RU2619208C2 - Method for prophylactic treatment in case ofneoplastic processes involving skin tissue, such as melanoma, basal cell and squamous cell carcinoma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for prophylactic treatment in case of neoplastic processes involving skin tissue, such as melanoma, basal cell carcinoma, squamous cell carcinoma, a pharmaceutical preparation is used comprising a protein-polypeptide complex (BPC) in the form of cosmetic compositions, such as day or night creams, gels, alone or in liposomal form, and also as a photoactive or photoprotective compositions as the active substance, daily at a concentration of 0.001-0.05 mg/ml 1-2 times a day with a dose of 10-20 days course not less than 4 courses per year. The active substance is a protein-polypeptide complex produced of hoofed farm animal's nerve and skin embryonic tissue homogenate and containing negatively charged weak-acid and neutral proteins and polypeptides with molecular weight from 5 to 200 kDa and containing an average molecular weight fraction within 10 to 120 kDa of not less than 80%, with the total protein concentration of 0.2-4.2 mg/ml, the ultraviolet spectrum absorption peak of the solution at wave length± 2805 nm, the UV-visible spectrum peak at wave length 274-284 nm, the presence of specific bands in the range of pl from 4.2 to 8.4 in isoelectric focusing in 5% polyacrylamide gel.

EFFECT: invention provides prevention of neoplastic skin diseases.

2 dwg, 6 ex

Description

The invention relates to medicine, in particular to oncology, pharmacology, veterinary medicine, and can be used for prophylactic treatment and prevention of neoplastic processes of skin tissue - squamous and basal cell skin cancer, as well as melanoma and other benign and malignant skin tumors, using a pharmaceutical preparation comprising a protein-polypeptide complex obtained from homogenates of the nervous and skin tissues of ungulates of farm animals.

Currently, against the background of a steady increase in diseases of the skin tissue of autoimmune, vascular, toxic, infectious, traumatic genesis, as well as iatrogenic complications after the use of anti-aging drugs, cosmetics and procedures, there are no drugs of the direct repair group specific to skin tissue.

Various forms of skin cancer (skin cancer) are known, such as, for example, melanoma, basal cell carcinoma (basal cell carcinoma), squamous cell carcinoma of the skin.

Treatment with melanoma, in particular, is a difficult task due to the rapid, early onset of dissemination. It should be carried out only in a specialized institution. Treatment for melanoma is predominantly surgical. At stages 1-2, a radical excision of melanoma is performed.

Known vaccines against melanoma, which are allogeneic tumor cells and their lysates, autologous tumor cells, various antigens (MAGE-1, MAGE-3, MART-1, gr100, etc.). These vaccines increase the activity of cytotoxic lymphocytes in relation to malignant cells, activate the proliferation of T-lymphocytes, tumor infiltration of CD8 +.

It is known that antitumor drugs according to the mechanism of action and chemical structure are divided into several groups: alkylating compounds, antimetabolites, antibiotics, substances of plant or animal origin, hormonal preparations, enzymes and others.

The antitumor effect of these compounds is based on the violation (suppression) of the exchange of nucleic acids, especially nuclear nucleotides, which leads to the cessation of cell pressure and tumor growth (patent US 4622325, 11.11.1986; copyright certificate SU 1208619, patent RU 2040255).

But, since known antitumor drugs only contribute to the suppression of cellular activity, without affecting the causes of the tumor, relapse of the tumor and metastasis are not excluded.

Basal cell carcinoma is a skin cancer with locally destructive growth that arises from the epidermis or hair follicles and rarely metastasizes (according to WHO 1974). According to a number of authors (Shanin A.P. Skin tumors, their origin, clinic and treatment, L., 1969, p. 154-172), basal cell carcinoma occurs more often than other skin neoplasms and occupies an intermediate position between benign tumors and squamous cell carcinoma. It accounts for 77 to 90% of malignant epithelial tumors of the skin (Venkey T., Shuvgar J. Malignant tumors of the skin, Budapest, 1962, p. 12-15). Known methods for treating skin basal cell carcinomas: surgical excision, electrocoagulation, cryodestruction, radiation therapy, laser therapy, chemotherapy, external treatment with anticancer drugs (Kogan M.G. ., 1984, p. 24) are not effective enough, cause relapses of the disease and are accompanied by late postoperative complications in the form of deep skin defects and.

From RU 2179000, 02/10/2002, a method for the complex treatment of skin basalomas is known.

It is proposed to begin treatment with the intake of human leukocyte interferon in the form of inhalations of 20 thousand units, every other day, for a course of treatment of 20 procedures. Against the background of immunocorrection, radio-wave removal of basal cells was performed using the Surgitron apparatus manufactured by Ellman international (USA). The method has a qualitatively new approach and high efficiency in the complex treatment of skin basal cell carcinomas and correction of immunological parameters, leads to a high percentage of cure, characterized by the absence of relapses and complications of therapy.

However, this method does not have a wide spectrum of action in the development of neoplastic and reparative processes of the skin and in the prophylactic treatment and prevention of various oncological skin diseases.

It is well known for a specialist in the treatment of various diseases, including cancer of the skin, i.e. with the development of neoplastic processes of skin tissue (melanoma, basal cell, squamous cell carcinoma), which is understood as:

- preventive treatment

- prevention in relation to treatment methods,

- in particular, the treatment of cancer of the skin, and their prevention,

prophylactic treatment and prophylaxis are understood in the initial development of neoplastic processes of skin tissue, such as melanoma, basal cell and squamous cell carcinoma.

A special direction in the treatment of malignant tumors is the development of methods for treating cancer using preparations from animal raw materials, mainly from the embryonic organs of animals and humans (DE 228739, 04.29.1983; DE 2600442, 08.01.1976).

Preparations obtained from animal tissues have a significant advantage over known cytostatics of chemical origin, as they have a weak hepatotoxic and teratogenic effect. However, the use of these drugs is constrained due to the limited raw material base and because of the danger of developing allergic complications due to the fact that the latter have visible antigenic specificity.

In particular, from the application of France No. 2451193, 03/13/1979, a method of treating cancer using embryonic tissue homogenate is known.

The known method consists in the following: with external localization of the tumor process, the patient is applied, in the form of applications, a homogenate (suspension) of embryonic tissue mixed with a suitable pharmaceutical excipient, and in the case of internal localization of the tumor, 10 ml of 10% aqueous saline homogenate of the embryonic tissue is administered parenterally 1-2 times a day for 4-12 weeks. An isotonic sodium chloride solution is used as a filler. After a month of treatment, the frequency of administration of embryonic tissue homogenate is gradually reduced to 1-2 times a week and treatment is continued for another 2-3 months. The method is especially effective in the external localization of the tumor, since the separation of the tumor from neighboring healthy tissues is often observed, which favors the successful surgical removal of the tumor. The nearest antitumor effect is observed 2-3 weeks after the start of treatment. The average duration of remission is 9 months.

The disadvantage of this method is that the use of xenogenic embryonic tissue is limited by species antigenic specificity. In addition, the proposed homogenate for embryonic tissue treatment is a mixture of an unknown composition containing fetal proteins, peptides, nucleic acids and other components. Therefore, the proposed tool can not be standardized in composition, since it will depend on the source tissue and during treatment, side complications may occur due to toxic reactions.

From RU 2105567, 02.27.1998, a method is known for treating malignant tumors, including cancer of the skin, lips, oral mucosa, with long-term healing wounds of trophic ulcers by administering alpha-fetoprotein (AFP) in a single dose of 500-5000 IU / kg of patient weight 1-2 times a day for 4-6 weeks using physiological saline (sodium chloride solution) as a carrier.

For ME (international unit) AFP take the amount of protein equal to 1.25 ng. Further on, all data will be given in ME.

AFP is an oncofetal protein that is obtained from human abortive material (natural alpha-fetoprotein) or from smallpox vaccine on human embryonic cells (recombinant alpha-fetoprotein). In the claimed method, any type of alpha-fetoprotein (natural or recombinant) can be used. AFP is not pyrogenic, does not cumulate in the body, does not have teratogenic, anaphylactogenic, embryotoxic, allergenic and histamine-like properties.

However, this method is based on the use of abortive human material, which limits its use.

From RU 2240810, November 27, 2004, a method is known for the prophylaxis and treatment of precancerous pathological conditions, including vaccination of persons belonging to high cancer risk groups according to test results with an embryonic antitumor modulator (EPOM), which is administered subcutaneously once a year at a dose of 0.02- 0.04 mg / kg. The EPOM modulator contains a wide range of oncofetal antigens. The preparation was obtained from human and animal embryonic tissues and contains a pool of glycoprotein proteins extracted by the method of alcohol deposition. However, this drug is not used for the treatment and prevention (prophylactic treatment) of skin cancer.

A protein-peptide antitumor preparation (composite) is known from RU 2283129, 09/10/2006, containing a heat shock protein of the HSP70 family and a tumor-specific peptide non-covalently linked to it, selected from the group: MAGE-A1, - A2, - A3, etc. 100 , HER2 / neu, EGFRvIII, or a fragment of any of them.

This cell preparation is used in a method of immunotherapy or immunoprophylaxis of tumors.

Thus, as follows from the prior art, various protein-peptide preparations are widely used in medicine and pharmacology.

So, in particular, from RU 2485132, 06/20/2013 known protein-polypeptide complex (BOD), which has antihypoxic, tissue-specific reparative action on the central and peripheral nervous system, and a pharmaceutical composition based on it.

BOD is obtained from the nervous and skin tissues of ungulates of farm animals.

It has not been used to treat skin cancer.

The technical task of the claimed invention is to increase the effectiveness of prophylactic treatment (prophylaxis) of neoplastic processes of skin tissue, as well as expanding the arsenal of means for the prophylactic treatment (prophylaxis) of these skin diseases using the above known embryonic protein-polypeptide complex with the properties of a cell differentiation regulator and a high overall biological activity.

The technical result in solving this technical problem is achieved by the method of prophylactic treatment of skin cancer with the development of neoplastic processes of skin tissue, such as melanoma, basal cell carcinoma, squamous cell carcinoma, i.e. at various early stages, the development of these diseases is possible, associated with exposure to the skin of various adverse factors.

So, the stated technical problem and the technical result achieved are achieved by the claimed method of prophylactic treatment in the development of neoplastic processes of skin tissue, such as melanoma, basal cell, squamous cell carcinoma, including the use of a pharmaceutical preparation, comprising a protein-polypeptide complex as an active active ingredient and pharmaceutically acceptable carriers, such as a diluent, possibly including a buffer solution, as well as traditionally used pharmaceutical highly acceptable carriers in the preparation of cosmetic compositions, such as day and night creams, gels alone or in liposome form, as well as photoactive or photoprotective compositions, daily, in a concentration of 0.001-0.5 mg / ml 1-2 times a day with a course dose of 10-20 days at least 4 courses per year, while the protein-polypeptide complex obtained from the homogenate of the embryonic nervous and skin tissues of ungulate farm animals and containing a negative charge is used as the active active ingredient weakly acidic and neutral proteins and polypeptides with a molecular weight of 5 to 200 kDa and with a content of a fraction with an average molecular weight in the range of 10 to 120 kDa of at least 80%, a total protein concentration of 0.2-4.2 mg / ml, s the maximum absorption of the ultraviolet spectrum of the solution at a wavelength of 280 ± 5 nm, the presence of a peak at a wavelength of 274-284 nm in the UV-visible region of the spectrum, the presence of specific bands in the range of pl from 4.2 to 8.4 with isoelectric focusing of 5% - Mr. polyacrylamide gel.

от 4,2 до 8,4 при изоэлектрофокусировке в 5%-ном полиакриламидном геле и фармацевтически-приемлемый носитель, например разбавитель (вода для инъекций, физиологический раствор раствор хлорида натрия), а также традиционные носители (фармацевтически приемлемые) при составлении мазей, гелей, кремов и т.д. Причем разбавитель может включать буферный раствор и вспомогательные вещества - высокомолекулярные соединения, относящиеся к классу консервантов (например, нипагин) и солюбилизаторов (например, полисорбат-80, ТВИН-10) в достаточных концентрациях. Субстанция получена путем ионообменной хроматографии фильтрата экстрагированного гомогената тканей кожи, головного и спинного мозга эмбрионов копытных сельскохозяйственных животных сроком гестации от середины первой трети до середины последней в присутствии буферного раствора, детергентов, ингибиторов протеолиза и солюбилизаторов.In the claimed invention, as the main active substance (biologically active substance), a known protein-polypeptide complex is used, which is a biologically active protein-polypeptide complex (hereinafter BOD) with a molecular weight of its constituents ranging from 5-200 kDa and the content of the average molecular fraction in the range from 10 to 120 kDa is not less than 80%, the total protein concentration of 0.2-4.2 mg / ml, with a maximum absorption of the ultraviolet spectrum of the solution at a wavelength of 280 ± 5 nm, the presence of a peak at no wavelength 274-284 nm in the UV-visible region of the spectrum, the presence of specific bands in the range of

from 4.2 to 8.4 when isoelectric focusing in a 5% polyacrylamide gel and a pharmaceutically acceptable carrier, for example, a diluent (water for injection, saline solution of sodium chloride), as well as traditional carriers (pharmaceutically acceptable) in the preparation of ointments, gels creams, etc. Moreover, the diluent may include a buffer solution and excipients - high molecular weight compounds belonging to the class of preservatives (e.g. nipagin) and solubilizers (e.g. polysorbate-80, TWIN-10) in sufficient concentrations. The substance was obtained by ion exchange chromatography of the filtrate of the extracted homogenate of skin tissue, brain and spinal cord of ungulates of farm animals, gestational period from the middle of the first third to the middle of the latter in the presence of a buffer solution, detergents, proteolysis inhibitors and solubilizers.

The biologically active protein-polypeptide complex used in the claimed invention and the method for its preparation are protected by the applicant of this invention and are described in RU 2428196, 09/10/2011.

Moreover, in RU 2445106, 03.20.2012, the applicant of the present invention protected a pharmaceutical composition intended for the treatment of diseases of the central and peripheral nervous system of the vascular, traumatic, hypoxic and autoimmune genesis, which contains the indicated biologically active protein-polypeptide complex as an active active substance.

The applicants of the claimed invention as a result of additional studies have revealed the possibility of using it as an active active substance in the method of prophylaxis and prophylactic treatment in the development of neoplastic and reparative processes of the skin, cancer of the skin, such as melanoma, basal cell carcinoma (basalt cell carcinoma), squamous cell carcinoma of the skin.

As a result of additional research and experiments, the applicant developed a regimen for the treatment of these diseases using the specified protein-polypeptide complex with certain specified characteristics.

Attempts to use extracts of embryonic tissues for the prophylactic treatment (prophylaxis) of cancer in general and the skin in particular have been known since time immemorial. All of them are united by one thing - the pronounced antitumor effect of proteins - differentiation factors produced by cells of embryonic tissues. The main function of these proteins - with the rapid and simultaneous division of a huge number of cells for active growth of the body as a whole - is to ensure the correct differentiation of cells and their timely apoptosis.

The main feature characterizing tumor cells is a violation of the final differentiation and loss of the ability to apoptosis.

Proteins and polypeptides are the main biological molecules that provide the main signaling to evolutionarily fixed pathways of tissue development and differentiation in mammalian embryogenesis. Obtaining fractions of native protein and polypeptide ensembles from embryonic tissues is a very difficult and expensive task, the solution of which depends on many sequential actions requiring the exact execution of each regulation item under strictly defined conditions, since the severity of the antitumor effect of the target fractions, their toxicity (allergenicity, immunotoxicity ) and stability directly depend on the degree of nativeness of the obtained protein-polypeptide molecules.

The most optimal solution for preserving the nativeness and stability of differentiation factors for their use in the treatment of neoplastic skin lesions turned out to be a method for producing a protein-polypeptide complex, which allows one to obtain a protein-polypeptide complex with the necessary properties that can be used in the claimed method of prevention and treatment in the development of neoplastic and reparative processes of the skin, cancer of the skin (a method of obtaining this protein-polypeptide complex ksa and the protein-polypeptide complex itself are described in patent RU 2428196, as mentioned above).

A pharmaceutical preparation is usually produced in the form of a solution, for example, as a 0.1 mg / ml preparation solution, on the basis of which pharmaceutical preparations are prepared in the form of solutions, gels, creams, liposomal emulsions of the required concentrations of BOD in them according to the claimed method in pharmaceutically acceptable carriers in combination, if necessary, with auxiliary additives (medicinal additives), traditionally used in medicine, in the pharmaceutical (including cosmetic) industry.

So, according to the claims used in the claimed "Method of preventive treatment ..." the pharmaceutical preparation can be used in the form of photoprotective and photoactive compositions, i.e. in the form of photoprotective, photoactive compositions (creams, ointments, gels, solutions, etc.). They contain various well-known photoprotective additives, photoactive additives, in addition to the indicated active active ingredient - protein-polypeptide complex (hereinafter - BOD) and a pharmaceutically acceptable (suitable) carrier, traditionally used in the preparation of pharmaceutical preparations of various diluents, as well as traditionally used carriers in the preparation of compositions ( as pharmaceuticals in the form of creams, ointments, gels, solutions) also various well-known photoprotective and photoactive additives, traditionally used at times personal, for example, photoprotective (in particular, sunscreen) creams, ointments and in photoactive compositions.

Such photoprotective known additives include, in particular, various carotenoids, beta-carotene, vitamins E, C, salicylic acid esters, i.e. substances (as target additives) that absorb UV rays.

In particular, it is well known that the photoprotective (and anti-inflammatory) effect is exerted by a complex of substances from plant extracts: tea, coffee, cocoa, calendula, frankincense, chamomile, eucalyptus; vegetable oils: sunflower, cloves, lavender, dill, mint and fir, as well as an extract of propolis, beeswax, vitamins A and E, glycerin and citric acid. The specified complex of substances leads to a synergistic photoprotective effect, thereby providing a high photoprotective effect of the hard UV region. The optimal light-filtering effect is provided in the range of 270-360 nm.

It is also well known that photoactive compositions include compositions capable of independently generating laser radiation under the action of laser light and containing various photoactive additives, for example 3,6-dianhydro-D-sorbitol (SORB additive), photosensitizers (used in radiation therapy, for example) in particular 5-aminolevulinic acid and other known photosensitizers, as photoactive additives.

In particular, it is well known that a photoactive preparation (compositions used in particular for photodynamic exposure) is exposed to light irradiation immediately before application to tissues using a special device. In the process of this, the composition is activated, transforming from photoactive to photoactivated form, acquiring new useful biologically active properties. Then the photoactivated composition is applied to the target tissue, where it has a therapeutic and / or cosmetic effect. A photoactive composition (photoactive agent) can have various liquid and soft forms: true, colloidal and micellar solutions, gels, creams, mono- and polydisperse systems, emulsions, vesicular and liposome systems, etc. In the proposed method, the tissues are exposed to already photoactivated product, which is in a biologically active state.

The protein-polypeptide complex can be introduced together with other immunomodulators, angiogenesis suppressors, substances that inhibit the growth of tumor cells, such as DNA and RNA polynucleotides, chaperones HSP 60, HSP 70, HSP 90, tumor-specific peptides from the MAGE group, such as MAGE-A1 , MAGE-A2, MAGE-A3, et al. 100, HER2 / neu, EGFRvIII or fragments thereof; cytostatics and other inhibitors of tumor growth in effective doses.

The protein-polypeptide complex is used in the form of an ointment or gel with the preservation of the indicated concentrations, volume ratios, single and course dosage regimens. As an ointment or gel base, or creams, including photoprotective (sunscreen) compositions (creams), a hydrophilic pharmaceutically acceptable carrier (for example, petrolatum, lanolin, cosmetic polyethylene glycol waxes, etc.) is used.

The protein-polypeptide complex in the treatment using applications can be used in the form of a liposomal emulsion, alone or as part of an ointment or gel, while maintaining the same concentration, volume ratios, single and course dosage regimens.

When implementing the method of introducing the protein-polypeptide complex through the skin, the method of electrophoresis, phonoresis, pharmacophoresis is used independently or as part of a liposomal emulsion, ointment or gel, while maintaining the same concentration and volume ratios, single and course dosage regimens.

Antineoplastic activity with the direct use of BOD or as part of pharmaceutical compositions, dietary supplements (biologically active additives) - is manifested in the activation of cascades of appoptosis and necrosis of tumor cells with simultaneous stimulation of autophagy processes. The mechanisms of action of the complex were proved in in vitro cell experiments on cell lines, in experiments with transplantable tumor cell lines in laboratory animals, in a clinic for patients with basal cell, squamous cell carcinoma of the skin and melanoma.

Below are examples illustrating the claimed group of inventions, but not limiting it.

In this case, for example, an indication of the concentration (content) of the protein-polypeptide complex (BOD) in the examples is not more than 0.1 mg / ml means 10 mg of BOD in 100 ml, i.e. 0.01% BOD solution (taking into account the error in the density of water (as a pharmaceutically acceptable carrier, diluent) or ointment, gel, etc. bases.

Example 1

Patient R., 49 years old, the patient considers herself about 4 months.

Turned to the clinic.

Statuslocalis

On the skin of the corner of the eye, a pink plaque d = 1.5 cm with a hemorrhagic crust in the center.

Preliminary diagnosis

Bazalioma of the angle of the left eye of the II art., II class. column

Scraping from a plaque of the left corner of the eye was performed. Cytology No. 1217/12,

conclusion: basal cell carcinoma.

Treatment: apply the protein-polypeptide complex in 50 mM glycine-phosphate buffer with a protein concentration of 0.1 mg / ml, and in an amount of 1.5 ml directly to the affected skin and around, up to 5 mm from its edge, daily with a course of 14 procedures , with a repeat of the course after 10-14 days. The complex was applied to a sterile four-layer gauze cloth, previously the skin of the focus area was cleaned with a 20% alcohol solution.

After a course of applications, the patient was referred for treatment to the department of radiotherapy, at the end of radiation therapy they repeated the same 2 courses of applications with an interval between courses of 14 days.

A 3-year follow-up by an oncodermatologist confirmed a complete remission of the disease.

Example 2

A 34-year-old patient, for 3 years, observes the appearance and increase in the form of a spot of pink formation up to 1.0 cm in diameter with the appearance of a red seal in the center.

Statuslocalis

On the skin of the forehead, in the center, closer to the scalp, a flat slightly depressed erosion of red color is determined, wet in the center up to 1.2 cm in diameter.

Preliminary diagnosis

Bazalioma of the skin of the forehead II tbsp.

An erosion scraping was performed. Cytology No. 292/13, conclusion: basal cell carcinoma.

Treatment: directly to the area of the affected skin and around, up to 5 mm from its edges, an ointment composition was applied, consisting of polyethylene glycol 400 and 1500 in a ratio of 3: 1 and a protein-polypeptide complex at a concentration of 0.1 mg / ml, 1 time per day 12 days with a repeat of the course after 7-10 days, up to 6 double courses per year. The composition was applied in a thin layer on a sterile gauze cloth, which was fixed with a patch. After an annual course, the tumor diameter decreased to 3 mm, the patient was referred for treatment to the department of photodynamic therapy, where 1 session with radochlorin was performed. After 3 weeks, after removing the bandage, a faint pink spot is noted at the site of the focus, slightly distinguishable from neighboring skin areas. A 2-year follow-up by an oncodermatologist confirmed a complete remission of the disease.

Example 3

Patient 45 years old; within 1 year, the appearance and increase in the form of a pink spot of the neoplasm up to 1.0 cm in diameter and compaction is observed.

Preliminary diagnosis: skin melanoma

Preventive treatment is carried out using a protein-polypeptide composition in the form of a pharmaceutical product containing BOD in a sterile solution (physiological solution - isotonic sodium chloride solution) with a protein content of 0.001-0.05 mg / ml (0.001 mg / ml, 0.003 mg / ml, 0 , 04 mg / ml) of the initial solution and three 10-fold diluted physiological solutions. Immediately after surgical treatment (removal of the tumor), the protein-polypeptide complex was systemically injected with subcutaneous injections for 14 days, when it was kept in solution (water for injection) 0.2 mg, 1 time per day, with a break between courses of 14 days, a total of 6 courses. The surgical wound healed by first intention, the sutures were removed on the 6th day, the suture is soft, inconspicuous.

A 2-year follow-up by an oncodermatologist confirmed a complete remission of the disease.

Example 4

Determination of the effect of BOD on the growth of normal human skin fibroblasts (FBC) and B16 melanoma cells.

Methodology

Cell culture. B16 melanoma cells were obtained from the collection of the Institute of Molecular Biology of the Russian Academy of Sciences. Skin fibroblasts of healthy people (FBCh), which were used in experiments only at 2-8 passages, were obtained from the biotechnology laboratory of the 1st Moscow State Medical University. THEM. Sechenov. Cell cultivation was performed under standard conditions (5% CO ₂ , 37 ° C). DMEM medium and fetal calf serum (ETS) were used (Gibco, USA, obtained through Invitrogen, Moscow).

To determine the cytotoxic antitumor activity of BOD, the cells were passaged in 96-well plates (Costar) in 100 μl of medium with a density of 5 × 10 ³ cells / well. Cells were preincubated in tablets for 24 hours to adapt before supplementation. BOD was added to the cells in the concentration range from 0.001 to 0.2 mg / ml in 4 repetitions and after 72 hours the experiment was stopped. The number of surviving metabolically active cells was measured using the MTT test. The method is based on the fact that mitochondrial dehydrogenases of only metabolically active cells convert the MTT reagent into stained formazan crystals. After 10 μl of a 0.5% MTT solution was added to the cultured cells, they were incubated for 3 hours at 37 ° C. Then, formazan crystals were dissolved in 0.1 ml of DMSO with shaking on a Titramax 101. The optical density of the solutions was measured on an EX multiscan (Lab. System, Finland) at a wavelength of 540 nm. The number of surviving cells was calculated as a percentage of the control, which were cells cultured without the addition of L-asparaginase preparations.

Flow-cytofluorometric analysis of the cell cycle and determination of the number of apoptotic cells after treatment with FBC and tumor cells of BOD was performed according to the standard method (Griffin C., Hamm C., McNulty J., Pandey S. Pancratistatin induces apoptosis in clinical leukemia samples with minimal effect on non -cancerous peripheral blood mononuclear cells // Cancer. Cell. Int. - 2010. - Vol. 10. - PP. 6. doi: 10.1186 / 1475-2867-10-6).

For this, fibroblasts and tumor cells (0.5 × 10 ⁶ ) were passaged into 25 cm ² mattresses containing 5 ml of growth medium and cultured under standard conditions. After 24 hours, BOD preparations were added to the medium. 72 hours later, a cell pellet was collected for analysis in a cytofluorimeter after treatment with propidium iodide (Sigma). For flow cytofluorimetry, a FACSAria I cytofluorimeter (Becton Dickinson, USA) was used.

Statistical processing of the results was carried out using the Statistica 6.0 program. The comparison was carried out using the nonparametric Mann-Whitney method.

Results.

The results are also illustrated in figures 1 and 2, where the abscissa axis represents the percentage (%) of surviving cells, and the ordinate axis shows the concentration of BOD in mg in the cell culture medium.

In FIG. 1 presents information about cytofluorometric studies of human fibroblast cells (FBC) 72 hours after adding the used preparation of this invention to the growth medium (trade name "Cellex").

In FIG. 2 presents information on cytofluorometric studies of B16 melanoma cells 72 hours after adding the used preparation of this invention to the growth medium (trade name "Cellex").

The results presented in FIG. 1, indicate that BOD added to human skin fibroblasts does not inhibit the growth of these cells after 72 hours.

At the same time, BOD after 72 hours inhibits the growth of B16 melanoma cells (Fig. 2). Cytofluorometric studies showed the absence of apoptotic cells in B16 melanoma samples after 72 hours of the experiment, most likely the cells died as a result of necrosis or autophagy.

Example 5

Patient B. 63 years. He considers himself sick for several months. The exact debut of the disease: January 30, 2012. Over the past few months, there has been an increase in pigmented neoplasm, the appearance of ulceration, erosion (primary treatment).

Complaints during treatment: for the presence of neoplasms on the skin of the chest, bleeding from the formation.

Statuslocalis

Primary elements of a rash: nodule.

Localization of rashes: chest.

Clinical diagnosis: melanoma of the skin of the chest T2N1M0. Immediately after surgical treatment (removal of the tumor), the protein-polypeptide complex was systemically injected with subcutaneous injections for 14 days, when it was kept in solution (water for injection) 0.2 mg, 1 time per day, with a break between courses of 14 days, only 6 courses . The surgical wound healed by first intention, the sutures were removed on the 6th day, the suture is soft, inconspicuous.

A 2-year follow-up by an oncodermatologist confirmed a complete remission of the disease.

Example 6

Patient L. 66 years.

Considers himself sick 7 years. Three years ago, independently treated the focus of "keratoma", (as it seemed to her) "superchistel", after inflammatory ulceration, the node was divided into two.

Statuslocalis

Localized rash. On the outside of the right thigh, the knot is about 1.5 cm in diameter, irregular in shape.

Clinical diagnosis: Nodular melanoma of the right thigh.

Histopathological conclusion:

Nodular melanoma from epithelial-like cells, with ulceration of the epidermis, with severe lymphoid infiltration, with invasion of the II-III level according to Clark and a thickness of up to 2.2 mm according to Breslow. The tumor is removed within healthy tissues.

Immediately after surgical treatment (removal of the tumor), the protein-polypeptide complex was systemically administered as subcutaneous injections in 14-day courses, 0.1 mg once a day, with a break between 14-day courses of only 4 courses and external use of 0.1% BOD to the area of the postoperative wound as part of a sterile hydrophilic ointment of polyethylene glycols 400 and 1200 in a ratio of 3: 1, respectively, for 7 days. The surgical wound healed by first intention, the sutures were removed on the 7th day.

A 3-year follow-up by an oncodermatologist confirmed a complete remission of the disease.

Thus, as follows from the above examples, the invention provides an implementation of a method for the prophylactic treatment of cancer using a protein-polypeptide preparation with certain characteristics and in certain dosages and a specific treatment regimen, ensuring the effectiveness of these methods, which clearly demonstrates the industrial applicability of the claimed invention.

Claims (1)

A prophylactic treatment method for the development of neoplastic skin tissue processes such as melanoma, basal cell carcinoma, squamous cell carcinoma, comprising the use of a pharmaceutical preparation comprising a protein-polypeptide complex and pharmaceutically acceptable carriers, such as a diluent, possibly including a buffering agent, as an active ingredient solution, as well as carriers traditionally used in the preparation of cosmetic compositions, such as day or night creams, gels, independently and and in liposomal form, as well as in the form of photoactive or photoprotective compositions, daily, in a concentration of 0.001-0.05 mg / ml 1-2 times a day with a course dose of 10-20 days, at least 4 courses per year, while as an active active substance, a protein-polypeptide complex is used, obtained from a homogenate of the embryonic nervous and skin tissues of ungulates in farm animals and containing negatively charged weakly acidic and neutral proteins and polypeptides with a molecular weight of 5 to 200 kDa and containing a fraction with an average molecular weight with a mass of 10 to 120 kDa of at least 80%, a total protein concentration of 0.2-4.2 mg / ml, with a maximum absorption of the ultraviolet spectrum of the solution at a wavelength of 280 ± 5 nm, a peak at a wavelength of 274-284 nm in the UV-visible region of the spectrum, the presence of specific bands in the range of values

from 4.2 to 8.4 with isoelectric focusing in a 5% polyacrylamide gel.

Images