RU2341272C1 - Nonspecific immunotherapy agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: declared is nonspecific immunotherapy agent containing embryonic antitumor modulator (EATM) as active agent and pharmaceutically acceptable carrier. EATM is made of embryonic substances by cryodestruction, extraction and sedimentation in spirits of increasing concentration and contains hyaluronic acid complex, tumour-associated antigens of normal embryogenesis. Agent can be produced in the form of solution, liniment, cream, tooth paste.

EFFECT: it is possible to use as highly effective agent for nonspecific immunotherapy.

5 cl

Description

The invention relates to the field of pharmacology, medicine. 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 non-specific immunotherapy.

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.

The prior art immunotropic agent obtained from plants with the addition of monofleur honey for non-specific immunotherapy (RU 2133616, 07.27.1999). However, this tool does not have high specificity.

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). 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 immunomodulating properties, antiviral and other activities that explain 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 diseases of various etiologies, other than tumor. This problem is solved by increasing the natural resistance of the individual organism.

To achieve the solution of the problem allows the use in the proposed method of a new embryonic antitumor modulator, which contains a wide pool of normal fetal proteoglycans.

The new embryonic antitumor modulator Mkrtchyan proposed in this invention has an interferonogenic and immunomodulating effect, as well as antiviral activity.

The reason for this conclusion is the data obtained by the author of the reaction of suppression of adhesion of leukocytes (RPPL), passive hemagglutination reaction (RPHA), complement fixation reaction (CSC) and delayed-type hypersensitivity reactions (RGT).

During the observation of patients it turned out that EPOM has a tonic and anti-aging effect on the body.

The following discloses a method of obtaining an embryonic antitumor modulator, providing a target product having the properties and qualities disclosed 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").

T.O. an aspect of the invention is the use of an embryonic antitumor modulator according to this invention for the manufacture of a non-specific immunotherapy agent.

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 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 RS-6 refrigerator centrifuge for 15 minutes at 6000 rpm.

96 ° wine alcohol is added to the transparent 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 a 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 (ALP) - 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-911 biochemistry analyzer, 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

Sa-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 kD.

Polyacrylamide gel electrophoresis - the presence of 5 protein fractions per mol. weighing 72, 67, 58, 50 and 40 kD. Protein fraction with mol. mass of 72 kD had 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 testing 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.

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.

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 solution; 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.

0.1 ml of a 0.1% solution of ninhydrin is added to 5 ml of solution A and heated to boiling. 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 inclusion 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).

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.

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.

The maximum possible doses were also used for the detection of LD ₅₀ EPOM in rats: sc - 5.0 mg EPOM in 1.0 ml of solution and ip - 5.0 mg 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 ranging from 2 to 5 g and in rats from 20 to 23 g was established. A general and biochemical analysis of blood 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. Animals were injected with 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.

Conclusion: 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 part. (-), 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).

The general conclusion is the lack of toxicity of EPOM in an experiment on dogs.

In this case, EPOM in the absence of clastogenic properties has a pronounced antimutagenic activity.

In addition, 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 used syringes and needles, as well as the drug and the solvent (saline) were sterile, EPOM in the 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.

Almost all patients after receiving subcutaneous injections noted improvement in well-being, some general strengthening and rejuvenating 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.

In addition, in people who for one reason or another refuse further chemoradiotherapy, the use of EPOM also has a beneficial effect. This circumstance is extremely important, because, unlike chemotherapy drugs, EPOM does not cause immunosuppression, but rather increases both specific and non-specific antitumor protection.

There was a significant increase after the introduction of EPOM, the number of T cells and a tendency to increase EKK. The introduction of EPOM did not significantly affect the relative and absolute indices of B cells. Oncofetal antigens have an immunosuppressive effect. The data indicate that our pool of embryonic proteoglycans did not have a suppressive effect on lymphocyte subpopulations.

EPOM application data and method of use in a clinic

The claimed agent as an active agent contains EPOM in an effective amount, and as a carrier for producing liniment it contains a base of substances selected from the group: tamoline, glycerin, 3-ethanolamine. Liniment is obtained by well-known methods. The tool has a stimulating effect on metabolic processes and restoring abilities of connective tissue, as well as integumentary epithelium of the skin and mucous membranes. It has antioxidant, antiviral and cytokinmobilizing activity. Experimental studies on rubbing liniment into the skin established a preventive effect on the development of transplantable tumors.

Indications: Gynecology. Cervical erosion, cervical dysplasia, cracks, vulvar kraurosis. Apply in the form of tampons or spongy applications for 2-3 weeks.

Dermatology. Non-healing trophic ulcers of the lower leg against venous insufficiency and induction of the skin. Adjuvant therapy for psoriasis, atopic dermatitis, herpes zoster.

Cosmetology. Smoothing wrinkles and crow's feet, increasing turgor of the skin of the face and neck, anti-aging effect, stimulation of natural antitumor resistance, especially in the perimenopausal period.

The cream product as a base contains lanolin and is obtained by well-known methods.

The toothpaste preparation is obtained by introducing EPOM into any toothpaste. Unlike existing toothpastes containing hyaluronic acid, the inventive paste includes a wide pool of embryonic proteoglycans that increase the regenerative potential of gum tissue and can be used to treat and prevent periodontal diseases. Periodontal disease was treated in 40 patients. Treatment in the form of intra-gum injections, curettage using a variable frequency ultrasound for rapid absorption. Assessment of treatment results was carried out on the basis of clinical, radiological and dopplerographic indicators. Of the 25 patients examined, in 11 cases the effect is good, in 9 - moderate, and in 5 - the state is unchanged. Positive dynamics was noted mainly in those patients who continued to use toothpaste enriched with EPOM. Studies were conducted at the University (YSMU) dental clinic.

Nonspecific immunotherapy of non-oncological diseases was performed as a single injection in 39 patients. A good effect was obtained in hospitalized patients with coronary artery disease, with pronounced changes in the coagulogram and resistant to therapeutic agents. We can definitely talk about improving the well-being of patients with hypertension, with emotional stress, migraine. With periodic illness (Mediterranean family fever), 26 patients received 1 injection of EPOM. 19 patients were followed up for 2 or more years, 11 of them did not have seizures within 2 to 4.5 months. In no case was colchicine used, and not a single patient was diagnosed with amyloid dystrophy.

There are no side effects of the agent in the effective dose of EPOM used, calculated from 0.02 to 0.04 mg / kg body weight, most preferably 0.03 mg / kg body weight.

Claims (5)

1. The tool for non-specific immunotherapy, characterized in that as the active agent contains embryonic antitumor modulator (EPOM) in an effective amount and a pharmaceutically acceptable carrier. 2. The tool according to claim 1, characterized in that it is made in the form of a solution, cream, liniment, toothpaste. 3. The tool according to claim 2, characterized in that in the form of liniment as the basis contains lanolin. 4. The tool according to claim 1, characterized in that in the form of liniment as the basis contains substances selected from tamoline, glycerol, 3-ethanolamine. 5. The tool according to claim 2, characterized in that in the form of a cream as a base contains lanolin.