer - 911, Japan).

An enzyme-linked immunosorbent assay based on monoclonal antibodies revealed the following cancer embryonic antigens (5 mg of lyophilized EPOM was dissolved in 2 ml of distilled water):

CEA - about 1.4-1.7 ng / ml

AFP - about 95.0-99.0 ng / ml

CG - about 2.2-5.5 mIU / ml

TWG - about 45.0-50.0 ng / ml

Ca-125 - about 13.5-14.3 U / ml

Ca-19.9 - about 79.8-102.2 U / ml

Abbreviations:

CG - chorionic gonadotropin

CEA - cancer-embryonic antigen

AFP - alpha-fetoprotein

TWG - trophoblastic β ₁ -glycoprotein

Ca-125 - carbohydrate antigen

Ca-19.9 - carbohydrate antigen

Embryonic antigens (EAG) in the composition of the drug are related to numerous tumor antigens and give them cross-immune responses. In addition to EAG, the presence of which determines the authenticity of EPOM, in each series of the drug there are other proteins that are associated with its interferonogenic, antiviral and antimutagenic properties. The following biochemical methods were used to identify these proteins: gel filtration computer chromatography, polyacrylamide gel electrophoresis, high-pressure liquid chromatography under pressure.

Gel filtration computer chromatography - was carried out on an apparatus of the company "Pharmacy" (France), on columns 5000 R 7.5 mm × 30 cm PB, 005.5%. Three peaks were obtained: the most significant third peak with a pier. mass of 150 KD and the second - with a pier. weighing 40 cd.

Polyacrylamide gel electrophoresis - the presence of 5 protein fractions per mol. weighing 72, 67, 58, 50 and 40 kD. Protein fraction with mol. with a mass of 72 kD had an electrophoretic activity characteristic of albumin and AFP, which are similar in molecular weights and biological properties (AFP - embryonic albumin).

Liquid chromatography under pressure. Structural analysis of the peptides contained in the preparation was carried out on a R-8000 chromatograph from Spectro-Physico (USA). Wavelength 214 nm. The most significant and clear peak is the 3rd with a delay time of 2679 m. The remaining peaks are less clear.

Electronic paramagnetic resonance (EPR). Given the important biological role of free radicals, samples of various series of the preparation were subjected to EPR analysis on a Varian apparatus with a sensitivity of 10 ¹¹ -10 ¹² spin. Records of the spectrum of the standard (manganese standard) and 7 samples of the preparation studied showed that the concentration of paramagnetic particles in all samples is lower than the sensitivity of the device, which indicates the absence of free radicals in the EPOM.

Quantification of protein. Activity is determined by the presence of proteins in the preparation. Determination of the protein pool was carried out according to the Lowry method, followed by determination of the staining intensity on a spectrophotometer (SF-26 LOMO) at a wavelength of 750 nm.

The presence of CEA was detected using enzyme-linked immunosorbent and radioimmune methods. ELISA was performed using available Roche test kits. The content of trophoblastic β ₁ -glycoprotein (TBH) was determined by the radioimmune method using a domestic kit.

Biological activity. Given the pronounced antitumor activity of the drug in an experiment on transplanted tumors (Ehrlich mouse ascites tumor, 37 mouse sarcoma, Seidel’s ascites hepatoma, Pliss lymphosarcoma and rat Walker carcinosarcoma) and in rat carcinogenesis induced by 7.12 A unit of activity the drug is taken in such quantity (single dose), which provides 50% inhibition of tumor growth. Pliss lymphosarcoma in rats is used as a test system. In the experiments, white rats were used, male Vistar line, quarantined for at least 10 days, the average weight of animals was 145 g. The drug was administered parenterally a week before the introduction of a suspension of tumor cells.

Additional characteristics of the target product

EPOM is a porous, soft mass of yellowish-white color, odorless and tasteless.

When 2 ml of an isotonic 0.9% NaCl solution is introduced into the vial, it is resuspended for about 15 minutes without the formation of unbreakable flakes and lumps.

The injection solution is preferably a 0.02% opaque homogeneous liquid with a yellowish tint. The time of complete deformation is about 15 minutes.

The color of the solution does not exceed the color standard N 50 (GF XI, issue 1, p. 194).

pH of the solution: 6.5-8.5 (determined potentiometrically RF XI, 1, p. 113).

Residual moisture. The determination was carried out from 0.1 grams of the drug at 100 ° C, for 1 hour (GF XI, 1). The moisture content is not more than 9%.

Solubility. The drug is hardly soluble in water and 0.9% isotonic NaCl; practically insoluble in 95% alcohol and ether (HF XI, 1).

Authenticity. 0.003 g of dry powder is dissolved in 10 ml of water (solution A).

3-4 drops of concentrated nitric acid are added to 5 ml of solution A: a flocculent precipitate (protein) immediately precipitates.

To 5 ml of solution A, 0.1 ml of a 0.1% solution of ninhydrin is added and heated to a boil. A blue-violet coloration appears (amino acid part).

Sterility test. Maintains the requirements specified in GF XI, issue 2, p. 187.

Mechanical inclusion. The determination of mechanical inclusions is carried out in accordance with the “Instructions for the control of mechanical inclusions of injection solutions” (I 42-3-85), approved by the Ministry of Health of the USSR 10.10.85.

Toxicity tests. The drug was administered to animals in two doses exceeding the therapeutic for humans 200 times (15 mg / kg) and 10 times (0.7 mg / kg).

Enzyme immunoassay for CEA in various series of the drug

Reaction principle: enzyme-linked immunosorbent assay (ELISA) is the immune response of test samples with monoclonal murine ATs to detectable AG and monoclonal ATs to these ATs covalently linked to horseradish peroxidase. In a single-stage solid-phase ELISA, the CEA required in the samples can react with specific monoclonal ATs, and simultaneously with conjugated anti-CEA ATs, forming a sandwich complex. After the end of the immune reaction and washing, it should be incubated with a solution of the working substrate - tetramethylbenzidine. Developing coloration results determine the amount of bound anti-CEA peroxidase conjugate. The concentration of the desired AH in the test samples is determined by the color intensity caused by the enzymatic reaction.

The reaction technique. To identify various CEA, lyophilized series of the drug are dissolved in physiological saline at a rate of 1 mg / ml, mixed well and kept, like other ELISA components, for 1 hour at room temperature.

Beads with monoclonal AT are placed in duplicate tubes, where test samples are added in a volume of 50 μl, the standards are determined by CEA, control - CEA of human serum, excluding one empty tube without a bead - an empty reagent. Then, in all tubes except the empty one, 250 μl of the conjugate against CEA AT were added and incubated in an incubator at 37 ° C, constantly shaking for 15 minutes. At the end of the immune reaction, the buffer in the tubes is washed.

After the end of the immune reaction, a solution of the working substrate is prepared according to the instructions attached to the kit. A working solution in a volume of 250 μl is added to all tubes. After 15 minutes of incubation at 37 ° C, with constant shaking and protection from bright light, the reaction is stopped by adding 1 ml of 5% sulfuric acid solution and mixing without spraying. Within 1 hour after stopping the enzymatic reaction, the adsorption of test samples, CEA standards and control of serum against an empty sample is measured on a spectrophotometer at 450 nm.

The level of the desired CEA is determined by a standard curve constructed from the absorbance values obtained for each of the CEA standards with a certain CEA concentration in IU / ml.

Unknown CEA concentrations in the test samples are determined from the standard curve using the average absorbance between the control and each sample.

To express the reaction results in ng / ml, those obtained in ME are multiplied by 1.25.

Quantification of protein. About 0.1 g of the powder of the drug (accurately weighed) is placed in a volumetric flask with a capacity of 200 ml and the volume is adjusted with an isotonic 0.9% sodium chloride solution to the mark. At the same time, 1 ml of the resulting solution and the working standard solution (Note 1) and water (control solution) are transferred to the flasks, then 1 ml of a 1.2% sodium hydroxide solution is added. All mixtures are left for 30 minutes. Then, 4 ml of solution B are added to these three flasks (Note 3), stirred and left for 10 minutes, after which 0.4 ml of Folin's reagent is added (Note 4), stirred and after 30 minutes on a spectrophotometer in cuvettes 1 cm thick. measure the optical density of the test and standard solutions with respect to the control solution at a wavelength of 750 nm.

The protein content in% (X), calculated by the formula

where Cn is the concentration of protein in a working standard solution of albumin; Cl is the concentration of the test solution; D1 is an indicator of the optical density of the test solution; Dn is an indicator of the optical density of a standard albumin solution; b - residual moisture.

The protein content in the preparation in terms of dry matter is preferably 42-57%.

Note:

1. Preparation of a working standard solution of albumin

0.05 g of bovine eligible albumin (Aibumin bovine ciryst. Rcascarch grade. “SERVA”) was dissolved in an isotonic 0.9% NaCl solution in a 100 ml volumetric flask and adjusted to the mark with the same isotonic solution. 1 ml of a standard solution contains 0.0005 g of albumin.

2. Preparation of solution A

2.5 ml of a 2% solution of CuSO ₄ × 5H ₂ O and 0.12 ml of a 1.2% sodium hydroxide solution (solution A) are added to 2.5 ml of a 2.5% solution of potassium tartrate and sodium.

3. Preparation of solution B

0.4 ml of solution A (solution B) is added to 20 ml of a 2% sodium carbonate solution. Solution B is prepared immediately before use.

4. Preparation of Folin's reagent

In a 300-500 ml round-bottom flask, 20 g of sodium tungstate is added and dissolved in 140 ml of distilled water. 10 ml of 85% phosphoric acid and 20 ml of concentrated hydrochloric acid are added to the solution. A reflux condenser was attached to the flask and the mixture was boiled for 10 hours. Then, 30 g of lithium sulfate (Li ₂ SO ₄ × H ₂ O), 10 ml of distilled water and a few drops of bromine are added to the flask. The mixture is boiled for 15 minutes to remove excess bromine without a refrigerator under draft. The solution is cooled, the volume of the reagent is adjusted to 200 ml. Reagent can be stored for a long time. Before use, the amount of Folin reagent required for work is diluted with distilled water twice.

According to the requirements for new drugs, we studied the possible acute and chronic toxicity of EPOM in various types of experimental animals: mice, rats, rabbits, guinea pigs and dogs.

Study of possible acute toxicity of EPOM

The studies were performed on 38 white mongrel mice, 35 syngeneic mice of the CBA line, as well as 38 white mongrel and 35 Wistar rats. Hematological and biochemical methods were used, morphological studies of internal organs were carried out, the state of the cardiovascular, nervous, excretory systems, and the activity of the gastrointestinal tract were studied.

Animals were distributed in 5-7 in each group. EPOM was administered sc and intraperitoneally. The initial body weight in white mice ranged from 19.0-24.4 g, of Wistar rats, on average, 140 g.

To determine the LD, ₅₀ mice were given the maximum possible doses: for sc administration, 2.5 mg of protein in 0.5 ml of solution and for iv administration, 2.5 mg of protein in 0.5 ml of solution. The introduction of these maximum doses did not cause the death of animals during the 2-week observation period.

To detect LD _{50 of} EPOM in rats, the maximum possible doses were also used: sc - 5.0 mg of EPOM in 1.0 ml of solution and ip - 5.0 mg of EPOM in 1.0 ml of solution. During the entire observation period, death of rats was never observed in either the experimental or control groups. Daily observation did not reveal changes in the behavior of animals compared with the control. Convulsions and impaired coordination of movements were not observed, skeletal muscle tone was normal. Reactions to painful, sound, tactile and light stimuli did not differ from those in control animals. Body hair remained unchanged, dyspeptic phenomena were absent, feed and water intake was normal. The body weight of the experimental animals was determined 2 times during the observation period. At the end of the experiment, an increase in body weight of both experimental and control mice was found to be in the range from 2 to 5 g and in rats from 20 to 23 g. A general and biochemical blood test in the experimental and control groups did not reveal deviations.

Study for possible chronic toxicity of EPOM

The study was performed on 180 outbred rats, divided into 2 groups. In the experimental group were 80 males and females, in the control - 10 males and females. The weight of the animals was 120-140 g. The animals were given EPOM for three days, the route of administration was s / c and / b. Observation of animals was carried out for 2 months. 3 doses were used - 2.5; 5.0 and 10.0 mg per kg of body weight, which were administered three times. Total doses, respectively 7.5; fifteen; 30 mg / kg of body weight.

Daily monitoring of the condition and behavior of animals showed that all animals tolerate the introduction of EPOM well, there were no changes in their behavior compared with the placebo control. None of the doses of EPOM used in rats caused changes in the hairline, disorders of the gastrointestinal tract and excretory system.

The corresponding table shows the number of red blood cells × 10 ¹² and white blood cells × 10 ⁸ , as well as the morphological composition of blood - basophils, eosinophils, neutrophils, lymphocytes, monocytes - after administration, on the 24th day and at the beginning of the experiment. Conclusion: the hematological parameters in rats of the experimental and control groups in the above observation periods were similar.

Blood sugar in the control at the end of the observation period was 81 mg /%, and in the experimental group 82.3 mg /%, residual nitrogen, respectively, 24.2 and 25.1 mg /%, total protein 6.3 and 6.1 mg /%, respectively.

The internal organs, as well as the brain of outbred rats, which received three times EPOM sc in a total dose of 30.0 mg, were subjected to histological examination using both conventional histological examination methods and some histochemical methods. In cardiomyocytes, hepatocytes and epithelium of the proximal nephron there is a mild protein dystrophy, as well as a mild congestive congestion of the lungs, liver and kidneys.

Similar data were revealed in the study of possible chronic toxicity in rabbits. All animals underwent electrocardiographic studies: ZCC - 150-160 per minute, PQ - 0.07 sec, QRS - 0.04 sec, QT - 0.14 sec. Prongs P _i frequent (-), P _{II, III} (+) - 0.03-0.04; P _i <P _II <P _III , r ₂ 0.07 -0.25; R ₃ > R ₂ , R ₃ 0.08 - 0.35, T _II - high, ST segment - on the isoline.

The respiratory coefficient is 0.83, the respiratory rate at rest is 0.97 Hz (75 per minute).

A thorough study of the possible chronic toxicity of the embryonic antitumor modulator in dogs has been carried out.

The experiments were conducted on 24 outbred mature dogs (12 males and 12 females) with an initial body weight of 15.2-18.1 kg according to the Rules for Preclinical Evaluation of the Safety of Pharmacological Products, Moscow, 1998.

After three weeks of quarantine and acclimatization, the dogs received EPOM in 1 therapeutic dose for humans (0.03 mg / kg), 10-fold therapeutic dose and 25-fold dose (0.75 mg / kg).

The following parameters were studied: body weight dynamics, heart rate per minute, respiratory rate, ECG, rectal temperature, peripheral blood picture, serum biochemical parameters (glucose, total protein, creatine, urea, cholesterol, alkaline phosphatase activity, LDH, ALT, ACT ), biochemical parameters of urine (protein, glucose, bilirubin, urea, electrolytes). The studies were conducted 2 times - before the introduction of EPOM (on the 8th and 15th day after placing the dogs in the vivarium), 3 times during the experiment (on the 7th, 15th and 29th day).

This section "Study of the pharmacological activity and harmlessness of EPOM" occupies 114 pages of the report and is equipped with 43 tables. In order not to clutter up the textual part of the patent with digital data, we only note the general conclusion - the absence of toxicity of EPOM in an experiment on dogs.

Study of possible clastogenicity, mutagenicity and gonadotoxicity of EPOM

According to the requirements of the National Cancer Institute of the United States, compounds that can be used for cancer chemoprevention should not be toxic and mutagenic, and at the same time have an antimutagenic effect (Boone CW, Kellofl GJ Development of surrogate endpoint biomarkers for clinical trials of cancer chemopreventive agents .-- J. Cell Biochem., 1994, Suppl. 19, 10-22).

EPOM was tested on 20 rats and 80 mice for the presence of clastogenic properties according to WHO requirements. Even high doses of EPOM with repeated administration did not induce a cytogenetic effect in rodent bone marrow cells.

The study of antimutagenic (anticlastogenic) activity of EPOM was carried out in rats, which were administered EPOM 15 and 5 days later (total dose of 5 mg protein) before exposure to benzo (a) pyrene (40 mg / kg) or 7.12 - dimethylbenz (a) anthracene ( 10 mg / kg). The study of rat bone marrow cells after 24 hours showed a significant decrease in the number of megakaryocytes with chromosomal aberrations (1.5 times in both cases; p <0.001) compared with the control (only carcinogens).

Determination of the duration of the antimutagenic effect caused by EPOM was carried out in mice. EPOM was administered to animals according to the scheme described above (total dose of 1 mg protein). Cyclophosphamide (30 mg / kg, ip) was used as a mutagen, which was administered to mice on days 6, 7, 8, 11, 13, 16, 21, and 36 after the second administration of EPOM. It was shown that EPOM significantly reduces the clastogenic effect, revealed by the presence of micronuclei in bone marrow erythrocytes from 6 to 11 days (by 29-50%).

Thus, EPOM in the absence of clastogenic properties has a pronounced antimutagenic activity.

When studying the possible gonadotoxicity of EPOM, using the method of dominant lethal mutations (DLM), 20 male non-linear rats weighing 130-150 g were used. EPOM was administered in a dose corresponding to 200 human doses. 3 females were planted for each male. The DLM method is based on the study of embryonic mortality in the offspring of males exposed to an agent. The method reflects sensitivity: I week - mature sperm; II week - late spermatids; III - a week of medium and early spermatids. A total of 180 females (140-160 g) were used in the experiments - 90 in the experiment and 90 in the control. Fertilized females were opened on the 19-20th day of pregnancy and the number of live and dead embryos, the number of corpus luteum in the ovaries, and mortality before and after implantation were counted. Statistical processing of the results was carried out according to the Student-Fisher method and the X-square criterion.

Based on the data obtained, it should be concluded that EPOM at a dose of 15 mg / kg (200 human doses) does not affect male germ cells, i.e. does not have gonadotoxicity and does not induce DLS in rats.

Allergy and pyrogenicity tests

Possible allergenicity of EPOM was studied in guinea pigs weighing 250-330 g. They were immunized with b / w 2.5 mg EPOM in a volume of 1 ml of saline. Cattle serum was introduced as a control. After 21 days, the animals were also given an i.v. resolving dose of EPOM equal to a sensitizing one (5.0 mg). Serum of cattle - 5.4 mg. There were 10 males, 8 females.

The introduction of a permissible dose of EPOM did not cause the death of animals and symptoms of anaphylactic shock. Signs of mild allergization were detected in only 20% of guinea pigs. They were expressed in short-term scratching of the muzzle, ruffled hair and single sneezing.

The data obtained make it possible to evaluate the allergic reaction to the introduction of EPOM as weakly positive (“+”). In the control group treated with cattle serum, all animals died from anaphylactic shock.

To determine the pyrogenic reaction of EPOM, rabbits weighing 1.5–2.0 kg were used. Within 3 days before the experiment, temperature measurements were carried out using a medical thermometer.

On the eve of the experiment, the remains of feed and water were taken from the animals in the evening. The syringes and needles used, as well as the drug and solvent (saline) were sterile. EPOM in an amount of 5 mg was dissolved in 2 ml of sterile non-pyrogenic saline. 30 minutes before the introduction of the solution was measured body temperature of the animals. The solution was heated to 37 ° C and injected into the ear vein of rabbits for 2 minutes. After the introduction of the EPOM solution, three times at intervals of 1 hour, the body temperature of the animals was measured.

After administration of the solution, none of the three rabbits showed a temperature increase of more than 0.2 ° C in any of the three measurements. Therefore, the EPOM solution does not have pyrogenic properties.

Some clinical observations

In the traditional form of fetal (cell) therapy, patients are treated with specially treated living cells taken from the embryonic tissues of animals or humans. At a certain stage of embryogenesis, these cells are not immunogenic, since they have not yet “crossed” the species specificity barrier. But despite this, cell embryonic therapy cannot be considered an absolutely harmless form of treatment for various diseases. In this regard, isolated humoral components of cell therapy and, above all, proteoglycans as carriers of embryonic properties deserve attention. With this formulation of the problem, it becomes especially urgent to obtain a protein extract in a state as close to native as possible, and, of course, to use such techniques that completely exclude the contamination of viral and other pathogenic agents.

For more than 5 years, we have been observing patients from high cancer risk groups who received an embryonic antitumor modulator in order to prevent a possible neoplastic pathology.

At the site of subcutaneous injection (the inner surface of the forearm far from the contours of the vessels) during the first 2 days there is hyperemia and infiltration, which pass without a trace after 48 hours. The dimensions of the indicated local reaction are on average 4.5 × 6 cm. Only in isolated cases has there been an increase in body temperature within subfebrile (in 2 out of 100 patients). The lack of a local reaction alarms us in terms of a predisposition to oncopathology, namely, the tolerance of the immune system of this individual to oncofetal antigens - tumor markers.

Almost all patients after immunization note improvement in well-being, some general strengthening and anti-aging effect. Moreover, there is a small number of observations of the adjuvant therapeutic effect of EPOM on the course of diabetes mellitus, hypertension and high blood coagulation potential in coronary heart disease.

EPOM has also established itself as a means of anti-relapse immunotherapy, especially in cases when cancer patients after radical surgery for one reason or another refuse further chemoradiation treatment. This circumstance is extremely important, because, unlike chemotherapy drugs, EPOM does not cause immunosuppression, but rather increases both specific and non-specific antitumor protection.

In different series of induced (7.12 - DMBA and benz / a / pyrene) carcinogenesis and in experiments on transplanted tumor models of different histogenesis, EPOM completely prevented the onset of malignant tumors in 40-70%.

In groups of people with a high risk of cancer, EPOM vaccine therapy did not affect the level of CD4 + cells, but showed a tendency to increase both relative and absolute indicators of CD4 / SD8 cells.

The dynamics of immunological parameters in the cancer risk group vaccinated with EPOM is shown in table 4.

A significant increase after the vaccination of the number of T cells and a tendency towards an increase in EKK were noted. Vaccination did not significantly affect the relative and absolute values of B cells. Oncofetal antigens have an immunosuppressive effect. The data in the table indicate that the pool of embryonic proteoglycans that we obtained did not have a suppressive effect on subpopulations of lymphocytes.

Data on the use of EPOM and a method of prevention in a clinic

The method is as follows. A risk group is formed, taking into account the age of the individual, profession, pre-tumor disease, smoking, family predisposition, hyperfibrinogenemia, the number of tumor markers in peripheral blood; calculate the level of tumor risk.

Given these indicators, the individual is vaccinated with EPOM from a rate of 0.02 to 0.04 mg / kg body weight, most preferably 0.03 mg / kg body weight.

In the course of a 17-year experimental study of the antitumor activity and the safety of EPOM on models of 7.12-induced DMBA and benzo (a) carcinogenesis pyrene, as well as on histogenetically different transplanted tumors (Ehrlich ascites tumor, 37 sarcoma, 180, sarcoma, hepatitis, and ascites hepatoma Seidel, Walker carcinosarcoma, Pliss lymphosarcoma), it was demonstrated that EPOM in 40-70% of cases completely prevents the occurrence of malignant neoplasms.

In patients with high oncological risk vaccinated with EPOM, no oncological pathology was revealed during long-term observations. The modulator showed healing properties in pre-tumor conditions of female genitalia and has been successfully used in gynecology. It is also indicated for anti-relapse immunotherapy. In the observed patients, the biologically active dose did not affect the populations of T-helpers and T-suppressors. Even in the case of a ten-fold excess of the dose, an increase in the number of T-suppressors was observed against the background of an unchanged number of T-helpers.

Example 1

Patient M., 55 years old. He smoked for a long time, the diet was wrong, by the nature of the work he was an administrative worker, hypodynamic, had prostate gland adenoma (the amount of prostatic antigen in the blood was 7.8 ng / ml, fibrinogen A - 540 mg /%, B - put).

The oncological risk level was calculated, which corresponded to the degree of moderate risk (23 units). Then vaccination with a modulator is made. Dose - 0.03 mg / kg. The skin reaction is positive (3 × 2 cm, slight infiltration, on the 3rd day the local reaction has passed). EPOM vaccination was carried out annually for 5 years. The lifestyle has not changed, humoral immunity indicators and tumor markers returned to normal, and no neoplasms were found.

Example 2

Patient K., 44 years old, nurse, smoked, malnutrition, mother died of ovarian cancer, blood count is normal. The pre-tumor state of the cervix was revealed: epithelial dysplasia of the I-II degree, leukoplakia of the vulva.

The level of oncological risk was calculated, which corresponded to the degree of moderate risk (21 units), then annual vaccinations with a modulator (5 years) were carried out. The lifestyle has not changed. Objectively: the cervical epithelium is in a normal state; no relapse was observed after the treatment; positive changes were detected in the immune system. No tumors were found.

Claims (4)

1. A method of obtaining an embryonic antitumor modulator, characterized in that the embryonic native tissue is removed, washed, kept in alcohol, crushed, frozen in liquid nitrogen, homogenized to a powder state, diluted with water and disintegrated in a cooled ball mill until revealing rectified cell nuclei (more 80%), the smallest cytoplasmic clippings and fragments of fibrous structures, process the resulting homogenate with ultraviolet light, centrifuge and process the resulting supernatant with alcohol to isolate glycoproteins in the precipitate, process the glycoprotein precipitate in a ball mill and separate them into the precipitate by centrifugation and then sterilize the filtration and lyophilization of the target product. 2. The embryonic antitumor modulator obtained by the method according to claim 1, containing, according to the data of analytical electrophoresis in a 10% Davis polyacrylamide gel, 5 protein fractions in an amount of from about 42 to 57 wt.%, As well as about 1.5 wt.% Free and about 8-10 wt.% bound carbohydrates, alanine aminotransferase (ALT) - about 1.4 u / l, aspartate aminotransferase (AST) - about 71.9 mmol / l, lactate dehydrogenase (LDH) - about 3.0 u / l, alkaline phosphatase (AL) - about 24.7 units, and trace elements: Mg - about 1.7 mmol / L, K - about 0.96 mmol / L, Ca - colo 0.65 mmol / L, P - about 0.52 mmol / L, (according to the Hitachi biochemical analyzer) and CEA - approximately 1.4-1.7 ng / ml, AFP - approximately 95.0-99.0 ng / ml, CG - approximately 2.2-5.5 mIU / ml, TWG - approximately 45.0-50.0 ng / ml, Ca-125 - approximately 13.5-14.3 U / ml, Ca- 19.9 - approximately 79.8-102.2 U / ml. 3. The embryonic antitumor modulator according to claim 2 for the manufacture of a medicament useful to individuals with a suspicion of a possible cancer disease to prevent the disease and its relapse. 4. A method for the prevention and treatment of precancerous pathological conditions, characterized in that it includes vaccination of persons belonging to groups of high cancer risk according to the test results, and the vaccination is carried out by the embryonic antitumor modulator described in paragraph 2, which is administered subcutaneously once a year in an effective amount, preferably at a dose of 0.04-0.01 mg / kg

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