WO2009054747A1 - Agent anti-métastatique - Google Patents

Agent anti-métastatique Download PDF

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Publication number
WO2009054747A1
WO2009054747A1 PCT/RU2008/000515 RU2008000515W WO2009054747A1 WO 2009054747 A1 WO2009054747 A1 WO 2009054747A1 RU 2008000515 W RU2008000515 W RU 2008000515W WO 2009054747 A1 WO2009054747 A1 WO 2009054747A1
Authority
WO
WIPO (PCT)
Prior art keywords
epom
embryonic
modulator
animals
solution
Prior art date
Application number
PCT/RU2008/000515
Other languages
English (en)
Russian (ru)
Inventor
Levon Nikitovich Mkrtchan
Original Assignee
Abramyan, Ara Arshavirovich
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abramyan, Ara Arshavirovich filed Critical Abramyan, Ara Arshavirovich
Publication of WO2009054747A1 publication Critical patent/WO2009054747A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention relates to the field of medicines, medicine, in particular to oncology.
  • the embryonic antitumor modulator, as well as the method for its preparation, can be used in the medical industry to obtain a highly effective antimetastatic agent.
  • Tsapg K.Y. 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 may only be used. in the treatment of such human tumors that cause this antigen.
  • OEA tumor embryonic antigen
  • the present invention is the result of lengthy and strictly controlled studies, which led to the discovery of a source of antimetastatic, immunomodulatory properties, antiviral and other activities.
  • An object of the present invention is to provide an antimetastatic agent.
  • Hyperfibrinogenemia is 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.
  • the new embryonic antitumor modulator Mkrtchyan proposed in this invention has antimetastatic action, interferonogenic and immunomodulatory effects, as well as antiviral activity.
  • 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.
  • the homogenate 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 again centrifuged 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.
  • 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.
  • alanine aminotransferase 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,
  • CEA - about 1.4-1.7 ng / ml AFP - about 95.0-99.0 ng / ml
  • EAG Embryonic antigens in the composition of the drug are related to numerous tumor antigens and give them cross-immune responses.
  • EAG Embryonic antigens
  • 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.
  • biochemical methods 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 P 7.5 mm x 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).
  • Electron paramagnetic resonance 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 p -10 12 spin. Records of the spectrum of the standard (manganese standard) and 7 studied samples of the preparation showed that the concentration of paramagnetic particles in all samples is lower than the sensitivity instrument, which indicates the absence of free radicals in the EPOM.
  • CEA was detected using enzyme-linked immunosorbent and radioimmune methods.
  • ELISA was performed using available Roche test kits. Content
  • TH trophoblastic ⁇ i-glycoprotein
  • the antitumor activity of the drug was revealed in an experiment on transplantable tumors (Ehrlich ascites tumor in mice, 37 mouse sarcoma, Seidel ascites hepatoma, lymphosarcoma
  • An additional characteristic of the target product EPOM is a porous, soft mass of yellowish-white color, odorless and tasteless.
  • 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 OD grams of the drug at 100 ° C 5 for 1 hour (GF XI, 1). The moisture content is not more than 9%.
  • mice mice, rats, rabbits, guinea pigs and dogs.
  • mice 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.
  • 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 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, AJIT, ACT ), biochemical parameters of urine (protein, glucose, bilirubin, urea, electrolytes).
  • 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, AJIT, 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).
  • EPOM 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.
  • EPOM does not cause immunosuppression, but rather increases both specific and non-specific antitumor protection.
  • induced (7.12 - DMBA and benz / a / pyrene) carcinogenesis and in experiments on models of transplanted tumors of different histogenesis, EPOM completely prevented the occurrence of metastases in 40-70%.
  • the method is as follows. Form a group, while taking into account the age of the individual, profession, pre-tumor disease, smoking, family predisposition, hyperfibrinogenemia, the number of tumor markers in the peripheral blood.
  • an EPOM is administered to an individual at a rate of 0.02 to 0.04 mg / kg body weight, most preferably 0.03 mg / kg body weight.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Reproductive Health (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Zoology (AREA)
  • Oncology (AREA)
  • Dermatology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne la biopharmacologie et la médecine. Le modulateur antitumoral embryonnaire de l'invention est utilisé comme un agent anti-métastatique. Ce modulateur antitumoral embryonnaire est obtenu à partir de substances embryonnaires par cryodestruction, extraction et précipitation dans des alcools de concentration croissante, et comprend un complexe de protéoglycanes de l'acide hyaluronique et les antigènes associés à la tumeur d'une embryogenèse normale.
PCT/RU2008/000515 2007-10-22 2008-08-12 Agent anti-métastatique WO2009054747A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2007139002 2007-10-22
RU2007139002/15A RU2341271C1 (ru) 2007-10-22 2007-10-22 Средство, обладающее противометастатическим действием

Publications (1)

Publication Number Publication Date
WO2009054747A1 true WO2009054747A1 (fr) 2009-04-30

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2008/000515 WO2009054747A1 (fr) 2007-10-22 2008-08-12 Agent anti-métastatique

Country Status (3)

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KR (1) KR20100111231A (fr)
RU (1) RU2341271C1 (fr)
WO (1) WO2009054747A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2240810C2 (ru) * 2002-01-30 2004-11-27 Левон Никитович Мкртчян Эмбриональный противоопухолевый модулятор мкртчяна, способ его получения и применения

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2240810C2 (ru) * 2002-01-30 2004-11-27 Левон Никитович Мкртчян Эмбриональный противоопухолевый модулятор мкртчяна, способ его получения и применения

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE PUBMED [online] Database accession no. 16391393 *
SAPIR T. ET AL.: "Facing the enigma of immunomodulatory effects of intravenous immunoglobulin.", CLIN REV ALLERGY IMMUNOL., vol. 29, no. 3, December 2005 (2005-12-01), pages 185 - 199 *

Also Published As

Publication number Publication date
RU2341271C1 (ru) 2008-12-20
KR20100111231A (ko) 2010-10-14

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