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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/12—Ketones
  • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
  • A61K31/125—Camphor; Nuclear substituted derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/13—Coniferophyta (gymnosperms)
  • A61K36/15—Pinaceae (Pine family), e.g. pine or cedar
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

• Antipyretic terpenoid pharmaceutical composition “ Abisilin ”with angiogenesis-inhibiting action”
• the invention relates to medicine and the pharmaceutical industry, in particular to the development of an antitumor drug for the treatment of oncological diseases of various origins, by inducing a cascade of anti-tumor processes: inhibiting angiogenesis, inducing an endogenous apoptosis system, inhibiting the proliferation of tumor cells, and activating the antirecurrent antimetosis when using a non-toxic polyactive agent that induces these processes - oral drug
• this invention can be used in the treatment of a large number of diseases associated with impaired regulation of angiogenesis, especially diseases caused by neovascularization of the eyes - retinopathy or age-related macular degeneration, as well as in proliferative disorders of mesangial cells - in cases of chronic and acute kidney diseases (glomerulonephritis), with psoriasis, development of atheromas, arterial restenosis, diabetes, rheumatoid arthritis, chronic asthma, endometriosis, autoimmune diseases , Arterial real or posttransplatatsionnom atherosclerosis and many others.
• diseases associated with impaired regulation of angiogenesis especially diseases caused by neovascularization of the eyes - retinopathy or age-related macular degeneration, as well as in proliferative disorders of mesangial cells - in cases of chronic and acute kidney diseases (glomerulonephritis), with psoriasis, development of atheromas, arterial restenosis, diabetes, r
• Antiangiogenic drugs blocking the formation of blood vessels in a tumor, slow down or stop its growth and prevent the development of metastases.
• Avastin is a monoclonal antibody from the pharmacological group with an anti-angiogenic mechanism that can reduce vascularization and inhibit tumor growth due to selective binding to biologically active vascular endothelial growth factor (VEGF) (US patent JVb 60542297; US certificate JN ° 6639055 )
• VEGF biologically active vascular endothelial growth factor
• angiogenesis inhibitors that block chemical signals that promote the growth of new blood vessels can be effective in any type of tumor, as these processes are common.
• Anthranilic acid and thioanthranilic acid N-arylamides are known to inhibit the activity of the tyrosine kinase Fit-1 VEGF receptor associated with neoplastic diseases and angiogenesis. When using them, insignificant therapeutic efficacy is noted.
• Paclitaxel which effectively suppresses the proliferation of endothelial cells, i.e. which is an inhibitor of angiogenesis (Klaubeg N. et al., Sapser Res., 57, 81-86, 1997).
• Taxol was developed on the basis of complex structure diterpenoids - taxanes isolated in 1971 from the bark of the Pacific yew tree (Takhis brevifolia Nitl).
• Currently known methods for the complete synthesis of Taxol and its analogues (USA Al, N 5488116, Jap. 1966, RU N 2196581 from 05/04/2000).
• Taxol - docetaxel isolated from the raw materials of the European yew (Tahis bassat ⁇ ), as well as R-tetraol created using native terpenoid complexes using conifers of coniferous species Ripis sibiibsa and / or Ripis setba, and / or genus Abies, and / or genus Larich (RU 2051900 Cl, 10.01.96) based on which it is obtained by isolating the active ingredient from a natural product or in a semi-synthetic way (RU 2196581, C2, 04.05.2000) using a fraction of labdanoid-diterpene acids with the addition of camphor and exposing the resulting mixture to ⁇ -radiation until the formation of the R-tetraol structure.
• an antiangiogenic agent include immunomodulatory, analgesic, antibacterial, anti-inflammatory and other types of activity;
• Antiangiogenic agents that are capable of simultaneously exerting such a variety of polyactive effects in the development of tumor processes are unknown and not obvious from the prior art.
• the aim of the present invention was the search and development of a tool that allows you to show selective inhibition or complete blocking of the neovascularization system using an antiangiogenic antitumor agent that is free of existing disadvantages of known agents, namely, it does not have toxic side effects with prolonged use, without contraindications that does not cause resistance (including apoptosis), as well as when used in combination with cytostatic drugs, does not not only increasing their effectiveness, but also helping to reduce their toxicity.
• the proposed method should have several advantages over all known blockers of angiogenesis - have a direct antitumor effect, be able to induce an apoptosis system in cells, have an antirecurrent and antimetastatic effect, while also exhibiting immunomodulating, antibacterial, wound healing, anti-inflammatory, analgesic and other painkillers.
• the polyactive active terpenoid pharmaceutical composition Abisilin which is an oral dosage form - 20% oil solution, with a new spectrum of pharmacological action and new possibilities for stopping pathological processes associated with impaired regulation of angiogenesis, in particular with the development of malignant neoplasms.
• the polyactivity of the terpenoid pharmaceutical composition Abisilin is achieved due to the fact that it uses a natural complex chemical complex of terpenes containing a provoked (in terms of yield and synthesis) composition for bioprotection against stress factors in representatives of the Ripaseae family, enriched with identical monoterpene compounds.
• the obtained antitumor oral polyactive drug Abisilin with an angiogenesis-inhibiting effect was investigated for the presence of a general toxic effect.
• Abisilin is administered orally at a dose of 80-100 mg / kg body weight.
• a preferred embodiment of the invention is also a method for inducing a selective cytotoxic effect on tumor cells and a method for inducing apoptosis death using an Abisilin dose of 1000 ⁇ g / ml, since the largest number of cells in the amount of positive AnV + Pl-, AnV + Pl + , AnV-Pl + , in relation to the Jukat cell line and amounted to 86.6%.
• Example 2 Example 2
• a preferred embodiment of the invention is a method for inducing the antitumor effect of the drug Abisilin when administered orally at a dose of 10-100 mg / kg body for 2-11 days, identified using the necessary set of transplantable solid tumors - melanoma B-16, Ca-755, LLC, PShM-5, Akatol and sarcoma MI. (Example 3)
• the preferred option which is the treatment of tumor diseases of various origins, is the use of the pharmaceutical composition
• Abisilin which may be in the form of a solution, tablets, gelatin capsules, dragees, syrups, suspensions, powders, emulsions, granules, microspheres or nanospheres, or other dosage forms, which allow for the controlled release of the active ingredients of the drug.
• a preferred embodiment of the present invention is a method of shifting the natural balance between pro-angiogenic and anti-angiogenic mediators towards the dominance of the latter when using Abisilin in ip vitro and ip vivo experiments. (Example 4)
• a preferred embodiment of the present invention is the use of Abisilin for the induction of antirecurrent and antimetastatic effects when administered orally in doses of 80-100 mg / kg body weight. (Example 5)
• a subchronic experiment was conducted on nonlinear male and female rats with an average mass of 165 ⁇ 3 g and l63 ⁇ 3 g, respectively.
• the studied drug was administered intragastrically daily once for 14 days at a dose of 100, 500 and 1000 mg / kg to males and at a dose of 100 and 1000 mg / kg to females.
• Abisilin in the doses studied did not cause significant changes in the integral parameters in the experimental animals (weight gain, behavioral reactions, food, water consumption), the peripheral blood cell composition, excretory, absorption, protein synthesizing, carbohydrate function of the liver, level serum cholesterol content, the functional state of the cardiovascular, excretory and nervous systems.
• T cell lymphoblastic leukemia - Jugkat B cell leukemia - Raji
• chronic myelogenous leukemia - K562 myelogenous leukemia - K562, melanoma - MeI P
• breast cancer - T 47 D ovarian cancer - SKOV-3
• prostate cancer - PC-3 T cell lymphoblastic leukemia - Jugkat
• B cell leukemia - Raji chronic myelogenous leukemia - K562
• myelogenous leukemia - K562 myelogenous leukemia - K562, melanoma - MeI P
• breast cancer - T 47 D ovarian cancer - SKOV-3
• prostate cancer - PC-3 prostate cancer - PC-3.
• Cell lines were grown in complete nutrient medium RPMI - 1640 containing 10% calf fetal serum, 2mM / ml glutamine, 0.1 mg / ml gentamicin, vitamins, sodium pyruvate, amino acids, at 37 ° C and in an atmosphere of 5% CCh .
• the cytotoxic activity of Abisilin was determined by the colorimetric method using the MTT test. 5 x tumor cells
• Identification of apoptotic cells was carried out by double intravital staining using annexin V-FITC in combination with a vital dye such as propedium iodide (PI).
• a vital dye such as propedium iodide (PI).
• Annexin-stained V-FITC and PI cells were evaluated according to the following criteria: living cells negative for Annexin V and PI (AnV " WG), early apoptotic cells positive for Annexin V and negative for PI (AnV + PI " ), late apoptotic or necrotic cells positive for Annexin V and PI (AnV + PI + ).
• As a control we used tumor cells that were incubated under the same conditions, but in the absence of the drug, as well as tumor cells that were incubated under the same conditions, but a solution of 1% DMSO was added instead of the drug.
• this aspect of the invention extends the scope of use of the well-known polyactive terpenoid substance Abisil and pharmaceutical compositions obtained on its basis, recommended for topical and external use, since it has established action spectra (antibacterial, immunomodulating, anti-inflammatory, wound healing, analgesic ) are added unexpectedly identified and having great therapeutic significance types of effects, namely: the ability to cause cytotoxic effects on some ny lines of tumor cells proceeding as apoptosis.
• the study of the antitumor effect of the oral pharmaceutical composition Abisilin on transplantable tumors The antitumor effect of the drug Abisilin was studied on transplantable tumors of mice, which are among the mandatory models of animal tumors that are used in the selection of new antitumor substances: melanoma B-16, Lewis epidermoid lung carcinoma (LLC), breast adenocarcinoma Ca-755, and cervical cancer PI -5, AKATOJl colon adenocarcinoma, MI polymorphic cell sarcoma.
• mice - first-generation hybrids BDF C BI / 6 x DBA / 2) and F (C Dl / 6 and CBA / 2)
• Mice and rats were obtained from the department of laboratory animals GU RONTs im. NN Blokhina RAMS and kept on a normal diet.
• the drug Abisilin was administered orally (re os) in the form of an oil solution at doses of 10, 100, 150, 500, 2500 mg / kg.
• an oil solution As a solvent for the preparation, vegetable sunflower oil was used.
• the drug Abisilin was administered to animals daily orally for 10 days, as well as in the regimes: on the 2nd and 6th days and 2nd and 11th days. Treatment was started 48 hours after transplantation of solid tumors (Ca-755, ACATOL, melanoma B-16,
• the drug Abisilin was administered to mice daily orally for 10 days 48 hours after transplantation of the tumor at a dose of 50 mg / kg, and the anticancer drug Cisplatin was administered once intraperitoneally (iv) at a dose of 5 mg / kg. Observation of animals with the above tumors was carried out before their death. The antitumor effects of the drug were judged by the inhibition of tumor growth (TPO,%) in experimental animals compared with the control.
• the toxicity of the drugs was evaluated by the early death of mice compared with the death of control animals and the state of their internal organs (spleen, liver, etc.), changes in body weight compared to the original.
• the drug was not effective at doses of 500 mg / kg after 10 days of administration and at a dose of 2500 mg / kg when administered twice with an interval of 10 days.
• the therapeutic dose of the drug Abisilin in experiments in the study of antitumor activity on transplanted tumors was 100 mg / kg with daily oral administration for 5 days.
• the therapeutic dose of the drug Abisilin with daily oral administration to animals with transplantable solid tumors was 100 mg / kg.
• the drug Abisilin did not cause toxic effects in a total dose of 5000 mg / kg, both when administered daily at a dose of 500 mg / kg for 10 days, and when administered twice at a dose of 2500 mg / kg with an interval of 4 and 9 days.
• the solution of the drug Abisilin was prepared in a stock concentration of 10 mg / ml (10% DMSO / PBS) in the laboratory for the development of dosage forms of the Research Institute of EDITO RONTs im. NN Blokhina RAMS on the day of the experiment.
• the drug to the concentration required in the study was diluted in 1% PBS.
• the antiangiogenic properties of the drug Abisilin can be demonstrated in vitro and in vivo according to the following procedure.
• the cytotoxic effect of Abisilin on endothelial cells was carried out during incubation of the drug for 24 hours with cells of the line SVEC-4-10, planted in high density.
• Abisilin causes the death of endothelial cells in the concentration range from 1 mg / ml to 0.125 mg / ml (Fig. 2).
• LD 5 o was 0.21 mg / ml.
• the antiproliferative effect of Abisilin was evaluated by studying the effect of Abisilin on the proliferative activity of activated bFGF endothelial cells SVEC4-10, planted in low density.
• Abisilin has been shown to block the proliferation of endothelial cells.
• Abisilin's ability to block the formation of tube-like structures of endothelial cells was evaluated in the dose range of 0.125 mg / ml to 0.015 mg / ml.
• the drug Abisilin has antiangiogenic properties in the experiment ip vivo and ip vitro.
• the data obtained are of practical importance, consisting in the possibility of using Abisilin according to the indicated method as an agent capable of blocking the angiogenesis of malignant tumors, all the more so since methods for producing such polyactive non-toxic agents with simultaneous antitumor and antiangiogenic effects are not known and obvious.
• mice and first-generation BDFl hybrids weighing 20-25 g with subcutaneous transplanted Lewis lung carcinoma were used. Mice were obtained from the department of the animal laboratory of the GU RONTs im. NN Blokhina RAMS and kept on a normal diet. All experimental procedures were carried out in accordance with international rules for working with animals.
• the drug Abisilin was administered orally in the form of an oil solution in the dose range from 60 to 400 mg / kg. Sunflower oil was used as a solvent for the preparation. In the control and in the group receiving treatment, there were from 7 to 10 animals.
• the proposed method of using the polyactive terpenoid drug Abisilin allows to achieve the desired result, namely: inhibit the process of artificial relapse of Lewis lung carcinoma and suppress the process of metastasis of recurrent tumors (by 25-46 % in different experiments).
• the best effect was achieved with the oral administration of the drug for 7-10 subsequent days at a dose of 100-120 mg / kg.
• a study of the activity of Abisilin in adjuvant therapy revealed its ability to inhibit the appearance and development of spontaneous relapse of Lewis lung carcinoma.
• the proposed oral method of using the polyactive terpenoid drug Abisilin unexpectedly made it possible to block the process of angiogenesis during the development of malignant tumors, as well as: - have an antirecurrent and antimetastatic effect;
• the proposed tool allows you to expand the scope of the well-known polyactive terpenoid substance Abisil and pharmaceutical compositions obtained on its basis, recommended for local and external use, as agents that can increase the effectiveness of surgical treatment of tumor diseases.
• the high therapeutic effect of the drug Abisilin is also due to the known polyactivity of its substance Abisil, namely: immunomodulating, antibacterial, wound healing, anti-inflammatory, analgesic, and, moreover, capable of stimulating the synthesis of specific endogenous humoral mediators of the nerve endings of the cerebral cortex.
• the proposed method for stopping the development of malignant neoplasms made it possible to achieve the goal - to identify and develop a tool that allows selective inhibition or complete blocking of the neovascularization system using the anti-angiogenic antitumor drug Abisilin, free of existing disadvantages of known drugs, namely, it does not have toxic side effects with prolonged use 1 , without contraindications, not resistant sti (including apoptosis), as well as with the combined use with cytostatic drugs, not only increasing their effectiveness, but also helping to reduce their toxicity.
• the proposed tool has several advantages over all known means of inhibiting the angiogenesis system - it has.

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• 2008
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