Biomarkers

FIELD: medicine.

SUBSTANCE: what is offered is applying an analysis of p53(TP53) gene status and/or expression level as a biomarker while evaluating sensitivity of an individual suffering a proliferative disease to treatment by an mTOR inhibitor combined with a cytotoxic agent or while selecting individuals sensitive to the specified combined therapy for the following treatment of the disease by this method. Thus sensitivity to treatment of the proliferative disease by the mTOR inhibitor combined with the cytotoxic agent is predicted if wild-type functionally active p53 gene is found in a sample taken from the patient.

EFFECT: higher analysis accuracy.

14 cl, 5 ex

 

The present invention relates to biomarkers for determining the sensitivity of proliferative diseases such as cancer, therapeutic agents, primarily to mTOR inhibitors used in combination with a cytotoxic agent.

Many inhibitors of mTOR have a strong antiproliferative effect, allowing their use for cancer chemotherapy, especially solid tumors, primarily developed solid tumors. Inhibitors of mTOR also combine with certain cytotoxic agents to further improve the effectiveness of treatment or to reduce side effects, for example as described in WO 02/66019. However, there is still a need to develop a targeted combination therapy based on the use of mTOR inhibitors, which requires the identification of patients likely to respond to treatment using such combinations of agents. Therefore, there is a need to develop biomarkers that can be applied, for example, in clinical experiments, allowing to predict the sensitivity of benign or malignant proliferative diseases such as tumor, in a patient for the treatment with the mTOR inhibitor in combination with a cytotoxic agent.

With the invention it has been unexpectedly found that suppressor gene is a tumor p53 wild-type (having the name of the gene TR) can be used as biomarkers for predicting sensitivity of the proliferative disease to treatment with a combination of mTOR inhibitor and a cytotoxic agent. In particular, it was found that the presence of p53 wild-type lines cancerous human cells correlates with increased apoptosis/programmed cell death/apoptosis that occurs during treatment with mTOR inhibitor in combination with a cytotoxic agent that violates or adversely affect the integrity of DNA. Thus, mTOR inhibitors in combination with cytotoxic agent, apparently, have more pronounced antiproliferative/leading to cell death activity in the treatment of cancer cells that retain wild type p53. Protein p53 (encoded by genome TR) is a tumor suppressor that plays a major role in the regulation of the termination of the cell cycle, physiological aging, differentiation and programmed cell death/apoptosis of mammalian cells. In particular, mediated p53 path induces the cessation of the cell cycle and/or apoptosis of mammalian cells subjected to stress (e.g., DNA damage, oncogenic stress, hypoxia, lack of survival signals). Mutations in TR occur in about half of all cases of cancer in humans, and in many cancer cells impaired ability to induce p53-response (Vousden and Lu, Nature Reviews, 2, 2002, s-604). The sequence of human p53 (mRNA [the coding sequence; 182 nucleotides] and protein [393 amino acids]) is available from GenBank under registration numbers NM 000546 or R. The full sequence of the human gene TR deposited in GenBank under the registration number U94788.

Thus, the present invention is the task of determining the existence of a gene p53 (TR) wild-type cells, which have a tendency to abnormal proliferation.

One of the objects of the present invention is the use of the presence of p53 gene (TR) wild-type (in contrast to the lack, deficiency, or deletion of the gene p53 [TR], or the presence of having the mutation of p53 gene [TR]) as a biomarker for assessment of the sensitivity of the proliferative disease to treatment with mTOR inhibitor in combination with a cytotoxic agent.

In the context of the present description, the term gene p53 (TR) wild type refers not only to the introns and exons, but also to associated and physically close to those regulatory areas, especially the areas closest to the 5' exon. It includes, for example, a full-sized DNA sequence naturally occurring gene and optionally a nucleotide change (including inversion), insertions and deletions of codons, provided that it ensures the expression of protein p53 wild-type or its functional equivalent, such as functional p53 protein which retains its ability to induce apoptos. On the contrary, lack, deficiency, deletion or mu is the situation of p53 gene (TR means of genetic or epigenetic changes, for example, amplification, methylation, polymorphisms, nucleotide mutations, deletions, inversion, or translocation and loss of heterozygosity (LOH), which leads to disruption of gene expression of p53 (THR) or expression of a mutant gene, for example, which lead to expression of mutant p53 protein with no more than the ability to induce apoptosis of cells.

The next object of the invention is a method of determining the sensitivity of the proliferative disease in an individual to treatment with mTOR inhibitor in combination with a cytotoxic agent, namely, that determine the status of p53 (TR) (wild type or mutant, or with deficiency of/absence status) in the sample taken from the individual.

Another object of the invention is the method of selection of individuals suffering from a proliferative disease, for treatment with mTOR inhibitor in combination with a cytotoxic agent, namely, that for every individual determine the sensitivity of the proliferative disease to the combined treatment, according to the above method, and selected for the specified combined treatment of individuals with gene p53 (TR) wild-type.

In the context of the present description, the term "mTOR inhibitor" refers (but not limited to rapamycin (sirolimus) or its derivatives. Rapamycin represents swesty macrolide antibiotic, produced by Streptomyces hygroscopicus. Suitable derivatives of rapamycin are, for example, the compounds of formula

in which

R1aadenotes CH3or3-C6quinil,

R2Adenotes H or-CH2-CH2-HE, 3-hydroxy-(2-hydroxymethyl)-2-methylpropanoyl or tetrazolyl and

XAAmeans =O, (N,N) or (N,IT)

provided that R2aawill not represent H, if XAAmeans =O and R1aadenotes CH3,

or the prodrug, when R2Adenotes-CH2-CH2-OH, for example it hydrolyzable under physiological conditions a simple ether.

The compounds of formula As described, for example, in WO 94/09010, WO 95/16691, WO 96/41807, USP 5362718 or WO 99/15530, which is incorporated into this description by reference. They can be obtained according to the described methods or analogously to the methods described in these references.

Representative derivatives of rapamycin of formula I are, for example, a 32-desoxidation, 16-Penta-2-ynyloxy-32-desoxidation, 16-Penta-2-ynyloxy-32(S or R)-dihydrocapsaicin, 16-Penta-2-ynyloxy-32(S or R)-dihydro-40-O-(2-hydroxyethyl)rapamycin, 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin (also called CCI779), 40-EPI(tetrazolyl)rapamycin (also called AVC). The preferred connection is e.g. the measures 40-O-(2-hydroxyethyl)rapamycin as described in example 8 of WO 94/09010, or 32-mesoxerophytic or 16-Penta-2-ynyloxy-32(S)-dihydrocapsaicin described in WO 96/41807. Derivatives of rapamycin may also include so-called rapelay, for example described in WO 98/02441 and WO 01/14387, for example AR, AR, AR or AR. Other examples of a derivative of rapamycin are the compounds described under the name TAFA-93 (prodrug of rapamycin), biolims-7 or biolims-9.

In each case, when specified in the description of the patent applications or scientific publications, it is understood that their essence regarding compounds included in the present description by reference. This applies equally to their pharmaceutically acceptable salts, the corresponding racemates, diastereoisomers, enantiomers, tautomers, as well as the corresponding crystalline modifications of the above compounds, if they exist, for example the solvate, hydrates and polymorphs of the present description. Compounds used as active ingredients in the combinations proposed in the invention, it is possible to obtain and enter according to the methods described respectively in the cited documents.

In the context of the present description, the term "cytotoxic agent" refers to an agent that has a deleterious effect on cell structure and function, such as usaet or damage the integrity of the DNA and ultimately can lead to cell death, for example, the antitumor drug, such as an agent having activity against microrobot or, especially, drug, damaging DNA, for example antineoplastics an antimetabolite, containing platinum compound, an alkylating agent or an inhibitor of topoisomerase I or II. The term "cytotoxic agent" refers to radiation therapy, which causes DNA damage, such as ionizing radiation, for example, with radioactive iodine. Such radiation therapy can also be used in conjunction with therapy with a cytotoxic agent. The term "cytotoxic agent" includes one, two or more cytotoxic agents that can be applied in the form of a combined ("cocktail") of therapy.

In the context of the present description, the term "inhibitor of topoisomerase I" includes (but is not limited to, topotecan, irinotecan, gimatecan, 9-nitrocamptothecin and macromolecular conjugate of camptothecin PNU-166148 (compound A1 in WO 99/17804). Irinotecan can be entered, for example, in the form in which it is sold, for example under the trademark CAMPTOSAR™. Topotecan can be entered, for example, in the form in which it is sold, for example under the trademark HYCAMTIN™.

In the context of the present description, the term "inhibitor of topoisomerases is II" refers to (but not limited to anthracyclines, such as doxorubicin (including preparations based liposomes, for example CAELYX™), daunorubicin, epirubicin, idarubitsin and nemorubicin, the anthraquinones mitoxantrone and losoxantrone, and podophyllotoxin etoposide and teniposide. Etoposide can be entered, for example, in the form in which it is sold, for example under the trademark ETOPOPHOS™. Teniposide can be entered, for example, in the form in which it is sold, for example under the trademark VM 26-BRISTOL™. Doxorubicin can be entered, for example, in the form in which it is sold, for example under the trademark ADRIBLASTIN™. Epirubicin can be entered, for example, in the form in which it is sold, for example under the trademark FARMORUBICIN™. Idarubitsin you can enter, for example, in the form in which it is sold, for example under the trademark ZAVEDOS™. Mitoxantrone can be entered, for example, in the form in which it is sold, for example under the trademark NOVANTRON™.

The term "agent that has activity against microrobot" refers to the stabilizing and destabilizing microtubules microtubules agents, including (but not limited to taxanes, such as paclitaxel and docetaxel, Vinca alkaloids such as vinblastine, especially vinblastine sulfate, vincristine, especially vincristine sulfate, and vinorelbine, d is stermole and epothilones and their derivatives, for example epothilone B or its derivatives. Paclitaxel can be applied, for example, in the form in which it is sold, for example under the trademark TAXOL. Docetaxel can be applied, for example, in the form in which it is sold, for example under the trademark TAXOTERE™. Vinblastine sulfate can be applied, for example, in the form in which it is sold, for example under the trademark VINBLASTIN R.P.™. Vincristine sulfate can be applied, for example, in the form in which it is sold, for example under the trademark FARMISTIN™. Discodermolide can be obtained, for example, according to the method described in U.S. patent 5010099.

In the context of the present description, the term "alkylating agent" includes (but is not limited to, cyclophosphamide, ifosfamide, melphalan or nitrosoanatabine (BCNU or Gliadel™). Cyclophosphamide can be applied, for example, in the form in which it is sold, for example under the trademark CYCLOSTIN™. Ifosfamide can be applied, for example, in the form in which it is sold, for example under the trademark HOLOXAN™.

The concept of "antineoplastics an antimetabolite" includes (but is not limited to, 5-fluoracil, tegafur, capecitabine, cladribine, cytarabine, fludarabine, fluorides, gemcitabine, 6-mercaptopurine, hydroxyurea, methotrexate and edatrexate and salts of these compounds and, in addition, ZD1694(RALTITREXED™), LY231514 (ALIMTA™), LY264618 (LOMOTREXOL™) and OGT719. Capecitabine can be applied, for example, in the form in which it is sold, for example under the trademark XELODA™. Gemcitabine can be applied, for example, in the form in which it is sold, for example under the trademark GEMZAR™.

In the context of the present invention, the term "containing platinum compound" includes (but is not limited to, carboplatin, cisplatin and oxaliplatin. Carboplatin can be applied, for example, in the form in which it is sold, for example under the trademark CARBOPLAT™. Oxaliplatin can be applied, for example, in the form in which it is sold, for example under the trademark ELOXATIN™.

Proliferative disease may represent a benign or malignant proliferative disease, such as benign hyperplasia of the prostate, or neoplastic disease, preferably malignant proliferative disease, for example cancer, such as tumor and/or metastases (regardless of their location), brain tumors or other tumors of the Central nervous system (e.g., shell, brain, brain, spinal cord, cranial nerves and other parts of Central nervous system, such as glioblastoma or blastoma medulla oblongata); cancer of the head or neck; SDA is Oli breast cancer; tumors of the circulatory system (e.g. heart, mediastina and pleura, and other intrathoracic organs, tumors of blood vessels and is associated with tumor vascular tissue); tumors of the urinary system (e.g., kidney, renal pelvis, ureter, bladder, and other nonspecific urinary system); tumors of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectal tract, rectosigmoid connection, rectum, anus and anal canal), tumors affecting the liver and intrahepatic bile ducts, gallbladder, and other nonspecific departments biliary tract, pancreas, and other organs and digestive system); head and neck; oral cavity (lip, tongue, gum, floor of mouth, palate and other areas of the mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform pocket, hypopharynx and other sites of lip, oral cavity and pharynx); tumors of the reproductive system (e.g., vulva, vagina, cervix, uterus, ovary and other areas of the female genital organs, placenta, penis, prostate, testicles and other areas associated with male sexual organs); tumors of the respiratory tract (e.g., nasal cavity and middle ear secondary sinus is artane, trachea, bronchus and lung, such as small cell lung cancer or non-small cell lung cancer); tumors of the skeletal system (e.g., bone and articular cartilage of limbs, bone articular cartilage and other areas); skin tumor (eg, malignant melanoma of the skin, skin cancer non-melanoma, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and tumors affecting other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneal space and peritoneum, eye and adjacent organs, thyroid gland, adrenal gland and other endocrine glands and related structures malignant tumor neoplasia and non-specific malignant tumor of lymph nodes, malignant tumor neoplasm of respiratory and digestive systems and malignant neoplasia in other areas, tumors of blood and lymphatic systems (e.g., Hodgkin's disease, non-Hodgkins lymphoma, Burkitt's lymphoma, lymphoma, AIDS-related malignant immunoproliferative disease, multiple myeloma and malignant neoplasm of plasma cells, lymphoid leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other views of the leukemia certain types of cells, the leukemia cells of undetermined type other and unspecified malignant neoplasm lymphoid, hematopoietic and related tissues, for example both diffuse lymphoma, T-cell lymphoma or T-cell lymphoma of skin cells). Myeloid cancer includes, for example, acute or chronic myeloid leukemia.

If above or below in the present description are referred to the tumor, a disease associated with a tumor, carcinoma, or cancer, then this means alternative or in addition, metastases in the specified organ or tissue and/or in other areas in which there is a tumor and/or metastases.

The term "cytotoxic agent" can also mean in the case of lymphatic or myeloid cancer, for example, busulfan, cytarabine, 6-tioguanin, fludarabine, hydroxyurea, procarbazine, bleomycin, or methotrexate. In the case of lymphatic or myeloid cancer as a cytotoxic agent are preferred inhibitors of topoisomerase II, such as daunorubicin or idarubitsin, or preferably compounds that treat, reduce or inhibit the activity DERIVED (platelet-derived growth factor) or members of the family with bI and their fused gene products, such as imatinib, inhibitors farnesyltransferase, Ara-C, VP-16, teniposide, mitoxantrone, carboplatin or Mido is taurine.

In accordance with the method proposed in the present invention, it is possible to select individuals suffering from such proliferative disease, to predict their sensitivity to the combined treatment with the mTOR inhibitor and a cytotoxic agent. The method can be carried out in vitro, for example, using a biological sample of tissue taken from the body of the individual. The sample can be any biological material isolated from the body of a mammal, such as tissue, cell line, plasma or serum, cell or tissue lysate, preferably tumor tissue.

The status of p53 gene (TR) analyzing the biological sample using any of the techniques, based on, for example, on DNA analysis of genetic and epigenetic changes such as DNA scan to determine amplification, methylation polymorphisms, nucleotide mutations (e.g. mutations of codons 175Arg, 245Gly, 248Arg, 249Arg, 273Arg, 282Arg and others), nucleotide deletions, inversions and/or broadcast and loss of heterozygosity (LOH). The status of p53 (TR) analyze biological samples using any of the techniques based on, for example, the expression of RNA using, for example, methods Northern blot or RT-PCR, or on the basis of analysis of gene/protein modifications using the-W, for example, methods, Western blotting, immunohistochemistry or enzyme-linked immunosorbent assay (ELISA), including using immunoassay, thus and electrophoretic analysis.

For example, to measure the protein levels of p53/phosphorylation/ubiquitinate/acetylation can be used in a standard format immunoassay antibodies specific for p53 protein or post-translational modifications such as phosphorylation (for example, phosphorylation of Ser46), ubiquitination or acetylation. To determine the protein levels of p53/post-translational modifications as biomarkers used as the analysis type ELISA (enzyme-linked immunosorbent assay), analysis on the basis of the thus, the standard analysis method, Western blotting and immunohistochemical analyses using, for example, monoclonal or polyclonal antibodies.

Polyclonal and monoclonal antibodies specific against protein p53/post-translational modifications, receive according to known methods of immunization or use commercially antibodies (for example, by Santa Cruz Biotechnology Inc. catalogue number sc6253).

The p53 status can also be assessed using two-dimensional (2-D) gel electrophoresis. Method 2-D gel electrophoresis is known in this field and, as a rule, is and is elektricheska focusing (IEF) in the first direction and the subsequent LTO-page (gel-electrophoresis in polyacrylamide gel with sodium dodecyl sulfate) in the second direction. Received electrophoregram analyze, for example, by the method of Western blot turns using antibodies.

In the present invention a method of selection of individuals suffering from a proliferative disease, in order to predict their sensitivity to the combined treatment with the mTOR inhibitor and a cytotoxic agent, namely, that determine the status of p53 (TR) as described above.

Another object of the invention is a method of treating a proliferative disease in an individual in need of this, namely, that define the status of the p53 gene (TR) or the level of expression of p53 and/or post-translational modification in the sample obtained from the organism of the individual using the above method, and accordingly process the individual mTOR inhibitor in combination with a cytotoxic agent.

Another variant embodiment of the invention relates to a method of increasing the activity of a cytotoxic agent or overcome resistance to the cytotoxic agent to an individual in need of this, namely, that determine the status of the gene/expression of p53 (THR) in the sample obtained from the organism of the individual, using the above method and introduce an individual an mTOR inhibitor in a therapeutically effective amount either simultaneously or on sledovatelno with the specified cytotoxic agent.

The status of p53 (TR) in a particular tissue taken from the patient, for example in the sample of tumor tissue can be compared with a control sample, for example with a sample of healthy tissue of an individual not suffering from a disease, or a model of a healthy (i.e. non-cancerous tumor) tissue of the same individual. The level of p53 status (TR) wild-type, which shows the use of mTOR inhibitor in combination with a cytotoxic agent, is a criterion for predicting a favorable therapeutic effect (i.e. antiproliferative and/or leading to increased cell death) of the combined treatment with the mTOR inhibitor and a cytotoxic agent.

Furthermore, the method can be applied to select the appropriate dose of the cytotoxic agent and/or inhibitor of mTOR with the aim of optimization of therapy for each individual patient. Depending on the status of p53 wild-type patients can be used in combination of lower doses of active substances, for example, often can be applied not only smaller doses, but also to introduce them less frequently or to reduce side effects when treating undesirable cell proliferation. In the context of the present description of the considered factors include, first of all, the particular condition to be treated, the particular mammal to be l the teaching, the clinical condition of the individual patient, the region of introduction of active substances, the specific type of active substances, method of administration, schedule of administration, the severity of the condition and other factors known to practitioners.

In the context of the present description of the concept of "co-administration" or "combined introduction, or the like, includes the introduction of a selected mTOR inhibitor and a cytotoxic agent to a specific patient, and it is implied that they are modes of introduction, in which the agents are not necessarily administered by the same or at the same time. For example, the mTOR inhibitor and a cytotoxic agent, you can enter the patient in the form of separate units at the same time, together or sequentially without fixed time intervals, provided that this introduction provides therapeutically effective levels of both compounds in the body.

A therapeutically effective amount of each active component of the composition should be determined in consideration of the above, and it represents the minimum amount necessary to prevent, alleviate or cure the disease. Such amount is preferably less than the quantity which is toxic to the host, or quantity, which makes the host more susceptible to infections.

Acceptable dose inhib the Torah mTOR specified, for example, in WO 02/66019, for example daily doses of approximately from 0.1 to 30 mg, for example from about 0.05 to 20 mg of the active substance with the introduction of r.o. as a single dose or in divided doses, or at periodic introduction of, for example once a week. Rapamycin or its derivative, for example a compound of formula A, you can enter any conventional route, in particular enterline, for example orally in the form of tablets, capsules, solutions for drinking or parenterally, for example in the form of solutions or suspensions for injection, containing, for example from about 0.1 to about 99.9 percent, preferably from about 1 to about 60%, the incumbent(s) of the substances(a).

Topotecan you can enter the person in the dose range from about 1 to 5 to 1 mg/m2/day. Irinotecan you can enter the person in the dose range from about 50 to 350 mg/m2/day.

Paclitaxel you can enter the person in the dose range from about 50 to 300 mg/m2/day. Docetaxel can enter the person in the dose range from about 25 to 100 mg/m2/day.

Cyclophosphamide can enter the person in the dose range from about 50 to 1500 mg/m2/day. Melphalan you can enter the person in the dose range from about 0.5 to 10 mg/m2/day.

5-Fluoracil you can enter the human in a dose of from about 50 to 1000 mg/m2/day, for example 500 mg/m2/day. Cap is citamin you can enter the human in a dose of from about 10 to 1000 mg/m 2/day. Gemcitabine hydrochloride, you can enter the human dose comprising about 1000 mg/m2/week.

Carboplatin you can enter the human dose of approximately from 200 to 400 mg/m2approximately every four weeks. Cisplatin can enter the human in a dose of from about 25 to 75 mg/m2approximately every three weeks. Oxaliplatin can enter the human dose of approximately from 50 to 85 mg/m2every two weeks.

Imatinib, you can enter the human in a dose of from about 2.5 to 850 mg/day, more preferably from 5 to 600 mg/day and most preferably from 20 to 300 mg/day.

The preferred combination, which is used in the method proposed in the invention is, for example, a combination of rapamycin, 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin 40-O-(2-hydroxyethyl)rapamycin and a cytotoxic agent such as gemcitabine or cisplatin. Another combination, which is used in the method proposed in the invention, is the combination of the mTOR inhibitor and a cytotoxic agent, such as described above gemcitabine or cisplatin, taken in quantities synergistic action.

Preferably TR is a human gene.

Preferably the methods proposed in the invention, carried out on tumor glue the hands, presenting the status of p53 (TR) wild-type.

In the development of another variant embodiment of the invention it has been unexpectedly found that increased apoptosis/programmed cell death/apoptosis caused by treatment with mTOR inhibitor in combination with a cytotoxic agent, for cells carrying p53 (TR) wild-type, associated with a pronounced weakening induced cytotoxic activity increases regulating the expression of P21 proteinWaf1/Cip1(with names CDKN1A, WAF1, CIP1, SDI1, SAR, MDA-6, P21), below in the present description identified as P21.

P21 is representative of the cip/kip family "inhibitors" cilincing, which plays a role in the passage of the cell cycle and prevent apoptosis. In the context of the present description clearly established function of P21 in relation to the cessation of cell growth in response to stress signals such as DNA damage, in response to activation of p53. Really found that increased expression of P21 protein allows you to survive so stressed cells, for example, allows you to complete the cell the process of DNA repair. Thus, the weakening of the enhanced expression of P21 in the treatment of cytotoxic agent may stimulate apoptosis/programmed cell death/apoptosis (Weiss, Cancer Cell,4, 2003, c.425-429). The sequence h is human P21 (mRNA [the coding sequence: 495 nucleotides] and protein product [164 amino acids]) is in GenBank under registration number NM 000389, NM 078467 or AAN. The full sequence of the human P21 gene is found in GenBank under the registration number NM 078467.

In addition, for some suffering of cancer patients has increased total or cytosolic expression of P21 in tumor cells, which is associated with low reliability prognosis and poor response to chemotherapy (Weiss,supra). Assessment of the level of expression of P21 in the body of a suffering cancer patient may be allowed to select patients for specific chemotherapeutic treatment, for example based therapy with mTOR in combination with one or more cytotoxic agents and optional radiation therapy.

Thus, in the present invention are also proposed:

I. application of P21 as a biomarker for determining the sensitivity or response of a proliferative disease in an individual for treatment with mTOR inhibitor in combination with a cytotoxic agent;

II. the method of selection of individuals suffering from a proliferative disease, with the aim of treatment with mTOR inhibitor in combination with a cytotoxic agent, namely, that determine the sensitivity of the proliferative disease to treatment with mTOR inhibitor in combination with a cytotoxic agent, each of the individual using the above method and selected individuals, coloradomarvin increased expression of P21, for combined treatment;

III. the method of determining the sensitivity or response of a proliferative disease in an individual for treatment with mTOR inhibitor in combination with a cytotoxic agent, which is determined in the sample taken from the individual, the level of expression of P21 before and/or after processing of the cytotoxic agent individually or in combination with mTOR inhibitor;

IV. method of increasing the activity of the cytotoxic agent or overcome resistance to the cytotoxic agent from the individual undergoing treatment cytotoxic agent, who is that

determine the level of expression of P21 in a sample taken from the body of the individual, using the above method,

if the expression of P21 is subjected to increasing regulation after the introduction of a cytotoxic agent, the individual is administered mTOR inhibitor in a therapeutically effective amount in combination with a cytotoxic agent,

again determine the level of expression of P21 in a new sample taken from the body of the individual after treatment with the combination of the mTOR inhibitor and a cytotoxic agent, and

if the expression of P21 is subjected to a down-regulation of the individual continues to be treated with mTOR inhibitor, entering it, either simultaneously or sequentially with a cytotoxic agent.

As noted above, ur the attention P21 protein can be determined according to the method described above for p53; however, it is understood that instead of using antibodies specific for p53, it is necessary to apply the antibody specific for P21, such as monoclonal or polyclonal antibody, for example marketed (e.g., supplied by the firm Oncogene Research products, clone EA, catalogue number OR).

The level found in a specific tissue taken from the body of the individual, for example in the sample of tumor tissue can be compared with a control sample, for example a sample of healthy tissue taken from an individual not suffering from a disease, or in the sample of healthy (i.e. non-tumor) tissue taken from the same individual. The absence of reducing the induction of P21 expression (when processing an mTOR inhibitor in combination with a cytotoxic agent compared with induction occurring when processing only one cytotoxic agent) allows us to predict a favorable therapeutic effect (i.e. antiproliferative/causes cell death action) mTOR inhibitor in combination with a cytotoxic agent. Evaluation of induction of P21 expression of the cytotoxic agent and/or reverse the effect of the mTOR inhibitor on the expression of P21 may be useful for adapting doses of the cytotoxic agent, for example to reduce the cytotoxic dose.

Below the present invention Ave is illustrated in the examples, not limiting its scope.

Example 1

Bearing p53 (TR) wild-type tumor cells of human adenocarcinoma line A (CCL-185) (American type culture collection, Rockville, PCs Maryland, USA) were seeded with a density of 2×103cells/100 μl per well in 96-well plates and incubated for 24 h at 37°C and 5% CO2. Cells incubated in the presence of suboptimal concentrations of gemcitabine (for example, from 5 to 17,5 nm) or in combination with 20 nm 40-O-(2-hydroxyethyl) - rapamycin, or media, representing DMSO, which is used as a control for 72 h To cells add dye UO-R (iodide, YO-PROR-1 [491/509], catalogue number Y3603, the company Molecular Probes) and use the tablet reader type Cytofluor II Fluorescence to assess cell death or cytotoxicity, and after cell lysis, to evaluate the relative cell proliferation. In this analysis, the mTOR inhibitor, e.g. 40-O-(2-hydroxyethyl)rapamycin causes a statistically significant increased leading to cell death actions suboptimal concentrations of gemcitabine (p<0,05; dispersion regression analysis (ANOVA) in conjunction with Tukey criterion). Results similar to those described above, is obtained using bearing p53 (TR) cell lines wild-type, non-human pulmonary adenocarcinoma A, such as the human line of glue is OK breast cancer MCF7 (HTB-22; American type culture collection).

The above procedure is repeated using lines of tumor cells bearing mutant/defective p53 (TR), such as prostate cancer cells human line RSM (which are sown with a density of 0.8×103cells/100 μl) or breast cancer cells human line MDA-MB231 (which are sown with a density of 2×103cells/100 μl; NTV-26; American type culture collection). In carrying mutant/defective p53 (TR) cell lines have not been expressed or naturally enhance cell death.

Cell lines A seeded with a density of 0.1×106cells/10 ml tablets 10 cm and incubated for 24 h at 37°C and 5% CO2. Cells incubated in the presence of suboptimal concentrations of gemcitabine (for example, from 5 to 12.5 nm) or in combination with 20 nm 40-O-(2-hydroxyethyl) - rapamycin, or the carrier DMSO, used as control, within 72 hours of Cell extracts containing 50 μg of total protein, analyzed by electrophoresis in 8% of the LTO-page and carry out the analysis using Western blot turns using rabbit polyclonal antibodies to poly-ADP-ribosomally (Poly (ADP-Ribose) Polymerase (PARP)) (company Cell Signalling Technology, catalogue number 9542). In this analysis the presence of the mTOR inhibitor, for example, 40-0-(2-hydroxyethyl)rapamycin, results in the increased cleavage of PARP (marker of apoptosis) when suboptimal concentrations of gemcitabine (compared with the use of gemcitabine or mTOR inhibitor individually in the same concentrations). This confirms the above results concerning the fact that in carrying p53 (TR) wild-type cell line A the presence of an inhibitor of mTOR leads to higher levels of cell death at suboptimal concentrations of gemcitabine.

Example 2

Bearing p53 (TR) wild-type tumor cells of human adenocarcinoma line A seeded with a density of 5×103cells/100 μl per well in 96-well plates and incubated for 24 h at 37°C and 5% CO2. Cells incubated in the presence of suboptimal concentrations of cisplatin (for example, from 3 to 10 µg/ml) or in combination with 20 nm 40-O-(2-hydroxyethyl) - rapamycin, or the carrier DMSO, used as control, within 24 hours carried out using YO-PRO®the analysis of cell death or cytotoxicity and after cell lysis, the evaluation of the relative cell proliferation. In this analysis, the mTOR inhibitor, e.g. 40-O-(2-hydroxyethyl)rapamycin causes a statistically significant increased leading to cell death actions suboptimal concentrations of cisplatin (p<0,05; ANOVA in conjunction with Tukey criterion). Subsequent analysis using bidirectional ANOVA indicates a high degree of reliability of the joint action RAD001 and cisplatin (p<0,001). Results similar to those described above, is obtained using chosen to replace the x p53 (TR) wild-type cells, cell lines that differ from human lung adenocarcinoma A, for example a human cell line breast cancer MCF7. In the latter case, the incubation in the presence of compounds should be performed within 30 PM

The above procedure is repeated using lines of tumor cells bearing mutant/defective p53 (TR), such as prostate cancer cells human line RSM (which are sown with a density of 3×103cells/100 μl) or DU145 (which are sown with a density of 5×103cells/100 ál:NTV-81; American type culture collection). Incubation with the compounds is carried out in this case for 22 h for DU145 or 30 hours for RSM. In carrying mutant/defective p53 (TR) cell lines have not been expressed or naturally enhance cell death.

Cell lines A seeded with a density of 0.1×106cells/10 ml in a Cup with a diameter of 10 cm and incubated for 24 h at 37°C and 5% CO2. Cells incubated in the presence of suboptimal concentrations of cisplatin (for example, from 0.5 to 4 μg/ml) or in combination with 20 nm 40-O-(2-hydroxyethyl) - rapamycin, or the carrier DMSO, used as control for 24 h Cell extracts containing 50 μg of total protein, analyzed by electrophoresis in 8% of the LTO-page and carry out the analysis using Western blot turns using rabbit p is likeonline antibodies to Poly (ADP-Ribose) Polymerase (PARP) and p53. In this analysis the presence of the mTOR inhibitor, e.g. 40-O-(2-hydroxyethyl)rapamycin, results in increased cleavage of PARP (marker of apoptosis) when suboptimal concentrations of cisplatin compared with cisplatin or mTOR inhibitor individually in the same concentrations). This confirms the above results concerning the fact that in carrying p53 (TR) wild-type cell line A, the presence of an inhibitor of mTOR leads to higher levels of cell death at suboptimal concentrations of cisplatin.

The status of p53 (THR) is a criterion for the prediction of sensitivity, for example, tumors in the individual action of the combination of the mTOR inhibitor and a cytotoxic agent. The p53 status can be assessed using DNA, RNA or protein obtained from tumor tissue according to the procedure described for predicting sensitivity to the combination of the mTOR inhibitor and a cytotoxic agent.

Example 3

Bearing p53 (TR) wild-type cell line A and MCF7 were seeded with a density of 0.3×103and 0.4×103cells/4 ml, respectively, in a Cup with a diameter of 6 cm and incubated for 24 h at 37°C and 5% CO2. Cells incubated in the presence of suboptimal concentrations of cisplatin (for example, from 0.5 to 4 μg/ml) or in combination with 20 nm 40-O-(2-hydroxyethyl) - rapamycin, or the carrier DMSO used the control within 24 h and 30 h, respectively. Cell extracts containing 30 μg of total protein, analyzed by electrophoresis in 15% of the LTO-page and carry out the analysis using Western blot turns using mouse monoclonal antibodies to P21 (firm Oncogene Research Products, clone EA, catalogue number OR). In both cell lines cisplatin individually causes increased expression of P21 protein, depending on the concentration. Unexpectedly, it was found that the presence of the mTOR inhibitor, e.g. 40-O-(2-hydroxyethyl)rapamycin, weakens induced by cisplatin improves the regulation of expression of P21 protein. In contrast, the expression of Bax protein, proapoptotic protein, which is regulated by p53, is not affected by the agent either for private use, or in combination. In this analysis causes the cytotoxic effect of the expression of P21 protein inhibited in the presence of the mTOR inhibitor. This helps to explain the increased leading to cell death/apoptosis effect caused by the combination of cisplatin and mTOR inhibitor.

Example 4

Bearing p53 (TR) wild-type tumor cells of human adenocarcinoma line A seeded with a density of 0.1×106cells/5 μl per well in a Cup with a diameter of 6 cm and incubated for 24 h at 37°C and 5% CO2. Cells leave retrospektivnyi or carry out crackower the military transfection with 100 nm siPHK, a target which is either human p53 (registration number: NM000546; target sequence: 5'-GCATCTTATCCGAGTGGA-3'), or control siPHK LacZ (registration number: M; target sequence: 5'-GCGGCTGCCGGA-3') using oligofectamine (firm Invitrogen, catalogue number 12252-011). After incubation for 30 h, cells incubated with increasing concentrations of cisplatin (for example, from 0.5 to 6 ág/ml) for 24 h Cell extracts corresponding to 30 μg (P21) and 50 μg (p53 and PARP) protein, subjected to electrophoresis in 15% of the LTO-page (P21) and 10% of the LTO-page (p53 and PARP) and carry out the analysis of the Western blot turns using the mouse monoclonal and rabbit polyclonal antibodies to P21 and p53/PARP, respectively. Treatment with cisplatin retrospectively or control transfection siPHK LacZ cells induces dependent on the concentration of the protein expression of p53 and P21, and at higher concentrations of cisplatin (from 2 to 6 μg/ml) is the cleavage of PARP (marker of apoptosis). Unexpectedly, it was found that in cells, transfection siPHK p53 is reduced cisplatin-induced expression of p53 protein, which correlates with a dramatic decrease in the expression of P21, PARP cleavage and loss of cell viability. The same effect on the expression of p53, the expression of P21 and the cleavage of PARP, the detection is but also when using the other two siPHK, target is a human p53 (target sequence: 5'-GGAAGACTCCAGTGG-3' and 5'-GATGAATGG-3'). These data clearly confirm the fact that the action leading to increased cell death/apoptosis caused by combinations of cisplatin and an inhibitor of mTOR, is due to p53-dependent mechanisms.

Example 5

Bearing p53 (TR) wild-type cell line A seeded with a density of 0.1×106cells/5 μl in a Cup with a diameter of 6 cm and incubated for 24 h at 37°C and 5% CO2. Cells leave retrospektivnyi or carry out short-term transfection with 100 nm siPHK, a target which is either P21 (registration number: NM000389; sequence-target: 5'-GTGGACAGCGAGCAGCTGA-3'), or, as described above, the control siPHK LacZ using oligofectamine (firm Invitrogen, catalogue number 12252-011). After incubation for 30 h, cells incubated with suboptimal concentrations of cisplatin (for example, from 1 to 2 µg/ml) for 24 h Cell extracts corresponding to 30 μg (P21) and 50 μg (PARP) protein, subjected to electrophoresis in 15% and 10% of the LTO-page, respectively, and carry out the analysis of the Western blot turns using the mouse monoclonal and rabbit polyclonal antibodies to P21 and PARP, respectively. Treatment with cisplatin retrospectively or control transfect the skilled siPHK LacZ cells induces dependent on the concentration of the protein expression of p53 and P21, moreover, the cleavage of PARP (marker of apoptosis) is poorly expressed. Unexpectedly, it was found that in transfection siPHK P21 cells is the attenuation of cisplatin-induced expression of P21 protein, which correlates with a pronounced induction of PARP cleavage. These data clearly confirm the fact that the weakening caused by the cytotoxicity of the protein expression of P21 leads to action, leading to increased cell death/apoptosis caused by combinations of cisplatin and mTOR inhibitor.

1. The application of the definition of the status of p53 (THR) in an individual suffering from a proliferative disease, as a biomarker to assess the sensitivity of the individual to treatment with mTOR inhibitor in combination with a cytotoxic agent, wherein the presence of wild type p53 predicts sensitivity to treatment with the mTOR inhibitor in combination with a cytotoxic agent.

2. The use according to claim 1, which is to use analysis of p53 gene (TR) and expression level/post-translational modification of p53.

3. The use according to claim 1, where the proliferative disease is a cancer.

4. The use of claim 1, wherein the mTOR inhibitor is rapamycin or a derivative of rapamycin.

5. The use according to claim 1, in which the derivative of rapamycin is a 40-O-(2-hydroxyethyl)rapamycin, 40-[3-hydro is si-2-(hydroxymethyl)-2-methylpropanoate]rapamycin or 40-EPI(tetrazolyl)rapamycin.

6. The use according to claim 1, in which the cytotoxic agent is selected from antineoplastics of antimetabolite containing platinum compound, an alkylating agent, an inhibitor of topoisomerase I or II with activity against microrobot agent and radiation.

7. The method of determining the sensitivity of the proliferative disease in an individual to combination therapy with an mTOR inhibitor and a cytotoxic agent, namely, that define the status of the p53 gene (TR) and/or expression level/post-translational modification of p53 in a sample taken from the body of the individual, in which the presence of wild type p53 predicts sensitivity to treatment with the mTOR inhibitor in combination with a cytotoxic agent.

8. The method according to claim 7, where the proliferative disease is a cancer.

9. The method according to claim 7, namely, that determine the genetic status of p53 (THR) and/or the level of p53 expression.

10. The method according to claim 7, in which the sample is taken from the individual tumor.

11. The method of selection of individuals suffering from a proliferative disease, combination therapy with an mTOR inhibitor and a cytotoxic agent, namely, that determine the sensitivity of the proliferative disease to a combined therapy in each individual with the help of the method, described in paragraph 7, and are selected for combination therapy of individuals having the status of p53 (TR) wild-type.

12. The method according to claim 7, in which the mTOR inhibitor is rapamycin or a derivative of rapamycin.

13. The method according to claim 7, in which the derivative of rapamycin is a 40-O-(2-hydroxyethyl)rapamycin, 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin or 40-EPI(tetrazolyl)rapamycin.

14. The method according to claim 7, in which the cytotoxic agent is selected from antineoplastics of antimetabolite containing platinum compound, an alkylating agent, an inhibitor of topoisomerase I or II with activity against microrobot agent and radiation exposure.



 

Same patents:

FIELD: medicine.

SUBSTANCE: for examination, blood is sampled from an elbow vein of an examined patient without regard to food, then blood is dissolved with sodium citrate 1 ml in the blood to sodium citrate relation 8:1. For tumour cell detection, an artificial medium is pressurised in order to enable the examined blood passing through a calibrated filter. The tumour cells are precipitated on the calibrated filter; the precipitate is applied on slides preliminary degreased and cooled for better tumour cell adherence and drying. Thereafter, the smear prints are fixed on the slides with a three-percent Leishman spirit for 2-4 minutes. It is followed by washing with distilled water and staining with an azure eosin mixture in the ratio 3:1 for 6-8 minutes. After staining, the slides are washed with distilled water, air-dried and investigated under microscope.

EFFECT: use of the method allows reducing analysis time, allows instant primary diagnosing in a patient.

6 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: tissue samples are incubated at 37°C for 4 hours with estradiol in the concentration 2.21x10-6 M, precipitated with dihitonine for one night at 4°C, and a homogenate of a check sample and a sample cultivated with estrogen is analysed for thyroid peroxidase (TPO) activity by spectrophotometry. If the enzymatic activity after co-incubation with estradiol is increased in the tumour tissue sample by at least 50 % in comparison with the reference, such growth is considered to be sensitive to estrogen action. Such conclusion is made regardless of the content of cytoplasmic receptors of steroid hormones in the analysed tissue.

EFFECT: use of the method allows evaluating estrogen-dependence of thyroid cancer in prescribing a conservative therapy of the disease.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns a method for detecting sensitivity of a biological sample containing human lung cancer cells, a combination of a epidermis growth factor receptor inhibitor and a chemotherapeutic agent, by analysing the biological sample for superexpression of phosphorylated ACT protein and phosphorylated MAPC protein.

EFFECT: extended range of methods for evaluating sensitivity to EGFR inhibitors therapy.

10 cl, 1 ex, 11 dwg, 3 tbl

FIELD: medicine.

SUBSTANCE: substance of the invention involves a method of detecting an antibody in a sample being tested containing a body fluid of a specified mammal where said antibody is a biological marker of a disease state or propensity for a disease where the method involves (a) contacting said sample being tested with a set of various amounts of an antigen specific to said antibody, (b) evaluating specific binding of said antibody and said antigen, (c) drawing a diagram or calculating a curve of said specific binding to the amount of the antigen for each amount of the antigen used at the stage (a), and (d) stating the presence or absence of said disease state or propensity for the disease by the specific binding of said antibody and said antigen for each individual antigen concentration used where the presence of said disease state or propensy for the disease is detected by screening a titration curve starting with the stage (c) for observing an S-shaped or sigmoid curve.

EFFECT: higher sensitivity of the diagnostic technique.

23 cl, 7 ex, 6 tbl, 15 dwg

FIELD: medicine.

SUBSTANCE: assessment of marker gene is carried out, or combination of marker genes selected from group including marker genes of epidermal growth factor, transforming growth factor alpha and HER2, or combination of marker genes including market gene of amphireguline and marker gene selected from group including marker genes of epidermal growth factor, transforming growth factor alpha and HER2, in biological sample taken from patient's organism. Response to patient's treatment with inhibitor of HER dimerisation is forecasted by assessment of results obtained at the first stage. Invention provides a new method to forecast response to treatment of HER dimerisation in patient with inhibitor.

EFFECT: improved response to treatment.

16 cl, 19 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: patients' venous blood taken over the 1st phase of menstrual cycle with no heparin added as an anticoagulant is analysed for R-protein titres of red blood cell membranes. That is ensured by agglutination reaction with anti R-serum of a rabbit immunised with (0) Rh+ human red blood cells. If R-protein titre of red blood cell membranes is 1:1280, proliferation risk group is detected, while R-protein titre of red blood cell membranes being 1:2560 - 1:10240 enables genetic mutation risk group to be detected in the breast cancer jeopardised patients.

EFFECT: use of the method allows detecting early deviations of general regulatory adaptation reserves in the pathological conditions of breast and molecular genetic mutations capable to cause breast cancer.

8 dwg

FIELD: medicine.

SUBSTANCE: biological samples taken from the patients are analysed for apolipoprotein AI (Apo-AI) isoforms with oxidised residues of amino acids W50, W 108 and M 112.

EFFECT: use of the technique enables diagnosing hepatocellular carcinoma.

13 cl, 2 ex, 2 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: invention concerns a method of neoplastic disease detection on the basis of the analysis of a solubilised physiological sample.

EFFECT: reliable early diagnostic technique of neoplastic disorders.

30 cl, 6 ex, 1 dwg, 7 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology. The compound- conjugate antibody-medicinal agent is proposed with formula I: Ab-(L-D)p, where one or more molecules of maytanazoid medicinal agents (D) are covalently binded by L to the antibody huMAb4D5-8 (Trastuzumab) (Ab), which binds to the HER2 receptor and inhibits tumor cell growth, overexpressing HER2 receptor.

EFFECT: also a pharmaceutical composition is proposed which contains the specified compound, and a method of treating malignant tumor using this composition.

20 cl, 8 tbl, 10 ex, 17 dwg

FIELD: medicine.

SUBSTANCE: determined are quantity of leukocytes, platelets, % of promyelocytes, APTT and prothrombin indices, fibrinogen concentration, AFMC in ethanol test, products of degradation of fibrin of D-demers and Amin elecrocoagulogram. In case of leukocytosis higher than 10×109/l, reduction of platelet number lower than 15×109/l, increase of promyelocytes higher than 50%, elongation of APTT index higher than 1.3±0.05, reduction of prothrombin index lower than 77.2±1.9%, fibrinogen concentration lower than 1.5+0.1 g/l, increase of D-dimers higher than 1000 ng/ml, Amin higher than 2.5±0.1, severe hemorrhagic complications of III and IV degree of severity are predicted with high degree of probability.

EFFECT: ensured is prediction of severe hemorrhagic complications in patients with acute promyelocytic leukemia in disease diagnostics.

3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: what is offered is a method of structure stabilisation of thrombin binding DNA-aptamers, and also DNA-aptamers stabilised in such a way. The presented method provides formation of an additional base-stacking system by means of heterocycles or their analogues by means of increasing a surface of an aromatic system of heterocycles or their analogues, owing to using methods of determining a tertiary structure or molecular simulation with stating the fact of contact formation of the aromatic system of heterocyclic bases or their analogues with a G-quadruplex quartet which is related to a lateral loop.

EFFECT: method allows more effective assembly of antithrombin DNA-aptamers and improved structural stability under physiological conditions.

7 cl, 7 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method involves allele-specific Nested-PCR with primers which are matched with nucleotide sequences coding amino acids in positions 70-71 of the amino acid sequence. The allele-specific primers E70f1 - 5'-AGAAGGAGATCCTGGAGGATAG - 3' and R71r1 - 5'-CCTGTCCACCTCGGCCCGCCTATC - 3' are matched with a part of BoLA-DRB3 gene located on chromosome 23 (localisation 23q21). They interact only with the nucleotide sequences coding alleles *11, *23, *28 =*7A causing genetic stability to cattle leukaemia. Then sequencing primer Zond 70/71 5'-GCCCGGCTACACCTGT - 3' is used to identify homo- or heterozygosity of an individual by the given alleles. If observing the primers interacting with alleles *11, *23, *28 =*7A, animals are considered to be leukaemia stable, while the absence of interaction with the same alleles can enable to refer to leukaemia unstable, and to neutral.

EFFECT: invention can be used for mass genetic typing of BoLA-DRB3 leukaemia tolerable animals in livestock and commodity economies for animal selection in a nuclear stock.

2 dwg, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: set contains species-specific oligonucleotide primer pairs and appropriate fluorescent-marked probes for conducting one-stage instant identification of several human-pathogenic Orthopoxviruses (VARV, MPXV, CPXV and VACV) by means of real-time multiplex PCR.

EFFECT: invention is intended for instant diagnostics of human and animal Orthopoxvirus infections by real-time multiplex PCR.

10 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: tissue is homogenised in a buffer and centrifuged at 105000 g for 60-90 min at 0-4°C to produce a cytoplasmic fraction which is then incubated with 10 mM of phosphocreatine and 10 mcg/ml of phosphocreatine kinase for 25-45 minutes at 35°C. Cytoplasmic fraction proteins are divided by ammonium sulphate at three stages, at the first stage ammonium sulphate is added to 38% of saturation and centrifuged to isolate a precipitate containing a 26S-proteasome pool, at the second stage, a supernatant is added with ammonium sulphate to 42 % of saturation and centrifuged to isolate a precipitate containing ballast proteins, at the third stage to the supernatant is added with ammonium sulphate to 70 % of saturation and centrifuged to isolate a precipitate containing a 20S-npoteasome pool. Ammonium sulphate is added in portions during 20 min on a magnetic stirrer and further mixed for 20 minutes.

EFFECT: invention allows dividing native 26S- and 20S-proteasomes and isolating them in those amounts they exist in living cells, with preserving at most an undamaged 26S-proteasome structure.

3 cl, 1 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.

EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.

87 cl, 37 dwg, 5 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: method includes analysing aliquots of said sample by one or more methods of protein description specified in the chromatography. The method is based on genetic analysis techniques specified in RFLP and T-RFLP. These methods can be applied both separately, and in a combination. The offered methods allow obtaining the information on the presence and fractions of various individual proteins or coding sequences. The obtained information can be used for evaluating stability of a polyclonal cell line in process, and also estimating a structure of various parties of end polyclonal products.

EFFECT: methods allow describing the composition consisting more than of 10, 20 or greater number of antibodies.

16 cl, 18 dwg, 18 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: composition includes at least three oligonucleotide probes and enables simultaneously determining a level of PSMB4, FCER2 and POU2F2 genes expression. The oligonucleotide composition under the invention is presented to be used, including as a part of a microchip, in a method for prediction of a developing disease in a subject suffering chronic lymphatic leukemia that involves analysing a level of expression of at least three named genes in patient's blood samples.

EFFECT: higher efficacy of the composition.

8 cl, 5 dwg, 16 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: method provides recovery of DNA of an analysed strain followed by PCR with using nucleotide primers of terC, ilvN and inv genes of the following sequences: 89-S - AATCAAATCTCGCCCAGC, 89-As -GCTGCGTATCATTTCACC; 45-S - AGTGGTCTGCTTCTCTGG, 45-As -CGGCATACACAGAATACC; inv839 - TACCTGCACTCCCACAAC, inv1007 -CCCATACGCTGATCTACC. The analysed strains are differentiated by matching the sizes of the derived fragments of terC, ilvN and inv genes with the similar fragments in typical strains of principal and nonprincipal plague agents.

EFFECT: invention allows quick, effective and reliable differentiation of the strains.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: what is offered is a method of marking a human 7th chromosome providing in situ hybridisation of metaphasic or interphasic cell chromosomes of a tested sample and a DNA probe presented by a marker plasmid alpha R1-13 which consists of EcoR 1-EcoR l DNA fragment of a pBR 325 vector of the value 5966 base pairs and EcoRl-EcoRl alphoid DNA fragment of the human 7th chromosome of the value 680 base pairs. The testing environment in which the used DNA-probe specifically interacts with a centromeric region of the 7th chromosome without cross hybridisation with other human chromosomes are developed.

EFFECT: more efficient identification of the presented chromosome and enabled application of the new method in medical diagnostics.

4 ex

FIELD: medicine.

SUBSTANCE: reactions are conducted in the same PCR-microtube on walls of which there is a probe immobilised. In comparison with standard, the PCR-tubes used have a feature of construction: an extended internal surface, and as consequence - a great sorption capacity. A process of selective amplificate sorption on the test tube walls occurs after each anneal stage: temperature decreasing leads to hybridisation of amplicon chain not only among themselves, and with probes preliminary immobilised on the test tube walls. Using the similar PCR-microtubes enables increasing sensitivity of an immune-enzyme assay of amplicons and decreasing a number of polymerase chain reaction cycles. The fixed analytical signal will characterise both the presence of the required DNA, and show its concentration in the sample.

EFFECT: offered test system can be adapted for any existing PCR-technique, does not require special instrumentation and is reliable, simple and efficient for all parameters.

6 cl, 1 dwg, 1 tbl, 1 ex

FIELD: medicine, psychiatry.

SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.

EFFECT: more objective prediction of disease development.

3 ex

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