Determination of cell sensitivity to treatment with b-raf inhibitor by detection of k-ras mutation and levels of rtk expression

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine. Claimed is method, including determination of presence or absence of K-rasG12D mutation. Presence of K-rasG12D mutation points to the fact that patient will not respond to treatment with B-Raf inhibitor.

EFFECT: invention provides effective method of identifying patient who does not respond to treatment with B-Raf inhibitor.

6 cl, 34 dwg, 12 tbl, 3 ex

 

CROSS-REFERENCE TO RELATED APPLICATIONS

According to this application is claimed under 35 U. S. C. § 119(e) priority of provisional applications U.S. serial number 61/236466, filed August 24, 2009, and 61/301149, filed February 3, 2010, which are contained herein by reference in full for all purposes.

The technical field TO WHICH the INVENTION RELATES

The invention relates to the diagnosis and treatment of cancer and, in particular, identification of mutations or sverkhekspressiya RTK, which are diagnostically and/or prognostically significant, and the choice of cancer treatment, depending on definition.

The LEVEL of TECHNOLOGY

Receptor tyrosine kinase (RTK) and their ligands are important regulators of the proliferation of tumor cells, angiogenesis and metastasis. For example, to the family of ErbB RTK include EGFR (HER1 and ErbB1), HER2 (neu or ErbB2), HER3 (ErbB3) and HER4 (ErbB4), and they have distinct ligand-binding and signaling activities. The ligands that bind to ErbB receptors include epidermal growth factor (EGF), transforming growth factor a (TGFa), heparin-binding EGF-like ligand (HB-EGF), amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), the epigenome (EPG), heregulin (HRG) and neuregulin (NRG). These ligands bind directly to EGFR, HER3, or HER4 and launch a large number of subsequent signaling cascades that about�worn path RAS-ERK and PI3K-Akt. EGF and other growth factors and cytokines such as platelet derived growth factor (PDGF), transmit the signal through Ras. Mutations in Ras firmly block the Ras in its active associated with GTP state (Wislez, M., et al.,Cancer Drug Discovery and Development: EGFR Signaling Networks in Cancer Therapy, Eds: J. D. Haley and W. J. Gullick, Humana Press, pp.89-95, 2008).

Another RTK is MET, the activation of which by means of its ligand - growth factor hepatocyte (HGF) - induced catalytic activity of the kinase MET, which launches transphosphorylation tyrosines Tyr 1234 and Tyr 1235. These two tyrosine launch a large number of signal transducers, thus initiating a range of biological activities caused by METH. HGF induces prolonged activation of RAS and thus prolonged activity MARK.

One of the ras gene is K-ras, which is mutated in a large number of types of cancer. Mutation of K-ras gene in codons 12 and 13 contributes to oncogenic transformation, leading to a functional modification of the protein P21-ras, K-ras gene, resulting in a transfer of surplus growth signals to the cell nucleus, stimulating the growth and division of cells. Therefore, the identification of mutations of K-ras gene has been widely used as convenient tools in the diagnosis of cancer, e.g., pancreatic cancer, colorectal and non-small cell lung cancer, and studies have shown that it could be associerad�on multiple phenotypes of tumors (Samowitz W. S., et al., Cancer Epidemiol. Biomarkers Prev. 9: 1193-1197, 2000; Andreyev, H. J., et al., Br. J. Cancer 85: 692-696, 2001; and M. Brink, et al., Carcinogenesis 24: 703-710, 2003).

Ras is essential in oncogenic transformation and the Genesis. Oncogenic H-, K - and N-Ras occur due to point mutations limited to a small number of sites (amino acids 12, 13, 59 and 61). Unlike normal Ras oncogenic ras proteins are deprived of their inherent GTP activity and therefore remain constantly activated (Trahey, M., and McCormick, F. (1987) Science 238: 542-5; Tabin, C. J. et al. (1982) Nature. 300: 143-9; Taparowsky, E., et al. (1982) Nature. 300: 762-5). The contribution of oncogenic ras in cancer person is estimated at 30% (Almoguera, C. et al. (1988) Cell. 53:549-54).

Mutations are often limited to only one of the ras genes, and the frequency is fabric and ofwholesale. The most commonly mutated oncogene in human cancer is K-ras, is particularly common mutation in codon 12. While the oncogenic activation of H-, K - and N-Ras, resulting from single nucleotide substitutions were found in 30% of cases of human cancer (Bos, J. L. (1989) Cancer Res 49, 4682-9), mutation of K-ras at codon 12 detected in more than 90% of pancreatic cancers (Almoguera, C. et al. (1988) Cell 53, 549-54; Smit, V. T. et al. (1988) Nucl Acids Res 16, 7773-82; Bos, J. L. (1989) Cancer Res 49, 4682-9). Ductal pancreatic adenocarcinoma, the most common pancreatic cancer, infamous because of its rapid onset and régis�intesti to treatment. High frequency of mutations of K-ras in tumors of the pancreas of a person indicates that continuous activation of Ras is crucial in the process of carcinogenesis of the pancreas. Adenocarcinoma of the exocrine pancreas is the fourth cause associated with cancer mortality in Western countries. Treatment have had limited success, and five-year survival remains less than 5% with an average survival of 4 months for patients with inoperable tumors (Jemal, A. et al. (2002) CA Cancer J Clin 52, 23-47; Burris, H. A., 3rd et al. (1997) J Clin Oncol 15, 2403-13). This point mutation can be identified early in the course of the disease with the progression of normal cuboidal epithelium of the pancreatic ducts in flat hyperplastic lesion, thought to be a cause of the disease in the pathogenesis of pancreatic cancer (Hruban, R. H. et al (2000) Clin Cancer Res 6, 2969-72; Tada, M. et al. (1996) Gastroenterology 110, 227-31). Regulation of signaling oncogenic K-ras in pancreatic cancer person, however, remains mostly unknown.

Mutations in K-ras are found in 50% of cases of cancer of the colon and lung (Bos, J. L. et al. (1987) Nature. 327: 293-7; Rodenhuis, S. et al. (1988) Cancer Res. 48: 5738-41). In cases of cancer of the urinary tract and bladder mutations are mainly located in the N-ras (Fujita, J., et al. (1984) Nature. 309: 464-6; Visvanathan, K. V. et al. (1988) Oncogene Res. 3: 77-86). Gene mutation of N-ras �strachotta in 30% of cases of leukemia and liver cancer. Approximately 25% of cases of skin lesions in humans involved the mutation of Ha-Ras (25% for squamous cell carcinoma and 28% for melanomas) (Bos, J. L. (1989) Cancer Res. 49: 4683-9; Migley, R. S, and Kerr, D. J. (2002) Crit Rev Oncol Hematol. 44: 109-20). 50-60% of cases of carcinoma of the thyroid gland is unique, having mutations in all three genes (Adjei, A. A. (2001) J Natl Cancer Inst. 93: 1062-74).

Constant activation of the Ras can be achieved by oncogenic mutations or through hyperactivemenu receptors of growth factors such as EGFR. Increased expression and/or amplification of EGFR family members, particularly EGFR and HER2 may be involved in a large number of forms of malignant tumors (as shown in the article Prenzel, N. et al. (2001) Endocr Relat Cancer. 8: 11-31). When some of these types of cancer (which includes cancer of the pancreas, colon, bladder, lung) overexpression of EGFR/HER2 exacerbated by the presence of oncogenic Ras mutations. Abnormal activation of these receptors in tumors can be explained by sverkhekspressiya, gene amplification, mutations, leading to permanent activation or autocrine loops of growth factors (Voldborg, B. R. et al. (1997) Ann Oncol. 8: 1197-206). Against receptors of growth factors, especially receptors EGFR, amplification and/or overexpression of these receptors is often found in cancer of the breast, ovary, stomach, esophagus, pancreas, easy�, of the colon and neuroblastoma.

The signaling pathway RAS-MAPK controls the growth, differentiation and viability of cells. This signaling pathway has long been considered a promising Avenue for cancer therapy due to its Central role in the regulation of growth and cell viability of a wide range of human tumors, and mutations in components of this signaling pathway underlie tumor initiation in mammalian cells (Sebolt-Leopold et al (2004) Nat Rev Cancer 4, pp 937-47).

The signaling pathway RAS-MAPK is activated by a large number of extracellular signals (hormones and growth factors) that activate RAS by replacement of GDP for GTP. Ras then attracts RAF to the plasma membrane where it is activated. As noted above, the basis of tumor initiation in mammalian cells are mutations in components of the signaling pathway leading to permanent activation. For example, receptors of growth factors, such as the receptor of epidermal growth factor (EGFR), in many types of cancer are amplifications and mutations, accounting for up to 25% of cases are non-small cell lung cancer and 60% of cases of glioblastoma. Also often mutated Braf, especially when melanoma (about 70% of cases) and carcinoma of the colon (approximately 15% of cases). Moreover, the most frequently mutated oncogene, present in approximately 30% section human cancer, is ras. Frequency and type of mutated ras genes (H-ras, K-ras or N-ras) vary widely depending on the type of tumor. K-ras, however, is the most frequently mutated gene, with the highest number of cases was detected in pancreatic cancer (approximately 90%) and colorectal cancer (approximately 45%). This makes it, as well as other components of the signaling pathway of a suitable target for anticancer therapy. Indeed, clinically tested small molecule inhibitors designed to target a large number of stages of this path. Moreover, it was recently approved for the treatment of renal cell carcinoma inhibitor of RAF kinases, resulting in inhibition of the transmission of the signal RAS, sorafenib (Nexavar.RTM., Bayer HealthCare Pharmaceuticals). On the basis of these data is increased interest in targeting the pathways RAS-MARK to develop improved cancer therapies.

Described in the article Downward, J. (2002) Nature Reviews Cancer, volume 3, pages 11-22, RAS proteins are members of a large superfamily of low molecular weight GTP-binding proteins that can be subdivided into several families according to the degree of conservatism sequences. Different families are important for various cellular processes. For example, a family of RAS controls the growth of cells, and RHO family control the actin cytoskeleton. Typically, the RAS family of�anywayt as consisting of three members of H-, N - and K-RAS, and K-RAS produces a large (4B) and small (4A) playeronly variant (Ellis, C. A. and Clark, G. (2000) Cellular Signalling, 12: 425-434). Discovered that members of the RAS family are activated by mutation in human tumors and have a strong transformative potential.

Members of the RAS are very closely related, having homology to the amino acid sequences of 85%. Despite the fact that RAS proteins function in very similar ways, recently became aware of some information on the subtle differences between them. Proteins H-ras, K-ras and N-ras is widely expressed, with K-ras is expressed in almost all cell types. Studies with knockout showed that for the normal development of the mouse are not required H-ras and N-ras either independently or in combination, whereas K-ras significant (Downward, J (2002) on page 12).

Moreover, as described in the article Downward, J (2002), the disturbed signal transmission through the way of the RAS occurs as a result of several different classes of mutational damage in tumor cells. The most obvious of these mutations are in the genes ras. Approximately 20% of human tumors have activating ras point mutations, often in K-ras (about 85%), then N-ras (15%), then N-ras (less than 1%). All these mutations kompromitirajuci GTP RAS activity, preventing GAP stimulated protein-coupled GTP hydrolysis on RAS and thus causing the accumulation of RAS in the GTP-related�Anna, the active form. Activation of almost all RAS in tumors due to mutations in codons 12, 13 and 61 (page 15 article Downward, J (2002)).

It would be effective if the cancer treatment could be selected for a particular cancer. In particular the present invention relates to methods of determining, not whether some approved and available methods of treatment is still ineffective for a particular type of cancer.

A BRIEF summary of the INVENTION

The present invention relates to prognostic methods for identifying tumors that are not sensitive to the treatment of inhibitor of B-Raf, by identifying mutations in the gene or the protein K-ras. The methods include determining the presence or absence in the sample of a mutated gene or protein K-ras, thus, identifying a tumor that does not respond to treatment by an inhibitor of B-Raf. To implement the methods described sets.

In another aspect, the present invention relates to prognostic methods for identifying tumors that are not sensitive to the treatment of inhibitor of B-Raf, by detecting aberrant levels of expression of the RTK. The methods include determining the expression levels of some RTK in the sample, and RTK overexpression correlates with lack of response to treatment by an inhibitor of B-Raf. Examples of RTK that correlate with response to treatment In a-Raf, are, �not limited to, about, EGFR and cMet. The methods also include determining the levels of induction of some of RTK ligands in the sample, and abnormally high levels of induction of ligands correlate with lack of response to treatment by an inhibitor of B-Raf. Examples of ligands that correlate with response to treatment In a-Raf, include, but not limited to, EGF and HGF. The methods also include determining in the sample the levels of Ras-GTP, and abnormally high levels of Ras-GTP correlate with lack of response to treatment by an inhibitor of B-Raf. To implement the methods described sets.

In another aspect, the present invention relates to methods of treating tumors that do not respond to treatment by an inhibitor of B-Raf. The methods involve the introduction of inhibitor of B-Raf in combination with the EGFR inhibitor.

DESCRIPTION of FIGURES

Figure 1 shows the biochemical enzymatic analysis. The data indicate that at physiological [ATP] only GDC-0879 is retained efficacy against B-RafV600Eand Raf WT isoforms.

Figure 2 presents the tests of viability on the lines of tumors with different status of mutation of Raf/Ras.

Figure 3 shows a prolonged induction rmec by Raf inhibitors only on the lines that are different from B-RafV600E. Levels rmec reach a certain level relative to IC50inhibitors against Raf WT.

On �figure 4 shows that C-Raf is an isoform of Raf, mainly responsible for the induction rmec Raf inhibitors on lines different from B-RafV600E.

Figure 5 shows the specific activity of C-Raf induced by both inhibitors only on the lines that are different from B-RafV600E. Was not reduce the levels of germination under conditions of induction of the Raf.

Figure 6 shows the lack of induction of pERK levels. The relative effectiveness of inhibitors is correlated with their biochemical IC50.

Figure 7 shows a bell-shaped effect on the levels rmek in initial conditions. The inhibitory effect of GDC-0879 prevails after stimulation with serum.

Figure 8A shows that the duration and magnitude of inhibition of the path determines the BRAF inhibitor B-Raf, the efficacy of GDC-0879 on the main xenograft models of human tumors. Graph the Kaplan-Meier showing the doubling time of tumor models derived from patient tumors: melanoma and non-small cell lung cancer treated daily with GDC-0879 (100 mg/kg or carrier. Genotypes BRAF, N-ras and K-ras are indicated. Statistically significant (P<0,05) slowing tumor progression was observed for tumors MEXF 989, MEXF MEXF 276 and 355. Introduction GDC-0879 significantly enhanced the growth of certain K-ras-mutant non-small cell of lung tumors, such as LXFA 1041 and LXF 983.

In figure 8B it is shown that treatment with GDC-0879 reduces the phosphorylation of ERK1/2 in a major xenotransplantation human tumors BRAFV600E. During the course of the pharmacodynamic studies, mice were treated with GDC-0879 (100 mg/kg and scored 1 or 8 h after the last dose (21-24 days). Presents immunoblot phosphorylated and total ERK1/2. Strong inhibition of phospho-ERK1/2, which lasted for 8 hours, significantly correlated with the status of BRAFV600Eand antitumor efficacy of GDC-0879. As a loading control for all samples was evaluated by the expression of total ERK1/2.

In the figures 9A, b, C and D shows that the lines of K-ras-mutant tumor cells have different sensitivity to the RAF inhibitor GDC-0879 and the MEK inhibitorin vivoandin vitro. In the figures A and b, inhibition of MEK, but not RAF, prevented the growth ofin vivoK-RAS-mutant tumors NST. Upon reaching a tumor size of ~200 mm3mice were selected randomly and treatment was started either inhibitor GDC-0879 (100 mg/kg (A) or an inhibitor of MEK at 25 mg/kg (MEK Inh; (B) on a daily chart. Points, mean; bars, SE. C, size EU50GDC-0879 130 cell lines are presented as a function of mutation status BRAF and K-RAS. GDC-0879-mediated inhibition of cell growth significantly in� extent correlated with BRAF mutation. D, scatter plot of the values of EC50the MEK inhibitor grouped according to genotype. Inhibition of MEK also influenced a significant fraction of cell lines expressing BRAF wild type. Data represent the mean of four measurements.

In the figures 10-18 shows the growth in the xenograft tumors of the lung after administration of doses of GDC-0879.

In the figures 19A and presented In Raf inhibitors induces RAS-dependent RAF translocation in wild-type plasma membrane in cells than B-RAFV600E. (A) MeWo Cells (RAS/RAFWT) were processed using GDC-0879 (2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-indene-5-yl]-3-(pyridin-4-yl)-1H-pyrazol-1-yl}Ethan-1-ol), PLX4720 (N-[3-[(5-chloro-1H-imidazo[2,3-b]pyridin-3-yl)carbonyl]-2,4-differenl]-1-propanesulfonate) or AZ-628 (3-(2-cyanoprop-2-yl)-N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin-6-ylamino)phenyl)benzamide) (all at concentrations of 0.1, 1, 10 mm) for 1 hour and fractionally membrane (P100) and cytosolic (S100) fraction. Aliquots of membrane and cytosolic fractions were subjected to immunoblotting with the indicated antibodies. (B) Cells NECT temporarily transfusional using Venus-C-RAF (green), CFP-K-RAS (red) and mCherry-H2B (blue). Labeled with Venus C-RAF localized near CFP-KRAS on the plasma membrane in cells treated for 4 hours with GDC-0879 or AZ-628, taken at a concentration of 10 mm, followed from�something viable cells, using confocal fluorescence microscopy. Translocation to the membrane is blocked by transfection of dominant negative CFP-labeled KRASS17N instead KRASWT (right panel).

In the figures 20A, b, C and D shows the importance of the role that active Ras plays in the activation of C-RAF and induction of phospho-MEK by RAF inhibitors. (A) Cells A (B-RAFV600E) was treated with GDC-0879 or PLX4720 for 1 hour and literally in hypotonia buffer for membrane fractionation. Both factions, membrane (P100) and cytosolic (S100), was subjected to immunoblotting with the indicated antibodies. (B) MeWo Cells were temporarily transfusional with KRASWT or KRASS17N were treated with GDC-0879 or PLX4720 (at concentrations of 0.1, 1, 10 mm) for 1 hour and fractionally membrane (P100) and cytosolic (S100) fraction. Aliquots of membrane and cytosolic fractions were subjected to immunoblotting with antibodies against phospho and anti - total MEK. (C) lysates From cells MeWo (RAS/RAFWT), A375 (B-RAFV600E) and H2122 (KRASMT) measured the levels of RAS-GTP using the ELISA Protocol Ras-GTP, using immobilized C-RAF-RBD as bait for the capture of RAS-GTP. Relative luminescence units represent the detection of RAS antibody against RAS associated with RBD. By RAS-GTP H2122>>Mewo>A375. (D) Transfection of mutant KRASG12D (but not KRASWT) in cells A (B-RAFV600E) enables the cells to induce heterodimer B-RAF With RAF and activate �inazu C-RAF in the presence of the RAF inhibitor GDC-0879 (dosed at 0.1, 1, 10 mm). C-RAF was immunoprecipitated from control and inhibitor-treated cells and analyzed in relation to the activity of the protein and heterodimerization B-RAF. Shown with WB total levels of C-RAF in immunoprecipitate indicate the loading of each lane.

In figures 21A, b, C and D presents the measurement of baseline and stimulated EGF knockdown of pERK with the help of Raf inhibitors in cell lines of B-RAFV600E and b-Raf WT. (A) table of genotype and EGFR levels among the tested lines. (B) Measurement of baseline and stimulated levels of pERK: cells were treated with compounds, taken at a concentration of 0.0004-10 mm in serum-free media for 1 hour. For stimulation for 5 min before lysis of the cells was added EGF (20 ng/ml Lysates was transferred into a die MSD, which measured the levels of phospho - and total ERK. (C) IC50 Data for pERK inflicted on the charts for two Raf inhibitors (CHR-265, 1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine, and GDC-0879) in baseline and stimulated with EGF conditions. (D) Graphs of the dose-response induction of pERK, with 1-hour processing of these lines, B-Raf WT using inhibitors of Raf.

In the figures 22A and b shows stimulation with EGF levels of phospho-MEK and cell proliferation of mutant cell lines with B-RAF V600E, resistant to the RAF inhibitor. (A) Cells were treated link�m at a concentration of 0.0004-10 mm in serum-free media for 1 hour. For stimulation for 5 min before lysis of the cells was added EGF (20 ng/ml Lysates was transferred into a die MSD, which measured the levels of phospho - and total MEK. The data on IC50 of phospho-MEK was put on the schedule for two of these Raf inhibitors in source and EGF-stimulated conditions. GDC-0879 is more effective in the knockdown levels of phospho-MEK, because it has a lower reported IC50 value against the isoforms of C-RAF and B-RAF wild type than PLX4720. (B) EGF Treatment identifies cells of B-RAFV600E resistant to RAF inhibitors, but the combination with Tarceva, and Masha (or MEK inhibitor, e.g., PD-0325901) overcomes this resistance. The cells were injected with the indicated inhibitors either alone or in combination in the presence of EGF in the medium at a concentration of 20 ng/ml.

Figure 23 shows stimulation of EGF induces the activity of B-RAF and C-RAF in mutant B-RAFV600E (LOX, 888 are melanoma, whereas NT29 is a cancer of the large intestine). All cell lines Express the levels of surface EGFR. 888 homozygote for an allele of B-RAFV600E, all other lines are heterozygous, therefore, also carry allele B-RAF wild type. Heterozygous cell lines induce the activity of B-RAF and C-RAF, whereas homozygous line only induces the activity of C-RAF. This activity RAF wild type could not be inhibited by selective inhibitors of RAF (B-RAF V600E, so in the e�their lines the levels of phospho-MEK, induced by EGF, resistant to RAF inhibition, whereas endogenous levels of phospho-MEK-induced B-RAF V600E, sensitive to selective inhibitors of RAF (B-RAF V600E.

Figure 24 shows a trend towards a negative correlation between high levels of EGF mRNA (x-axis) and the value of IC50 of RAF inhibitor (μm along the y-axis). The data on cell performance is shown for cell lines of melanoma B-RAF V600E and represent the RAF inhibitors, which are biochemically selective in respect of isoforms of B-RAF V600E with less relevant biochemical and cellular effects in respect of the isoforms of RAF wild type.

Figure 25 shows the levels of RAS-GTP in a large number of types of tumors. The levels of RAS-GTP are low in tumors K-RASWTand high in tumors carrying mutant K-RAS, for example, tumors N. The levels of Ras-GTP were determined using analysis of RBD-Elisa.

Figure 26 shows the levels of Ras-GTP in cells B-Raf V600E in the presence (+EGF) and in the absence (NI) EGF induction. Stimulation of EGF in cells BRAF V600E increases the levels of Ras-GTP.

Figure 27 shows the levels of pERK in cells B-Raf V600E in the presence (stim) and without (unstim) EGF induction. Stimulation of EGF in cells BRAF V600E increases the levels of Ras-GTP, resulting in an increase in pERK levels in cell lines with B-RAF V600E via activation of C-Raf (see activation of C-Raf, shown in figure 23). All 4 cell lines are mutant B-Raf V600E, but among them A itself has�e low levels of Ras-GTP (the lowest levels of active Ras) and she has not seen a steady induction levels rmec and pERK in response to EGF. It is known that cells A sensitive to inhibitors of Raf.

Figure 28 shows the levels rmek in cells B-Raf V600E in the presence (stim) and without (unstim) EGF induction. Stimulation of EGF in cells BRAF V600E increases the levels of Ras-GTP, resulting in an increase in pMEK levels in cell lines with B-RAF V600E via activation of C-Raf (see activation of C-Raf, shown in figure 23).

Figure 29 summarizes the effects of some inhibitors of RAF (GDC-0879, PLX-4720 and “Raf inh a”, which represents a 2,6-debtor-N-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-3-(propylsulfonyl)benzamide) to block cellular induction of pERK in response to stimulation with EGF. Cells BRAF V600E expressing EGFR, were placed in serum-free medium and then either left without stimulation (-EGF) or stimulated with EGF (+EGF) in the presence of these RAF inhibitors at different doses. Built graphs of inhibition of pERK and graphically represented values IC50. GDC-0879, as shown in figure 1, can effectively block the transfer of signal through RAF wild type, while the other two inhibitors are selective against BRAF V600E.

Figure 30 shows how the stimulation of HGF (+HGF) results in induction of pERK in cells, sverkhekspressiya with-METH. This induction is not blocked by inhibitors of RAF. However, the source levels of pERK, which are called BRAF V600E, efficiently blocked by inhibitors of RAF. E�about demonstrates that the signal transmission through the C-MET also occurs with the participation of the RAF isoforms of wild-type.

Thus, aberrant expression of receptor tyrosine kinases (RTK), which include EGFR, or aberrant induction of appropriate ligands can identify the cells that are resistant to RAF inhibitors.

Figure 31 shows how among cells of B-RAFV600E expression of EGFR is associated with resistance to RAF inhibitors. This graph represents the values of the EU50, reflecting cell viability, (μm) of mutant cell lines with B-RAF V600E melanoma and colon, which was treated with a RAF inhibitor for 4 days to determine viability. The EGFR levels were determined by Western-blot and was classified as negative in the absence of the bands at carrying out a Western blot of cell lysates with an antibody against EGFR. Between the EGFR-positive cell lines, there is a continuum of expression from low to middle and high. The only EGFR-negative cell line, which is stable (>20 µm / EC50), is a line of cells PTEN null.

In the figures 32A-C shows a combined investigation of RAF inhibitor and EGFR inhibitor (Tarceva, and Masha) on tumors of the large intestine with different levels of EGFR expression.

Figure 32A Western-blot of lysates of two lines of large intestine cancer BRAF V600E shown their different levels of total EGFR: COLO201 has low� levels of EGFR, while the CX-1 has a relatively high levels of EGFR.

Figure 32V shows the effect of combined treatment of COLO201 cells or RAF inhibitor alone, Tarceva, and Masha alone or in combination of a RAF inhibitor and Tarceva, and Masha.

Figure 32C shows the effect of the combined treatment of cells CX-1 or RAF inhibitor alone, Tarceva, and Masha alone or in combination of a RAF inhibitor and Tarceva, and Masha. Neither the RAF inhibitor alone or Tarceva, and Masha does not itself supression proliferation as effectively as the combination. Both inhibitors in a joint introduction into the cells of the CX-1 is observed satisfactory synergies.

Thus, among BRAFV600E cells expressing EGFR, high levels of EGFR predict a strong synergism between inhibitors of RAF and EGFR inhibitors. In particular, in cancer of the colon, when among tumors with BRAFV600E prevalent high expression of EGFR, the combination of these inhibitors of RAF and Tarceva, and Masha is observed synergy in terms of inhibition of proliferation of tumor cells.

Figure 33 shows the mechanistic basis of synergism between inhibitors of RAF and Tarceva, and Masha in the cells of BRAFV600E tumors expressing high levels of EGFR. Western blot was performed by taking the cells treated with either 1 hour or 24 hours with or without inhibitors (rows 1, 5, 9, 13), or an inhibitor of the RAF samostojatel�but (ranks 2, 6, 10, 14), Tarceva, and Masha independently (rows 3, 7, 11, 15) or a combination of a RAF inhibitor and Tarceva, and Masha (rows 4, 8, 12, 16) at a concentration equal to the value of their cellular EU50. Temporary point 24 hours shows that the phosphorylation of ERK in mutant cells (B-RAFV600E with high EGFR expression (CX-1) reduced sensitivity to inhibition by inhibitors of the RAF, and for maximum efficiency requires a combination of a RAF inhibitor and EGFR inhibitor. Part of the activation of ERK signal comes from RAF wild type, which is activated after EGFR and cannot be blocked by a selective inhibitor of BRAF V600E.

In figures 34A-C shows the results of the interaction and efficacy of a RAF inhibitor and erlotinib (Tarceva, and Masha), administered in combination to mice NCR nude (Taconic) bearing subcutaneous xenograft colorectal carcinoma human HT-29 BRAF V600E. Figure 34A RAF inh a received 100 mg/kg with increasing doses of Tarceva, and Masha. Figure 34B all animals received Tarceva, and Masha with increasing concentrations of RAF inh a. Increased efficiency was observed after the introduction of the two compounds in combination. Figure 34C lysates of tumors treated with the indicated doses of inhibitors in figures 34A and b was analyzed by Western blot in the levels of phospho-ERK (pERK). The RAF inhibitor and and Tarceva, and Masha in mice by co-administration acted synergistic in terms of reduction in tumors UB�her phospho-ERK.

DETAILED description of the INVENTION

In one of the embodiments of the subject matter described herein relates to a method of identification of a patient not responding to treatment with an inhibitor of B-Raf, comprising determination of the expression or induction of RTK and/or their ligands. The methods include determining in the sample the levels of expression or induction of some RTK and/or their ligands, and overexpression of RTK and/or their ligands correlates with lack of response to treatment by an inhibitor of B-Raf. In one of the embodiments of the sample expresses mutant B-Raf V600E. Examples of RTK that correlate with response to treatment of B-Raf, include, but not limited to, EGFR and cMet. The methods also include determining in a sample the expression levels of some of RTK ligands, and abnormally high levels of expression of ligands correlate with lack of response to treatment by an inhibitor of B-Raf. Examples of ligands that correlate with response to treatment of B-Raf, include, but not limited to, EGF and HGF.

In one of the embodiments of the subject matter described herein relates to a method of identification of a patient not responding to treatment with an inhibitor of B-Raf, comprising determining in a sample the amount of Ras-GTP, and increased amounts indicate that the patient will not respond to treatment �specified inhibitor of B-Raf. In one of the examples of increased amount higher than the amount found in normal not stimulated samples. Ways to measure the levels of Ras-GTP in the sample is known, for example, using ELISA assays (e.g., ELISA assays of Ras-GTP the products of our company Upstate, Inc.). In one example of method additionally includes the introduction does not meet the specified patient an effective amount of an inhibitor of MEK or ERK. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR in combination with an inhibitor of B-Raf.

In one of the embodiments of the subject matter described herein relates to a method of identification of a patient not responding to treatment with an inhibitor of B-Raf, comprising determining in the sample the expression level of EGF or EGFR, and sverkhekspressiya levels of either EGF or EGFR indicate that the patient will not respond to treatment specified inhibitor of B-Raf. In one of the examples determine the number of EGF mRNA. Ways to measure the expression levels of EGF and EGFR in the sample is known, for example, using ELISA immunoassays (e.g., QUANTIKINE immunoassays®the products of our company R&D Systems, Inc.). In one example of method additional�but includes the introduction does not meet the specified patient an effective amount of an inhibitor of MEK or ERK. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR in combination with an inhibitor of B-Raf.

In one of the embodiments of the subject matter described herein relates to a method of identification of a patient not responding to treatment with an inhibitor of B-Raf, comprising determining in the sample the expression level of HGF or quotes, and sverkhekspressiya levels of either HGF or estimates indicate that the patient will not respond to treatment specified inhibitor of B-Raf. In one example, the patient expresses B-Raf V600E. In one of the examples to determine the amount of HGF mRNA. Ways to measure the expression levels of HGF and estimates in the sample is known, for example, used quantitative PCR analyses RT-RealTime. In another example, using ELISA immunoassays (for example, kits for carrying out ELISA cMET PhosphoDetect®the products of our company EMD Chemicals, Inc or to perform ELISA Human cMET, the products of the company Invitrogen, Inc.). In one example of method additionally includes the introduction does not meet the specified patient an effective amount of an inhibitor of estimates or HGF. In another example, the method additionally comprises administering an effective amount of an inhibitor of estimates of HG or in combination with an inhibitor of B-Raf.

In one of the embodiments of the subject matter described herein relates to a method of identification of a patient not responding to treatment with an inhibitor of B-Raf, comprising determining the presence or absence of mutations in K-ras, and the presence of mutations in K-ras indicates that the patient will not respond to treatment specified inhibitor of B-Raf. In one example of method additionally includes the introduction does not meet the specified patient an effective amount of an inhibitor of MEK or ERK. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR in combination with an inhibitor of B-Raf.

In some embodiments, the object of the invention described herein relates to a method of determining, whether the tumor to treatment with an inhibitor of B-Raf, comprising determining in a sample of the tumor for the presence of the mutant protein or K-ras gene, wherein the presence of the mutant protein or K-ras gene indicates that the tumor will not respond to treatment by an inhibitor of B-Raf. In one example of method additionally includes the introduction of specified do not meet the tumor an effective amount of an inhibitor of MEK or ERK. In another example, the method will complement�flax comprises administering an effective amount of an inhibitor of signal transmission through the EGFR. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR in combination with an inhibitor of B-Raf.

In some embodiments, a method of prediction, of whether the patient to treatment by an inhibitor of B-Raf. In some embodiments, the method comprises determining the presence or absence in the patient's tumor mutations in K-ras, where the mutation of K-ras is in codon 12 or codon 13. In some embodiments, in the presence of mutations in K-ras predicts that the patient will not respond to treatment by an inhibitor of B-Raf.

In some embodiments, a method of prediction, whether the tumor to treatment with an inhibitor of B-Raf. In some embodiments, the method comprises determining the presence or absence in the sample of the tumor mutations in K-ras, where the mutation of K-ras is in codon 12 or codon 13. In some embodiments, the presence of mutations in K-ras indicates that the tumor will not respond to treatment by an inhibitor of B-Raf.

In some embodiments, a method is proposed for the classification of the individual person in the treatment Protocol. The method comprises determining the presence in the sample of the individual mutant K-ras gene or its protein, and the presence of the mutant gene or protein K-ras indicates that the individual will not respond to treatment by an inhibitor of B-af, and exclusion of individual treatment by an inhibitor of B-Raf. This method may include the assignment of an individual to a particular subgroup, for example, in a clinical trial. In another embodiment of the method additionally includes the introduction of a specified individual, having specified the mutant gene or protein K-ras, an effective amount of an inhibitor of MEK or ERK. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR in combination with an inhibitor of B-Raf.

In one embodiment, the implementation of the proposed method for classification of tumors of the breast, lung, colon, ovarian, thyroid, melanoma or pancreatic tumors. The method comprises the steps: obtaining or delivery of the tumor sample; determining in the sample the expression or activity of (i) a gene encoding a mutant B-Raf V600E, and (ii) a gene encoding a mutant k-Ras. The method may further include classifying the tumor as belonging to a subclass of tumors on the basis of the results of the identification; and selection of treatment based on stage classification, where said treatment is different from treatment for a specific inhibitor of B-Raf V600E with sverkhekspressiya in the�asanam sample of the tumor indicated mutant of an effective amount of an inhibitor of signal transmission through the EGFR. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR in combination with an inhibitor of B-Raf.-RAS. In one example of treatment includes the introduction of specified do not meet the tumor an effective amount of an inhibitor of MEK or ERK. In another example, the method additionally includes the introduction

In another embodiment, the implementation of the proposed method of treatment of colorectal cancer or lung cancer. The method includes determining, whether the cancer is called K-ras or B-Raf, and in the treatment of this cancer, which determine how the induced K-ras, treatment does not include an inhibitor of B-Raf. In one example of treatment includes introduction to the specified called K-ras cancer an effective amount of an inhibitor of MEK or ERK. Also proposed is a set containing a specific material to determine whether the cancer is called K-ras or B-Raf, and instructions to identify the patient or tumors that do not respond to treatment by an inhibitor of B-Raf.

In some embodiments, determining the presence or absence of one or more individual mutations of K-ras includes determining in the sample the individual the presence or amount of expression of the mutant polypeptide K-ras. In some embodiments, determining the presence or absence of one or more individual mutations of K-ras includes definitions� in the sample of the individual the presence or amount of transcription or translation of a mutant polypeptide K-ras.

In some embodiments, determining the presence or absence of one or more individual mutations of K-ras includes determining the presence or amount of expression of the polypeptide containing at least one amino acid sequence selected from the group consisting of the following SEQ ID nos listed in US2009/0075267: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16. In some embodiments, determining the presence or absence of one or more individual mutations of K-ras includes determining in the sample the individual the presence or amount of transcription or translation of a polynucleotide encoding at least one amino acid sequence selected from the group consisting of the following SEQ ID nos listed in US2009/0075267: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:16.

In some embodiments, the implementation of the proposed determination of the presence or absence of a polynucleotide encoding a mutant polypeptide K-ras. In some embodiments, the method for determining the presence or absence in the sample polynucleotide encoding a mutant polypeptide K-ras, includes (a) the exposure of the sample with the probe to hybridise to the polynucleotide coding region of the mutant polypeptide K-ras, where the region contains at least one mutation K-ras, selected from G12S G12V, G12D, G12A, G12C, G13A, and G13D, and (b) determining the presence or absence in the sample polynucleotide encoding a mutant polypeptide K-ras. In some embodiments, the method for determining the presence or absence in the sample of a mutant polypeptide K-ras includes (a) the exposure of the sample with the probe to hybridise to the polynucleotide coding region of the mutant polypeptide K-ras, where the region contains at least one mutation K-ras, selected from G12S, G12V, G12D, G12A, G12C, G13A, and G13D, and (b) determining the presence or absence in the sample of a mutant polypeptide K-ras.

In some embodiments, the implementation of the proposed determination of the presence or absence of a polynucleotide encoding a mutant polypeptide of B-Raf. In some embodiments, the method for determining the presence or absence in the sample polynucleotide encoding a mutant polypeptide of B-Raf, includes (a) the exposure of the sample with the probe to hybridise to the polynucleotide coding region of the mutant polypeptide of B-Raf, where the region contains the V600E mutation, and (b) determining the presence or absence in the sample polynucleotide encoding a mutant polypeptide of B-Raf. In some embodiments, the method for determining the presence or absence in the sample of a mutant polypeptide of B-Raf includes (a) the exposure of the sample with the probe to hybridise to the polynucleotide coding region� mutant polypeptide of B-Raf, where the region contains the V600E mutation, and (b) determining the presence or absence in the sample of a mutant polypeptide of B-Raf.

In some embodiments, a set for the determination of the individual polynucleotide encoding a mutant polypeptide K-ras. In some such embodiments, the kit contains a probe that hybridise to the polynucleotide coding region of the mutant polypeptide K-ras, where the region contains at least one mutation K-ras, selected from G12S, G12V, G12D, G12A, G12C, G13A, and G13D. In some embodiments, the kit further comprises two or more primers for amplificatio. In some embodiments, the kit further comprises a component for detection. In some embodiments, the kit further comprises a component for processing nucleic acids. The kit can optionally contain a material to determine the mutations of B-Raf. These materials are known in this field. Combination set, capable of detecting mutant genes or proteins K-ras and B-Raf, can be used in particular in the treatment of cancer of the colon and lung. The set includes instructions to identify the patient or tumors that do not respond to the inhibition of B-Raf, when the cancer is called K-ras. Called RAS cancer known in this field. Called Ras cancer is a favourite of RA� or tumor the distorted activity of Ras protein leads to the production of transformed cells or the formation of cancer or tumor.

In some embodiments, the implementation for these samples, tumors, cancers, individuals or patients that are identified as not meeting the inhibitor of B-Raf, the methods further comprise the introduction does not meet the specified samples, tumors, types of cancer, individuals or patients of an effective amount of a MEK inhibitor.

In some embodiments, the implementation for these samples, tumors, cancers, individuals or patients that are identified as not meeting the inhibitor of B-Raf, the methods further comprise the introduction does not meet the specified samples, tumors, types of cancer, individuals or patients of an effective amount of the ERK inhibitor. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR. In another example, the method additionally comprises administering an effective amount of an inhibitor of signal transmission through the EGFR in combination with an inhibitor of B-Raf.

Signalling via EGFR can be inhibited by a large number of ways, which include the inhibition of kinase activity of EGFR, binding to the extracellular domain of EGFR to inhibit activation or by inhibiting the activity and signal transmission with ligand GF.

In this area known inhibitors of signal transmission through EGFR, and these include, for example, erlotinib (Tarceva, and Masha®), gefitinib (IRESSA®), lapatinib, pelitinib, cetuximab, panitumumab, zalutumumab, nimotuzumab and matuzumab and medications, as described in U.S. patent No. 5747498.

In this area known inhibitors of B-Raf, and these include, for example, sorafenib, PLX4720, PLX-3603, GSK2118436, GDC-0879, N-(3-(5-(4-chlorophenyl)-1H-imidazo[2,3-b]pyridine-3-carbonyl)-2,4-differenl)propane-1-sulfonamide and medications, as described in WO2007/002325, WO2007/002433, WO2009111278, WO2009111279, WO2009111277, WO2009111280 and U.S. patent No. 7491829.

In this area known inhibitors of estimates, including, but not limited to, AMG208, ARQ197, ARQ209, PHA665752 (3Z)-5-[(2,6-dichlorobenzyl)sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-it, N-(4-(3-((3S,4R)-1-ethyl-3-foreperiod-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridin-4-carboxamide and SU11274 and medications, as described in U.S. patent No. 7723330.

In this area known MEK inhibitors, including, but not limited to, ARRY-162, AZD8330, AZD6244, U0126, GDC-0973, PD184161 and PD98059 and described in WO2003047582, WO2003047583, WO2003047585, WO2003053960, WO2007071951, WO2003077855, WO2003077914, WO2005023251, WO200501300, WO2005051302, WO2007022529, WO2006061712, WO2005028426, WO2006018188, WO20070197617, WO2008101840, WO2009021887, WO2009153554, WO2009027560, WO2009129938, WO2009093008, WO2009018233, WO2009013462, WO2008125820, WO2008124085, WO2007044515, WO2008021389, WO2008076415 and WO2008124085.

In this area known inhibitors of ERK, and these include, but are not limited to, FR180204 and 3-(2-aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione and medicines described in WO2006071644, WO2007070398, WO2007097937, WO2008153858, WO2008153858, WO2009105500 and WO2010000978.

For the method described herein, any suitable known method for detecting mutant gene or protein K-ras. Specific mutations identified in exon 1, are: G12C; G12A; G12D; G12R; G12S; G12V; G13C; G13D. Methods of determining the presence of mutations in K-ras are also similar to the methods used for identification of mutations of K-ras and EGFR, for example, the oligonucleotide K-ras for PCR, are listed as SEQ ID NO:55, 56, 57 and 58, described in published patent application U.S. No. US2009/0202989A1 cited herein as references in full. As an example, other methods of detecting mutant gene or protein K-ras and primers, oligonucleotides SEQ ID NO described in published United States patent application No. US2009/0202989A1, US2009/A, US20090143320, US20040063120 and US2007/0003936. Techniques and procedures are generally performed according to accepted methods, well known in this field and described in a large number of General and more specific references that are cited and discussed throughout the present description. See, NRA�emer, manual Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is included herein by reference.

In this field there are some known methods of detecting mutations in the polynucleotide. Some typical methods include, but are not limited to, sequencing, reactions of elongation of the primers, electrophoresis, picogreen assays, assays ligation oligonucleotides, hybridization assays, TaqMan assays, assays and SNPlex assays are described, for example, in U.S. patents№№ 5470705, 5514543, 5580732, 5624800, 5807682, 6759202, 6756204, 6734296, 6395486 and patent publication U.S. No. US 2003-0190646 A1.

In some embodiments, the detection of a mutation in the polynucleotide includes a first amplification of the polynucleotide, which may contain a mutation. In this field there are some known methods of amplification of the polynucleotide. These amplification products can be used for detecting mutations in the polynucleotide by any of the methods described herein or known in this field.

Some methods for detecting mutations in the polynucleotide are known in this field. Some typical methods include, but are not limited to, detection using an agent for specific binding with the mutant polypeptide. Other ways of detecting mutant polypeptide include, noimi not limited to, electrophoresis and peptide sequencing.

Some of the typical methods for the detection of mutations in the polynucleotide and/or polypeptide are described, for example, in articles Schimanski et al. (1999) Cancer Res., 59: 5169-5175; Nagasaka et al. (2004) J. Clin. Oncol., 22: 4584-4596; PCT publication no WO 2007/001868 A1; patent publication U.S. No. 2005/0272083 A1; and article Lievre et al. (2006) Cancer Res. 66: 3992-3994.

In some embodiments, offers microchips containing one or more polynucleotides encoding one or more mutant polypeptides of K-ras. In some embodiments, offers microchips containing one or more polynucleotides complementary to one or more polynucleotides encoding one or more mutant polypeptides of K-ras. In some embodiments, offers microchips containing one or more polynucleotides encoding one or more mutant polypeptides of B-Raf. In some embodiments, offers microchips containing one or more polynucleotides complementary to one or more polynucleotides encoding one or more mutant polypeptides of B-Raf.

In some embodiments, using the technology of microchips, in two or more samples of cells or tissues to determine the presence or absence of one or more mutant polynucleotides K-ras In some embodiments, using microarray technology, in two or more samples of cells or tissues to determine the quantity of one or more mutant polynucleotides K-ras.

In some embodiments, using the technology of microchips, in two or more samples of cells or tissues to assess the presence or absence of one or more mutant polynucleotides of B-Raf. In some embodiments, using the technology of microchips, in two or more samples of cells or tissues to determine the quantity of one or more mutant polynucleotides of B-Raf.

In some embodiments, using the technology of microchips, in two or more samples of cells or tissues to determine the presence or absence of one or more mutant polypeptides of K-ras. In some such embodiments, first, the sample of cells or tissue mRNA extracted and then converted to cDNA, which hybridized with a microchip. In some such embodiments, the presence or absence of cDNA, which specifically binds with a microchip, indicates the presence or absence of the mutant polypeptide K-ras. In some such embodiments, the expression level of one or more mutant polypeptides of K-ras evaluated by calculating the amount of cDNA that specifically binds with a microchip.

p> In some embodiments, using the technology of microchips, in two or more samples of cells or tissues to determine the presence or absence of one or more mutant polypeptides of B-Raf. In some such embodiments, first, the sample of cells or tissue mRNA extracted and then converted to cDNA, which hybridized with a microchip. In some such embodiments, the presence or absence of cDNA, which specifically binds with a microchip, indicates the presence or absence of the mutant polypeptide of B-Raf. In some such embodiments, the expression level of one or more mutant polypeptides of B-Raf estimate, calculating the amount of cDNA that specifically binds with a microchip.

In some embodiments, offers microchips containing one or more agents specifically binding to one or more mutant polypeptides of K-ras. In some such embodiments, determining the presence or absence in a cell or tissue of one or more mutant polypeptides of K-ras. In some such embodiments, estimate the number in a cell or tissue of one or more mutant polynucleotides K-ras.

In some embodiments, offers microchips containing one or�how many agents, specifically binding to one or more mutant polypeptides of B-Raf. In some such embodiments, assess the presence or absence in a cell or tissue of one or more mutant polypeptides of B-Raf. In some such embodiments, estimate the number in a cell or tissue of one or more mutant polynucleotides of B-Raf.

All references cited herein, including patents, patent applications, publications, manuals, and the like, and the references cited in them, in those cases where they have not previously presented, is hereby reproduced in this document as a reference in full. If one or more of the documents listed as references, define the term in contradiction with the definition of this term in the present application, this application form is a control. The headings of the sections used herein are for organizational purposes only and should not be construed as limiting the described subject matter.

Definition

Unless otherwise stated, the scientific and technical terms used in connection with the present invention, will have the meanings commonly understood by experts in this field. In addition, unless the context requires a different, in terms of ed�stvennom the number will include the plural, and terms in the plural will include the singular.

The term "inhibitor of B-Raf" refers to any compound or agent that inhibits, reduces the activity of the kinase B-Raf. Such an inhibitor may also inhibit other kinases, including other kinases raf. "A specific inhibitor of the kinase B-Raf" refers to the inhibitor, which is selective for the mutant B-Raf, such that has a mutation in velinova residue at amino acid position 600, e.g., a V600E mutation, compared with B-Raf wild type. This inhibitor at least twice, usually at least three or more times more effective inhibitor of B-Raf wild type. Efficiency can also be mapped in the values of IC50for cellular assays, which measured the inhibition of growth.

The term "treatment Protocol" refers to a treatment regimen or course of introducing one or more agents for the treatment of disorders or diseases. This includes clinical trials.

The terminology "X#Y" in the context of the mutation in the polypeptide sequence adopted in this area, where "#" indicates the location of the mutation with regard to the sequence number of the amino acid polypeptide, "X" indicates the amino acid found at that position in the amino acid sequence of wild-type, and "Y" indicates Mutan�ing the amino acid at this position. For example, the expression "G12S" with reference to a polypeptide K-ras indicates that amino acid number 12 sequence of K-ras wild type is glycine, and that in the mutant sequence K-ras glycine is replaced by serine.

The terms "mutant polypeptide K-ras and mutant protein K-ras" are used interchangeably and refer to a polypeptide K-ras containing at least one mutation K-ras, selected from G12S, G12V, G12D, G12A, G12C, G13A, and G13D. Some typical the mutant polypeptides of the K-ras include, but not limited to, allelic variants, spliced variants, derivative variants, the variants with substitutions, variants with deletions and/or variants with inserts, fused polypeptides, orthologs, and interspecies homologs. In some embodiments, the mutant polypeptide K-ras includes additional residues at the C - or N-end, such as, but not limited to, the remains of the leader sequence, is aimed residues aminobenzene methionine residues, lysine residues, labeled residues and/or residues fused protein.

The terms "mutant polypeptide of B-Raf and mutant protein B-Raf" are used interchangeably and refer to a polypeptide B-Raf containing the V600E mutation. Some typical the mutant polypeptides of B-Raf include, but not limited to, allelic variants, spliced variants, derivatives of varia�you variants with substitutions, variants with deletions and/or variants with inserts, fused polypeptides, orthologs, and interspecies homologs. In some embodiments, the mutant polypeptide of B-Raf includes additional residues at the C - or N-end, such as, but not limited to, the remains of the leader sequence, is aimed residues aminobenzene methionine residues, lysine residues, labeled residues and/or residues fused protein.

The terms "mutant polynucleotide K-ras", "mutant oligonucleotide K-ras and mutant nucleic acid K-ras" are used interchangeably and refer to a polynucleotide, codereuse polypeptide K-ras containing at least one mutation K-ras, selected from G12S, G12V, G12D, G12A, G12C, G13A, and G13D.

The terms "mutant polynucleotide of B-Raf", "mutant oligonucleotide B-Raf and mutant nucleic acid of the B-Raf" are used interchangeably and refer to a polynucleotide, codereuse polypeptide B-Raf containing the V600E mutation.

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract derived from biological materials.

The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition, method�Oh when administered to the patient properly to induce a desired therapeutic effect. Other chemical terms used in this document according to the application, for example, proposed a dictionary of chemical terms McGraw-Hill (Parker, S., Ed., McGraw-Hill, San Francisco (1985)), provided herein as a reference.

The term patient includes individuals people and animals.

The terms "mammal" and "animal" for purposes of treatment refers to any animal classified as a mammal, which includes Pets or farm animals zoos and animals, sports animals, animals-Pets, such as dogs, horses, cats, cows, etc Preferably, the mammal is a human.

The term "disease state" refers to the physiological state of a cell or only mammal in which there is a delay, cessation, or disorder of the functions of the cell or organism, system, or organs.

The term "treatment" refers to therapeutic treatment and prophylactic or pre-emptive measures when the subject has a warn or slow down (lessen) undesired physiological change or disorder, such as the development or spread of cancer. For the purposes of the present invention to favorable or desired clinical results include, but is not limited to, alleghany� symptoms reduced incidence of disease, stabilized (i.e. not worsening) state, delay or slowing of disease progression, alleviation or relief of the disease state, and remission (partial or complete), detected or redaktiruem. "Treatment" can also mean the prolongation of life compared with the expected term of life without treatment. To those who are in need of treatment include individuals who already have the condition or disorder, and also tend to buy the condition or disorder or those who have the condition or disorder must be prevented.

The term "responsible" as used in this document, I understand that the patient or the tumor after administration of an agent observed a complete response or partial response according to RECIST criteria (criteria for assessment of response in solid tumors). The term "not responsible" as used in this document, I understand that the patient or the tumor after administration of the agent is observed stable disease or progressive disease according to RECIST. Criteria RECIST described, for example, the guide Therasse et al., February 2000, “New Guidelines to Evaluate the Response to Treatment in Solid Tumors,” J. Natl. Cancer Inst. 92(3): 205-216, which is reproduced herein as references in full.

"Disorder" is any condition on the cat�Roy would have a positive impact one or more methods of treatment. It includes chronic and acute disorders or diseases, which includes such pathological conditions that trigger the mammal considered a disorder. It is a non-derogable examples of disorders which could be treated herein include benign and malignant tumors, leukemias and malignant neoplasms of lymphoid tissue. "Tumor" includes one or more cancer cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant neoplasms of lymphoid tissue. To more specific examples of such cancers include squamous cell cancer (e.g., cancer of the squamous epithelium), lung cancer, which includes small cell lung cancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric cancer, refers to gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, carcinoma of the endometrium or uterine carcinoma of the salivary glands, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal CT�inoma, penile carcinoma, and head and neck cancer. In particular, the present method is suitable for the treatment of breast cancer, colorectal cancer, ovarian cancer, pancreas or lungs. More specifically, the cancer is a cancer of the colon, the lungs or the ovaries. Cancer can be a cancer caused by Ras.

To a disease or condition associated with a mutant K-ras include one or more of the following diseases: disease or condition caused by a mutant gene or protein K-ras; a disease or condition, the development of which was facilitated by the mutant gene or protein K-ras; and a disease or condition associated with a mutant gene or protein K-ras. In some embodiments, the disease or condition associated with a mutant K-ras, is a cancer.

"The disease or condition associated with a mutant polypeptide K-ras" include one or more of the following: a disease or condition caused by a mutant polypeptide K-ras; a disease or condition, the development of which was facilitated by the mutant polypeptide K-ras; a disease or condition that causes a mutant polypeptide K-ras; and a disease or condition associated with a mutant polypeptide K-ras. In some embodiments, the disease or condition, �knitted mutant polypeptide K-ras, can exist in the absence of the mutant polypeptide K-ras. In some embodiments, the disease or condition associated with a mutant polypeptide K-ras, can be compounded by a polypeptide having a mutant K-ras. In some embodiments, the disease or condition associated with a mutant polypeptide K-ras, is a cancer.

The following examples, including the experiments and the results obtained are only offered for illustration purposes and should not be construed as limiting the invention.

EXAMPLES

Example 1

Deletion and pharmacological inhibition of B-RAF increases the carcinogenesis caused by K-ras

Family GTP Ras controls a large number of subsequent signaling cascades under the influence of signals that regulate cellular processes, including proliferation and survival. AlthoughRasrepresents one of the main targets for mutation type gain-of-function in human tumors, it is unclear how the effector pathway of Ras in oncogenesis caused by mutant K-ras. As important to the function of K-ras is the activation of B-Raf canonical signaling pathways MARK, the authors proposed a study to determine the role of B-Raf in the stimulation and maintenance of tumors caused by the mutant K-ras. In some tumors with mutant K-ras inhibited�e B-Raf not only did not lead to any beneficial effect on the tumor, it even increased the growth of tumors. Cm. Fig. 8A, which shows the doubling time of the tumor.

In the lungs of mice genetically engineered with a conditional allele ofK-rasG12D(K-rasLSL-G12D) and either 0, 1 or both copies of the geneB-rafflanked by sites ofLoxP(B-rafCKO), delivered adenovirus expressing the recombinasesCre. This procedure leads to expression of a mutant K-rasG12Din the presence or in the absence of lung mouse of one or both of the deleted alleles ofB-raf. Unexpectedly, deletion of B-raf significantly increases the number of tumors in the lungs and the severity of the disease and reduces overall survival. When using highly specific molecular inhibitor that targets B-Raf, on-line non-small cell lung carcinoma mice carrying the mutation of K-rasG12Dthe authors found increased cell proliferation and the formation of colonies on soft agar. Further study revealed that treatment of cells expressing K-rasG12Dusing the inhibitor of B-Raf increased the phosphorylation of MEK and Erk. Thus, these data suggest that deletion of B-Raf, not only did not inhibit the initiation of tumors caused by K-ras, and the progression of the disease, but its presence can be key in establishing a negative feedback for constant activity mu�antago K-ras.

Example 2

Understanding of signal transmission through RAF in mutant B-RAFV600Ecompared with wild-type tumors

To understand the implications of the way the Raf with different mutations in Ras and Raf, the authors described two selective low-molecular inhibitor of Raf with clear profiles of effectiveness against B-Raf and c-Raf wild-type (WT) in the case of mutant (MT) B-RafV600E. Despite their biochemical differences, they had identical cell profiles, acting against tumors of B-RAFV600Ebut not WT or Ras MT. Both inhibitors induced the activation of the path of Raf/MEK/ERK in the lines than BRAFV600Emainly using isoform of C-Raf. On the contrary, according to their predicted biochemical effects they inhibited the activity of the Raf/MEK/ERK stimulated with phorbol ester and growth factor. Thus, the cell specificity of selective Raf inhibitors against B-RAFV600Enot a simple reflection of their selectivity in respect of isoforms of B-RAFV600Ebut rather reflects the complex regulation of Raf activity in various conditions in the cell.

Biochemical selectivity against B-RafV600Eis not the only incentive for efficiency profiles in the cell of Raf inhibitors. Inhibitors induce levels rmec selectively in non-V600E mutant lines through the C-Raf. Inhibitors induce specific activity�ü C-Raf levels and rmec fast and dose-dependent manner according to its effectiveness. Under normal conditions bell curve for GDC-0879 indicates dual stimulatory versus inhibitory effect on C-Raf. The status for paths In a - and C-Raf in various situations determines the pharmacodynamics inhibition of Raf. Results characteristics shown in Fig. 1-7.

Example 3

The growth in the xenograft tumors of the lung after administration of a dose of inhibitor of B-RAF

The data presented below and in Fig. 10-14 for the experiment n and Fig. 15-18 for the experiment N.

1. The method of identification of a patient not responding to treatment with an inhibitor of B-Raf, comprising determining the presence or absence of mutations in K-rasG12Dand the presence of mutations in K-rasG12Dindicates that the patient will not respond to treatment specified inhibitor of B-Raf.

2. A method according to claim 1, where the specified inhibitor of B-Raf is a specific inhibitor of B-Raf kinase.

3. A method according to claim 2, where MC�linked inhibitor of B-Raf kinase is GDC-0879.

4. A method according to claim 1, where the presence of mutations in K-ras is determined by nucleic acid amplification of K-ras from said tumor or its fragment suspected to contain a mutation, and sequencing indicated amplified nucleic acids.

5. A method according to claim 1, where the presence of mutations in K-ras is determined by nucleic acid amplification of K-ras from said tumor or its fragment suspected to contain a mutation, and comparing the electrophoretic mobility of the amplified nucleic acid to the electrophoretic mobility of the corresponding nucleic acid or fragment of K-ras wild-type.

6. A method according to claim 1, where determining the presence or absence of mutations in K-ras in the tumor contains the definition of the sample of the tumor mutant polypeptide K-ras.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to oncology, and can be used for detecting endogenous intoxication (EI) symptoms in the patients suffering colorectal cancer. That is ensured by studying the complete blood count and deriving an integral intoxication index (III) by formula: EI=(Im+St+Seg+Myel+Plas)*10(Bas+Eos+Lym+Mon)*Er*CC, wherein III is the integral intoxication index; Im is immature leukocytes, %; St is stab neutrophils, %; Seg is segmented neutrophils, %, Myel is myelocytes, %; Plas is plasma cells, %; Bas is basophiles, %; Eos is eosinophils, %; Lym is lymphocytes, %; Mon is monocytes, %; Er is absolute erythrocytes to the power of 1012; CC is an absolute colour characteristics.

EFFECT: invention provides diagnosing the absence (III=4,6) or presence (III=10,4) of the EI symptoms and associated respiratory disorders and enables assessing the therapy efficacy.

FIELD: medicine.

SUBSTANCE: predicting heart rhythm disorder in the patients with pre-excitation syndromes is ensured by measuring patient's blood plasma matrix metalloproteinase-9 (MMP-9), and if the measured value is less than 90.6 ng/ml, the favourable clinical course accompanied by no heart rhythm disorders (HRD) is predicted, whereas the MMP-9 concentration of more than 90.6 ng/ml enables predicting HRD.

EFFECT: method enables predicting HRD in the patients with pre-excitation syndromes by high sensitivity and specificity of the method with its low invasiveness and no counterindications.

3 ex

FIELD: medicine.

SUBSTANCE: technique consists in measuring a pinopod count in the germinal epithelium, an expression level of progesterone and oestrogen alpha receptors in the endometrial stroma, calculating their ratio, and determining an expression level of implantation LIF factor on the 21st-24th day of the menstrual cycle ('implantation window'). If the pinopod count is less than 35%, the expression ratio of progesterone and oestrogen alpha receptors in the endometrial stroma less than 3.5, as well as the LIF expression level of less than 2 points, disturbed fertility associated with uterine myoma is diagnosed.

EFFECT: technique enables applying the differentiated approach to managing the patients planning pregnancy, suffering uterine myoma undeforming the uterine cavity, as well as with myomatous nodule up to 5,0 cm in diameter.

2 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: method involves sampling a biological tissue, introducing a biologically active substance therein, keeping the prepared mixture in a thermostat at 37°C and incubating, performing microscopic examination for identifying the forms of trichomonads. The sampled biological tissue represents the urogenital mucosa that is incubated for 24 h in a nutrient medium. The prepared sediment is separated and added with the biologically active substance nitrosoguanidine in an amount of 500 mcg/ml. That is followed by incubating for 15 min at +37°C; a phosphate buffer with pH 5.6 heated to +37°C is added and centrifuged twice at 1000 rpm for 10 min. The material is placed into the nutrient medium for trichomonad growing, incubated for at least 24 hours, and microscopic examination is used to identify atypical nonflagellate and typical round and amoeboid forms of trichomonads.

EFFECT: using the declared invention enables the effective, fast and reliable identification of typical changed and atypical forms of trichomonads.

3 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: method involves measuring pregnant women's blood anti-cytomegalovirus antibody titre and glutathione reductase activity in erythrocytes of the obstetric patient suffering the cytomegaloviral infection. If the anti-cytomegalovirus antibody titre is 1:1600, whereas the measured glutathione reductase activity is below 4.48±0.22 units/gHb, the aggravated cytomegaloviral infection is diagnosed.

EFFECT: higher diagnostic technique.

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, cosmetology, production of food products, vitamins, food supplements, drugs and describes versions of device for realisation of non-invasive potentiometric determination of oxidant/antioxidant activity of biological tissues, which includes device for measuring potentials and double-sided electrode, made in form of plate with similar working surface, covered with electricity-conducting gel, containing mediator system. Electrodes are fixed on biological tissue in such a way that one working surface, playing role of measuring electrode, is in direct contact with biological tissue via gel, second working surface pale role of comparison electrode. Electrodes contact with each other via gel, with oxidant/antioxidant activity being determined by formulae with application of difference between final and initial potentials.

EFFECT: simplification, as well as increase of accuracy and reliability of determination, is achieved.

14 cl, 3 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: invention represents a method for the prediction of preeclampsia in the second trimester of pregnancy by blood examination, differing by the fact that the activity of acid and neutral proteinases is measured in blood serum of the women 7-8 weeks pregnant; if the activity of acid proteinases is more than 5.6 mcmole/l, while the activity of neutral proteinases is more than 3.9 mcmole/l, the preeclampsia progression in the second trimester of pregnancy is predicted.

EFFECT: higher accuracy and specificity of the method for the prediction of gestational toxicosis.

2 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to a method of automated morphometric myelofibrosis diagnostics. The method essence consists in the fact that overview images of zones with different optical properties with determinable fibrous and heamopoietic properties of a biological tissue are performed. Ratios of areas of the said zones of at least three paraffin cuts of trepanobiopsy samples are calculated. The coefficient (Cop) is calculated as the ratio of the fibrous tissue area to the area of the heamopoietic tissue by formula. If the value Cop ≥14.5%, myelofibrosis is diagnosed.

EFFECT: application of the claimed method makes it possible to increase the accuracy and improve the efficiency of myelofibrosis diagnostics.

7 cl, 1 tbl, 4 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: testicular germ cells are measured quantitatively. That is ensured by 50-day oral administration of selexen and ascorbic acid into male white rats in doses 1.5 and 500 mg/kg of animal's body weight respectively once a day. 14 days later, administering the selenium-containing biocomplex is accompanied by the 30-minute daily exposure to microwave radiation at 42 GHz (λ=7.1 mm) for 30 days. Once the experimental exposures are completed, the corrective properties of the biocomplex as having an effect on the morphofunctional state of epididymal sperm cells are assessing by formula: MFSI=A+B, wherein MFSI is a morphofunctional state index, A is a portion of normal sperm cells in relation to the reference, and B is a portion of moving sperm cells in relation to the reference. If the MFSI value is 1.3 or more, the spermatogenesis correction is considered to be ineffective, while the MFSI value being more than 1.3 shows the effective spermatogenesis correction if exposed to microwave radiation.

EFFECT: invention enables assessing the spermatogenesis correction efficacy with underlying administration of the biocorrector.

2 tbl, 3 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular hepatology. Method of quantitative estimation of histological activity in liver biopsy materials in case of chronic diffuse liver diseases results in protocol of counting in absolute numbers, percent and points of pathological signs, identified in the course of morphological analysis of liver biopsy materials from patients with chronic diffuse liver diseases, by sections: necrosis of hepatocytes, globular and atomised fatty hydropic degeneration of hepatocytes, small cell dysplasia of hepatocytes, portal, periportal, intralobular and central infiltration. After that conversion of absolute values into point system is performed by means of said protocol, and histological activity is determined by number of points.

EFFECT: method makes it possible to create quantitative unification of obtained results in case of said diseases.

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

FIELD: medicine, urology.

SUBSTANCE: one should conduct subcutaneous prevocational tuberculin test and, additionally, both before the test and 48 h later it is necessary to perform the mapping of prostatic vessels and at decreased values of hemodynamics one should diagnose tuberculosis. The information obtained should be documented due to printing dopplerograms.

EFFECT: more reliable and objective information.

1 ex, 1 tbl

FIELD: molecular biology.

SUBSTANCE: the suggested innovation deals with the fact that nucleic acids should be isolated directly out of the sample without pipetting stage but with the help of interconnected reservoirs being prepared beforehand. The above-mentioned vessels should be applied either separately or being interconnected according to standard microtitrating format. The sample should be mixed with a lyzing buffer and nucleic acids are bound with matrix in closed system including, at least, two interconnected reservoirs. Forced movement of sample's mixture and buffer back and forth from one reservoir into another one for several times through narrow passage provides their thorough intermixing. The method provides quick and safe isolation of nucleic acids.

EFFECT: higher efficiency.

44 cl, 4 dwg, 1 ex

FIELD: medicine, phthisiology, microbiology.

SUBSTANCE: diagnostic material is poured preliminary with chlorohexidine bigluconium solution, homogenized, kept at room temperature for 10-12 h and centrifuged. Precipitate is poured with Shkolnikova's liquid medium, incubated at 37oC for 3 days, supernatant part of Shkolnokova's medium is removed, fresh Shkolnikova's medium is added, and precipitate is stirred and inoculated on the dense cellular egg media. Sensitivity of the strain is determined in 3 weeks by the presence of growth in the control tube only. Invention provides enhancing precision and reducing time for assay. Invention can be used in assay for medicinal sensitivity of tuberculosis mycobacterium.

EFFECT: improved assay method.

3 ex

FIELD: medicine, biotechnology, pharmacy.

SUBSTANCE: invention relates to agents used for treatment of pathological states associated with disorder of synthesis of neuromediating substances. Method involves the development of pharmaceutical composition and a method for it preparing. Pharmaceutical composition represents subcellular synaptosomal fractions: synaptic membranes, "light" synaptosomes and "heavy" synaptosomes prepared from gray matter of cerebral hemispheres from experimental animals based on the goal-seeking modification of humoral mediators of nerve endings transformed to synaptosomes in development and regression of malignant processes. The composition provides inhibiting the growth of tumor cells, to elevate span-life of patients with ascite Ehrlich's sarcoma, breast adenocarcinoma Ca-755, Wolker's carcinosarcoma-256.

EFFECT: valuable medicinal and anti-tumor properties of composition.

12 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method involves carrying out microscopic examination of blood serum samples taken from femoral vein and cubital vein. Femoral vein sample is taken on injured side. The examination is carried out before and after treatment. The blood serum samples are placed on fat-free glass slide in the amount of 0.01-0.02 ml as drops, dried at 18-30°C for 18-24 h. The set of pathological symptoms becoming larger or not changed after the treatment in comparison to sample taken before treatment, and morphological picture of samples under comparison taken from the cubital vein showing no changes or being changed to worse, the treatment is considered to be effective.

EFFECT: enabled medicamentous treatment evaluation in course of treatment to allow the treatment mode to be changed in due time; avoided surgical intervention (amputation); retained active life-style of aged patients.

4 dwg

FIELD: medicine, clinical toxicology.

SUBSTANCE: at patient's hospitalization one should gather the data of clinical and laboratory values: on the type of chemical substance, patient's age, data of clinical survey and laboratory values: body temperature, the presence or absence of dysphonia, oliguria being below 30 ml/h, hemoglobinuria, erythrocytic hemolysis, exotoxic shock, glucose level in blood, fibrinogen and creatinine concentration in blood serum, general bilirubin, prothrombin index (PTI), Ph-plasma, the state of blood clotting system. The state of every sign should be evaluated in points to be then summed up and at exceeding the sum of points being above "+20" one should predict unfavorable result. At the sum of "-13" prediction should be stated upon as favorable and at "-13" up to "+20" - prediction is considered to be doubtful.

EFFECT: higher accuracy of prediction.

2 ex, 3 tbl

FIELD: medicine, juvenile clinical nephrology.

SUBSTANCE: disease duration in case of obstructive pyelonephritis should be detected by two ways: either by detecting the value of NADPH-diaphorase activity, as the marker of nitroxide synthase activity in different renal department and comparing it to established norm, or by detecting clinico-laboratory values, such as: hemoglobin, leukocytes, eosinophils, urea, beta-lipoproteides, lymphocytes, neutrophils, the level of glomerular filtration, that of canalicular reabsorption, urinary specific weight, daily excretion of oxalates, arterial pressure, and estimating their deviation against average statistical values by taking into account a child's age.

EFFECT: higher efficiency of detection.

7 dwg, 1 ex, 6 tbl

FIELD: medicine, urology.

SUBSTANCE: the present innovation deals with differential diagnostics of prostatic cancer and other prostatic diseases at the stage of primary inspection. The method includes the detection of PCA and calculation of probability coefficient for prostatic cancer (PCC) by the following formula: where e - the foundation of natural logarithm (e=2.718…), PCA - the level of total blood PCA in ng/ml, V - patient's age in years. At PCC value being above 0.2 one should diagnose prostatic cancer and to establish final diagnosis one should perform polyfocal prostatic biopsy. The method enables to increase accuracy of diagnostics at decreased number of unjustified prostatic biopsies.

EFFECT: higher efficiency of diagnostics.

2 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

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