Methods of treating and diagnosing cancer. RU patent 2509809.

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically methods of forecasting development of cancer and can be used in medicine. The method of performing forecast for a patient suffering from NSCLC involves determining the expression level of ChoK beta or ChoK beta and ChoK alpha in a sample collected from said patient. Low levels of ChoK beta relative to the levels in a standard sample indicate a bad forecast for the patient. Low levels of ChoK alpha and high levels of ChoK beta relative to the expression levels of said proteins in the standard sample indicate a good forecast for the patient.

EFFECT: invention enables to forecast probability of survival, for example, survival without relapses for a patient with NSCLC.

6 cl, 16 dwg, 2 tbl, 3 ex

The scope of the invention

The present invention relates to the treatment of cancer and, in particular, treatment of cancer, based on the introduction of activity Kalinkina-beta (hereafter ChoKβ), as well as ways to develop a personalized treatment methods and definitions reaction to the agent that to induce Kalinkina-beta (hereafter ChoKβ), to treat cancer, as well as ways to determine the prognosis for a patient on the basis of determination of expression levels ChoKβ, as well as on the basis of determining the ratios of the levels of expression of

ChoKβ and ChoKα. Finally, the invention relates to methods of determining the response of the patient who is suffering from cancer, inhibiting ChoKα agents on the basis of determination of expression levels PEMT and/or ChoKβ.

The background to the invention

Kalinkina (ChoK) is the first enzyme in the so-called path Kennedy biosynthesis of phosphatidylcholine (PC), which is the main lipid membranes of eukaryotic cells. In particular, ChoK catalyzes the reaction of transformation of choline (Cho) in phosphocholine (PCho) using ATP molecules and Mg 2+ as a cofactor. The way Kennedy continues with the action of the enzyme on PCho CDP-postcholecystectomy (CT), giving CDP-choline, and then DAG-cholinesterase (CPT), resulting in a PC (figure 3). Although activity ChoK forms the first stage in the synthesis of PC, it is considered that limiting or regulating stage of biosynthesis PC stage is catalyzed by CT.

Amino acid sequence that forms domains Kalinkina, well kept in all eukaryotic organisms, for example homology between mouse and human genes is 85-88%. In mammals family Kalinkina encoded in two different genes: CHKA and CHKB, located in the chromosomes 11q13.2 and 23q13.33 respectively (Ensembl Genome Browser v48, gene view: http://www.ensembl.org/). Due to their high homology assume their appearance in the process of gene duplication and subsequent divergence from common prototype. The expression of these genes gives the translation of three proteins domain choline/ethanolainines: ChoKα1, ChoKα2 and ChoKβ1 (labeled ChoK-like). Alpha-isoform has two variants generated by alternative splicing primary mRNA: ChoKα1 from 457 amino acids (AA) and ChoKα2 (439 AK, from which it differs only 18 AK in the N-terminal region. Beta-isoform also has two different versions with alternative splicing, only one of which, ChoKβ1, has kinase activity. ChoKβ1 has 395 AK and differs from alpha isoforms about 40% of its sequence (Aoyama at al., 2004) (figure 4). Finally, the shift reading frames in the transcript ChoKβ gives the appearance ChoKβ2, shorter protein (127 AK), which has no domain choline/ethanolainines and which differs from option 1 to its C-end. It is unknown what role can play this option, however, identified mouse option from 164 AK with similar characteristics, identified as ChoKα3.

In addition to its role in the lipid metabolism, there is an important sign, which indicates that ChoK involved in carcinogenesis. The first sign that ChoK can play an important role in carcinogenesis, arises from the observation that during the transformation of the cell-mediated oncogene

RAS, there is an increase in PCho. Later it was demonstrated that the cause of the increase PCho is increased activity ChoK (Ramirez de Molina at al., 2001, Biochem. Biophys. Res. Commun. 285: 873-879), mediated by two of the most famous effectors of oncogene

RAS, PI3K and Ral-GDS (Ramirez de Molina at al., 2002, Oncogene 21: 937-946). Also described the production PCho as an essential process in the growth of cells, induced by growth factors in a mouse fibroblasts and in different systems of human cells, in which the processing ChoK-specific drugs is blocking the synthesis of DNA, induced by various factors, such as EGF, PDGF or HRG.

ChoK excessively is expressed in the large number of cell lines allocated from human cancers, as well as in various tissues tumors of the breast, lung, colon, bladder and prostate person (Nakagami at al., 1999, Jpn. J. Cancer Res 90: 419-424; Ramirez de Molina at al., 2002, Oncogene 21: 4317-4322; Ramirez de Molina at al., 2002, Biochem Biophys. Res. Commun. 296: 580-583). These types of tumors represent more than 70% of the total number of cancer cases in developed countries. Biochemical data show the activation of an enzyme in a large number of cases with acceleration improve ChoK in tumor States transcriptional and post-translational levels (Ramirez de Molina at al., 2002). The degree of excessive expression or excessive activity ChoK in tumors these types are usually very high, accounting for 40 to 60% (Ramirez de Molina at al., 2004, Cancer Res 64: 6732-6739; Ramirez de Molina at al., 2002, Biochem Biophys Res Commun 296: 580-583). In cases that were analyzed, highlights the relationship between the activation ChoKα and degree of malignancy (Ramirez de Molina at al., 2002, Oncogene 21: 4317-4322). In conclusion, a recent study on 167 samples from patients with small cell lung cancer (NSCLC), which showed patients, tumor which demonstrate a high expression of ChoKα, have a much worse prognosis of disease, which may have important clinical consequences (Ramirez de Molina at al., 2007, Lancet Oncol 8: 889-897). All of these studies were conducted for alpha-isoforms.

Significant feature shows the change ChoK in the carcinogenic process, indicating this enzyme as a target for the development of anticancer strategy, based on the specific inhibition of its activity. First of all, various in vitro studies in cells transformed with oncogenes, show that there is a high correlation between the inhibition of the enzyme inhibition of cell proliferation without mortality associated with taking hemikalii-3 (Campos at al., 2000, Bioorg Med Chem Lett 10: 767-770; Cuadrado at al., 1993, Oncogene 8: 2959-2968; HeRNAndez-Alcoceba at al., 1997, Cancer Res 59: 3112-3118; Jimenez at al., 1995, J Cell Biochem 57: 141-149). Also successfully conducted in vivo studies inhibition of cell growth in xenografts of tumor cells epidermoid carcinoma, adenocarcinoma of the colon and adenocarcinoma of the breast of a man, generated in Nude mice (HeRNAndez-Alcoceba at al., 1999, Cancer Res 59: 3112-3118; Lacal, 2001, IDrugs 4: 419-426; Ramirez de Molina at al., 2004, Cancer Res 64: 6732-6739). In conclusion, demonstrated in vivo specificity MN58b relatively ChoK-target in the xenografts of cancer cells in the breast and colon by NMR, and determined that after cancer treatment through MN58b affected only the levels PCho, but not other phosphomonoesterase (Al-Saffar at al., 2006, Cancer Res 66: 427-434).

However, despite deep knowledge of the mechanisms of action of inhibitors ChoKα still unknown whether you can apply ChoKβ as a target for the development of anticancer drugs or can I use the specified enzyme as a biomarker to respond to anticancer drugs or to predict in case of patients suffering from cancer.

Short description of the invention

In the first aspect of the present invention relates to a method of determining the prognosis for a patient suffering from cancer, which includes the determination of the levels of expression of ChoKβ in the sample from specified patient, which lowered levels ChoKβ relative to the levels in the reference sample indicate that the patient shows a poor prognosis.

The second aspect of the present invention relates to a method of determining the prognosis for a patient suffering from cancer, which includes the determination of expression levels ChoK ascension and ChoKβ in the sample from the patient, which decreased levels

ChoKα and high levels ChoKβ relative to the levels of expression of these proteins in the reference sample indicate that the patient shows a good prognosis.

In the third aspect of the present invention relates to a method of determining the reaction of the patient with cancer treatment inhibitor

ChoKα, including the definition of the sample from the patient of expression levels of protein, selected from the group: PEMT and ChoKβ, which increased levels of expression of PEMT or increased levels of expression of ChoKβ relative to the levels in a standard sample indicate good response inhibitor

In the final aspect of the invention refers to the agent, inducing activity ChoKβ, with a view to its use in the treatment of cancer.

Description of figures

Fig 1. mRNA levels ChoK α increased in tumor lines, while ChoKβ they unchanged or decreased. The results of quantitative PCR for ChoKα (grey) and ChoKβ (white) in the tumor lines person: (A) the lung, (B) the bladder and C) of the breast. mRNA levels of tumor lines compared with normal epithelial line of the same origin method 2-ΔCt . Used for the normalization of endogenous gene is a 18S.

2. An example of gene expression isoforms ChoKα and ChoKβ in samples from patients diagnosed with non-small cell lung cancer. The levels of expression of mRNA for ChoKα (A) or ChoKβ (B) in samples of lung tumor compared to commercial normal lung tissue obtained with the use of method 2-ΔCt . The results correspond Log10RQ (relative amount) α or β isoforms regarding the expression of endogenous gene (18S). In each case, the first column shows the expression for normal tissue.

3. The induction of apoptosis in response to MN58b. Cells Hek293T, Jurkat, SW70 and H1299 treated with 20 microns MN58b within 0 hour, 24 hours and 48 hours and for the control of the same cells left untreated during the same time periods. Cell extracts of these lines divided by PAGE-SDS and transferred to nitrocellulose membrane for their immunodetection antibodies. Shows examples of photos as a result of immunodetection on different cell lines: (A) (PARP and B) Caspase 3, degradation which is an indicator of apoptosis. GAPDH used as loading control.

Figure 4. Increase transcription ChoKβ in response to MN58b. Cells Hek293T, Jurkat, SW70 and H1299 treated with 20 microns MN58b within 24 hours and 48 hours and for the control of the same cells left untreated during the same time periods. Then extracted all the RNA of these cells and perform quantitative PCR. In response to medicinal product receive increasing levels of mRNA ChoKβ in all considered cases. Presents Log10RQ obtained by means of 2-ΔΔCt , interval RQ max-RQ min corresponds to error.

Figure 5. Co-expression Chokα and ChoKβ has the opposite effects on the levels of intracellular ethanolamine and choline. Cells Hek293T transferout vectors expression ChoKα, ChoKβ, ChoKα/ChoKβ simultaneously or an empty vector pCDNA3b. Cells mark in equilibrium 14 C-choline or 14 C-ethanolamine within 24 hours. Extracted lipids. Shows the number of intracellular PEtn or PCho relative to the total lipids. Common over-expression of ChoKα and ChoKβ reduces PCho achieved by excessive expression ChoKα separately, although there is an increase in the intracellular levels of PEtn. The results shown correspond to the mean value±standard deviation of 3 independent experiments carried out in three repetitions. * Statistically significant fluctuations (p<0.05).

6. ChoKβ inhibits oncogenic ability ChoKα. Cells Hek293T transferout vectors expression ChoKα, ChoKβ together (ChoKα/ChoKβ) or an empty vector pCDNA3b as a negative control. After transfection inocularea 106 cells subcutaneously in the back of Nude mice (nu/nu-). Found that the formation of tumors in the case ChoKα is statistically significant (p < 0,001). Stimulation of tumors caused ChoKα, completely void if at the same time overly expressed ChoKβ.

Fig.7. ChoKβ inhibits oncogenic ability ChoKα (II). Cells ADJ of tumors generated by excessive expression ChoKα, transferout vectors expression ChoKβ or an empty vector pCDNA3b as a negative control. After transfection inocularea 106 cells subcutaneously in the back of Nude mice (nu/nu-). A) comparison of the growth of tumors generated through ADJ/ChoKβ and ADJ/pCDNA3b, B) pictures of xenografts in the case of Nude mice, which made the injection of cells ADJ/ChoKβ (upper photo) and ADJ/pCDNA3b (bottom photo) (4.5 weeks).

Fig. Excessive expression ChoKβ in cells ADJ derived from Hek293T, inhibits cell proliferation. Cells ADJ, stable in relation to the expression of ChoKα, transferout via vectors expression ChoKβ or an empty vector pCDNA3b as a negative control. Cells were seeded at the 24-hole tablets with density 104 cells per well and incubated for 16, 48 and 96 hours in optimal growth conditions, determine the optical density after application crystal violet. Growth of cells transfected by ChoKβ, significant after 96 hours. Statistically significant consider result when p<0.05 marked with an asterisk.

Fig.9. Quantitative determination of expression ChoKβ in samples of tumors in patients with NSCLC and compared with the expression in the commercial normal tissue used as a reference sample.

Figure 10. Chart Kaplan-Meier for expression ChoKβ and survival, General and without recurrence for patients with NSCLC.

11. Chart Kaplan-Meier for expression ChoKβ and survival for patients with stage I NSCLC.

Fig. Chart Kaplan-Meier for expression ChoKβ and survival for patients with squamous cell carcinoma.

Fig. Chart Kaplan-Meier estimates for the combined effect of expression ChoKα and ChoKβ on the survival of patients with NSCLC.

Fig. Strengthening transcription PEMT in response to MN58b. Cells Hek293T, Jurkat, SW780 and H1299 treated with 20 microns MN58b within 24 h and 48 h, and the same cells left untreated during the same time periods as control. Then extracted all RNA these cells and perform quantitative PCR. Depicted Log10RQ obtained by means of 2-ΔΔCt , interval RQ max-RQ min represents an error. Arrow in the case of cell lines Hek293T and SW780 as a picture of the magnitude of the error indicates that the control is not the expression of PEMT and that expression starts in the treated cells, therefore, comparison extrapolate to the maximum number of cycles PCR.

Fig. Strengthening transcription PEMT in response to excessive expression ChoKβ. Cells Hek293T transferout expression vector ChoKβ or an empty vector pCDNA3b as a control. The expression PEMT, the enzyme that is not expressed in this system, in normal conditions, as observed in the case of control, induce as transcriptional response to excessive expression ChoKβ. The figure shows the relative amount of mRNA PEMT in Log10RQ obtained by means of 2-ΔΔCt . The arrow indicates that although the expression control specified gene is not shown, compared to extrapolate the maximum number of PCR cycles.

Detailed description of the invention

The method of determining the prognosis for a patient suffering from cancer, is based on the use of expression levels ChoKβ or on the ratio of expression levels ChoKα and ChoKβ

The authors of the present invention has established that the levels of expression of ChoKβ correlate with survival of patients with cancer. In particular, lower levels ChoKβ defined in the sample of the patient's tumor, indicate that the patient shows a poor prognosis. Thus, you can use ChoKβ as biomarkers in order to predict prognosis for the subject who is suffering from cancer.

In addition, the authors of the present invention has demonstrated that the combined determination of the levels of expression of two isoforms ChoK (ChoKα and ChoKβ) is a prognostic factor with greater prognostic likely than the definition of each of them separately. In particular, the authors of the present invention observed that patients who both have high levels ChoKα and lowered levels ChoKβ demonstrate a worse prognosis, characterized by survival or frequency of relapses.

Thus, in another aspect of the invention relates to a method of determining the prognosis for a patient suffering from cancer (hereafter, the second method of forecasting the invention), including the identification of expression levels

ChoKα and ChoKβ in the sample from the patient, in which lowered levels ChoKα and high levels ChoKβ relative to the levels of expression of these proteins in the reference sample indicate that the patient shows a good prognosis.

In the present invention, the expression "forecast" is understood as the expected progression of the disease, and it deals with estimation of probability, under which everyone suffers from disease, and assessing its beginning, the state of development, progression or regression and/or predict the course of the disease in the future. As will be clear to the experts, this assessment cannot usually be valid for 100% of subjects exposed diagnosis, although it is mostly correct. However, the term requires a statistically significant part of the subjects can be identified as suffering from disease or having a predisposition to it. If the part is statistically significant, specialist in this field can easily be detected using some well-known methods of statistical estimation, for example the determination of confidence intervals, the determination of the values of p, t-test t-test, Mann-Whitney test and other Full details can be found at Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%at least 60%, at least 70%at least 80%at least 90%, at least 95%. The values of p preferably be 0.2, 0.1, the 0,05.

Predicting clinical outcome can be executed using any of the evaluation criteria used in Oncology and well-known expert in the field. The estimated parameters are suitable to describe the progression of the disease include:

the progression without the disease, which is used in the present invention, describes the proportion of subjects in a state of complete relapse, who had no recurrence of the disease during the study period;

objective reaction, which is used in the present invention, describes share treatment of people who have a complete or partial response;

tumour control, which is used in the present invention relates to the share of treated people who have a complete response, partial response, a low response or stable disease & GE 6 months;

survival without disease progression, which is used in the present invention is defined as the time from the beginning of treatment to first determine the growth of cancer cells;

survival without disease progression within six months or the degree of "PFS6", which, as used in this the invention relates to the percentage of people who lack the progression during the first six months after the start of therapy;

the average survival, which is used in the present invention refers to the time in which half of the patients involved in the study, still alive, and

time of progression, as used in the present invention refers to the time after which diagnose the disease (or treat) to worsening of the disease.

In particular embodiment, the invention determine the clinical outcome as the surviving entity or the survival without relapse.

Used in this description, the term "entity" means all of the animals that are classified as mammals, and includes, but is not limited to, domestic and agricultural animals, apes and humans, such as humans, primates other than humans, cows, horses, pigs, sheep, goats, dogs, cats or rodents. The preferred subject is male or female of any age or race.

For the implementation of methods for predicting the invention get a sample from the subject under study. Used in this description, the term "sample" refers to any model which you can get from the patient. The presented method can be applied to any type of biological samples from the patient, such as a biopsy, tissue, cellular sample or sample fluids (serum, saliva, sperm, sputum, cerebrospinal fluid (CSF), tears and mucus, pot, milk, extracts of the brain and the like). In particular embodiment, the specified pattern is a sample of tissue or part of a tissue, preferably a sample of the tumor tissue or part of such tumor tissue. The specified pattern can be obtained by conventional means, such as a biopsy, using the methods are well known to specialists in the areas related to medical technologies. Ways of getting biopsy sample include dividing tumor on large pieces, or microsurgery, or other ways to separate cells, known in this area. In addition, tumor cells can be obtained by aspiration Cytology using a thin needle. To facilitate storage and handling of samples it can be fixed in formalin and add paraffin or first frozen and then pour kreaturecave environment, for example OCT connection, by immersing in vysokoskrostnoy environment that allows for rapid freezing.

As a specialist in this area, the levels of expression of ChoKβ and/or ChoKα can be determined by measuring the levels of mRNA encoded by these genes, or by measuring the levels of the proteins encoded by these genes, i.e. ChoKβ or ChoKα protein.

Thus, in particular embodiment, the invention determine the levels of expression of ChoKβ and/or ChoK alpha , measuring the levels of mRNA expression encoded by the genome ChoKβ and/or ChoKα. For this purpose the biological sample can process up to the physical or mechanical destruction of the structure of tissues or cells with the release of intracellular components in aqueous or organic solution, getting nucleic acids for additional analyses. Nucleic acid is extracted from the sample methods, well-known experts in this field and are available commercially. Then RNA extracted from frozen or fresh samples in any of a typical for this area of knowledge of ways, for example as described Sambrook, J. at al., 2001 Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3. During the extraction process, preferably exercise care to prevent the degradation of RNA.

In particular embodiment, the level of expression can be identified using mRNA received from the tissue sample, fixed in formalin, filled with paraffin. the mRNA can be distinguished from the typical pathological sample or biopsy sample, which first deparaffinized. The usual way of deparafineerimine includes washing the sample in paraffin with an organic solvent, such as xylene. Dewaxed samples can be regidratirtee aqueous solution of lower alcohol. Suitable lower alcohols include, for example, methanol, ethanol, propanol and butanol. Dewaxed samples can be regidratirtee, for example, by successive washing solutions lower alcohols with lower concentrations. Alternatively, the sample deparaffinized and rehydration at the same time. Then the sample are lysed and extracted from the sample RNA.

Although the methodology for determining the profile of gene expression (RT-PCR, SAGE or TaqMan) suitable for use if all the previous aspects of the invention, the levels of mRNA expression is often defined by reverse transcription-polymerase chain reaction (RT-PCR). In particular embodiment, the levels of mRNA expression ChoKβ and/or ChoKα determined by quantitative PCR, preferably PCR in real time. The determination can be also performed in individual specimens or micromeria tissue.

When the mean value is set, the level marker expressed in tumor cells of patients can be compared with the average value and, therefore, be defined as "high" or "low" level of expression. Because of the variability among actors (such as aspects related to age, race and other) very difficult (if not practically impossible) to establish absolute reference values expression

ChoKβ and/or ChoKα. Thus, in particular embodiment, reference values for "high" or "low" expression ChoKβ and/or ChoKα determined by calculating percentiles of the normal ways, which include assessment of groups of samples isolated from normal subjects (i.e. people without cancer diagnoses), levels of expression of ChoKβ and/or ChoKα. Then the "lower" levels ChoKβ can preferably be set for samples in which the levels of expression of ChoKβ equal to or less than the 50th percentile in the normal population, including, for example, levels of expression, equal to or below the 60th percentile in the normal population equal to or less than 70 percentile in the normal population, equal to or less than 80 percentile in the normal population equal to or less than the 90th percentile in the normal population and equal to or less than 95 percentile in the normal population. Then the "elevated" levels ChoKα can preferably be set for samples in which the levels of expression of equal ChoKα or more of the 50th percentile in the normal population, including, for example, levels of expression, equal to or greater than 60 percentile in the normal population, equal to or greater than 70 percentile in the normal population, equal to or greater than 80 percentile in the normal population, equal to or greater than 90 th percentile in normal populations, and equal to or greater than 95 percentile in the normal population.

Alternatively, in one embodiment, the levels of expression of ChoKβ and/or ChoKα can be determined by measuring the levels of the proteins encoded by these genes, i.e. protein ChoKβ and/or ChoKα, and the levels of their varieties.

The definition of levels of protein expression can be performed by immunological methods, such as ELISA, immunoblot analysis or immunofluorescence. The immunoblot analysis is based on the detection of proteins previously separated by gel electrophoresis in the conditions of denaturation and immobilized in the membrane, usually nitrocellulose, by incubation with specific antibody and manifest system (for example, chemoluminescence). Analysis by immunofluorescence assay requires the use of antibodies specific relatively protein target for analysis of gene. ELISA is based on the use of antigens or antibodies labeled enzymes, so that conjugates formed target labeled antigen and antibody result in the formation of enzyme-active complexes. Given that one of the components (labeled antigen or antibody) immobilized on the substrate complexes antigen-antibody immobilized on the surface and thus can be detected at addition of the substrate that is converted by the enzyme product that is detected, for example, by spectrophotometry or fluorimetry.

If applied immunological method for detection of the number of target proteins you can use any antibody or reagent for which it is known that it is associated with a protein target with high affinity. However, it is preferable to use antibodies, for example polyclonal sera, supernatants hybrid or monoclonal antibodies, fragments of antibodies, Fv, Fab, Fab' and F(ab') 2 , scFv, diatel, Triatel, tetratel and humanized antibodies.

In addition, the definition of the levels of expression of a protein can be, making micronor tissues (TMA)containing combination samples from subjects, and determining the levels of protein expression by immunohistochemical techniques, well-known in the modern state of science.

Although methods of forecasting the invention can normally be used for tumors of any type in a preferred embodiment, for tumors characterized by high levels of expression of ChoKα use both ways of forecasting according to the invention, the first and the second.

The definition of high levels ChoKα, the way of determining levels and the reference sample, suitable for the definition of these levels are explained in detail in the context of a therapeutic method according to the invention.

In particular embodiment, methods of forecasting on the invention applied to cancer of the lung, breast, bladder or colorectal cancer.

How to determine the response of the patient with cancer treatment inhibitor ChoKα based on use levels PEMT and/or ChoKβ

The expression "determination of patient's response" refers to the evaluation of the results of treatment of the patient who is suffering from cancer, response to therapy based on the use of inhibitors

ChoKα. To use biomarkers for the invention to control the effectiveness of treatment can also ways of selecting and screening of drugs with potential anticancer activity. This method includes a) introduction to the subject (preferably animal) medicinal products subject to research; (b) identification of biological specimens of animals in different points of the study (before, during and/or after the introduction) and the definition of the levels of the marker according to the present invention, and c) comparison of definitions conducted on samples at the different phases of the treatment, and their comparison with the control group of animals, such as untreated animals.

In the context of the present invention under PEMT mean protein phosphatidylethanolamines capable of catalyzing the transformation of phosphatidylethanolamine in phosphatidylcholine by double methylation.

As described in respect methods of forecasting on the invention, the definition of the levels PEMT and ChoKβ can be performed by determining appropriate levels of polypeptides, which apply a standardized methodology, for example, Western-blotting or immunoblotting, ELISA (adsorption enzyme-linked immunosorbent assay), RIAa radioimmunoassay), competitive EIA (competitive enzyme-linked immunosorbent assay), DAS-ELISA (sandwich method is a double antibody ELISA), immunocytochemical and immunohistochemical techniques, methods based on the use of micronorb proteins or biochips, including specific antibodies, or studies based on the precipitation of colloids, for example, in the format of strips.

Alternative, determining levels PEMT and ChoKβ may be achieved through the definition of corresponding mRNA level, for which you can apply standard methods such as PCR in real time, SAGE, TaqMan RT-PCR and the like.

If PEMT also possible to determine the levels of expression by determination of enzyme activity of the corresponding protein, which apply the usual methods, such as methods based detection inclusion of methyl groups, labeled phosphatidylethanolamine, using for this purpose [methyl-3H]AdoMet as a donor of methyl groups, as originally Ridgway described and Vance (Methods Enzymol. 1992, 209, 366-374), Zhu at al. (Biochem. J., 2003, 370, 987-993) and Song at al. (FASEB j, 2005, 19: 1266-1271).

In the context of the present invention, the expression "inhibitor ChoKα" is understood as any connection capable of reducing the activity ChoK, including compounds that prevent gene expression ChoKα, resulting in decreased levels of mRNA or protein ChoK, as well as compounds that inhibit ChoK, causing a decrease of enzyme activity.

Connection capable of preventing the expression of a gene ChoKα, can be identified using standard tests to determine the levels of mRNA expression, such as RT-PCR analysis using protection RNA, Northern technique, in situ hybridization, methodology with micronaire and such.

Connection, which give lower levels of the protein ChoK, can be identified using standard tests to determine the levels of expression of the protein, such as the Western blot turns or Western blot, ELISA (adsorption enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme-linked immunosorbent assay), DAS-ELISA (sandwich method is a double antibody ELISA), immunocytochemical and immunohistochemical techniques, methods based on the use of micronorb proteins or biochips that include specific antibodies, or research, is based on the deposition of colloids in formats of strips.

Determination of the inhibitory capacity of biological activity Kalinkina conducted using standard measurements activity Kalinkina, for example, the methods based on the detection of phosphorylation of choline, labeled [ 14 C]-in the presence of ATP purified recombinant Kalinkina or enriched kalinkinsky faction and subsequent detection of phosphorylated choline using standard analytical techniques (such as TLC), as described in EP1710236.

Specific inhibitors Kalinkina I-XVIII, which can be used in the first compositions of the present invention, described in table 1.

Inhibitors ChoKα

Connections are described in the patent application US 20070185170, having the General formula:

where Q is the basis of conjugated with pharmaceutically suitable organic or inorganic acid; R 1 and R' 1 are independent from each other, aryl radical, not necessarily replaced by halogen atom, trifluoromethyl, hydroxyl, 1-6 C-alkyl amino or arcoxia; R 2 and R' 2 are independent from each other, aryl radical, not necessarily replaced by halogen atom, trifluoromethyl, hydroxyl, 1-6 C-alkyl amino or arcoxia; R 3 and R' 3 are independent from each other, or radical, selected from the group consisting of an H atom, halogen atom, triptorelina, hydroxyl, amino, alkoxyl and C 1-6 alkyl, not necessarily replaced by trifluoromethyl, hydroxyl, amino or arcoxia, or together with R 4 and R' 4 , respectively, and independently from each other, the radical-CH=CH-CH=CH-not necessarily replaced by halogen atom, trifluoromethyl, hydroxyl, 1-6 C-alkyl amino or arcoxia; R 4 and R' 4 are independent from each other, or radical, selected from the group consisting of H and C 1-6 alkyl, not necessarily replaced by halogen atom, trifluoromethyl, hydroxyl, amino or arcoxia, or together with R 3 and R' 3 , respectively, and independently from each other, the radical-CH=CH-CH=CH-not necessarily replaced by halogen atom, trifluoromethyl, hydroxyl, 1-6 C-alkyl amino or arcoxia; A spacer elements is the group that contains any of bivalent organic structure that communicates between the two pyridinium groups that operate in a structure defined by the formula I, in particular, ferrous molecules with the structure,

selected from the group:

where m, n, and p are equal to integers, which can have the following values: m is 0, 1; n is 0, 1-10; p 0, 1; provided that m, n and p not vanish simultaneously.

Preferred connection in this group include compounds, in which the Deputy NR 1, R 2 , R 3 , R 4 and A are the following:

Preferred connection in this group include 4-(4-chloro-N-methylaniline)chinoline and 7-chloro-4-(4-chloro-N-methylamino)the quinoline with patterns:

, respectively .

Connections are described in the international patent application WO 9805644 with common structural formula:

where n is 1, 2 or 3, Z means any structural group, selected from the group:

where Y is chosen from the group: -H-CH 3 , -CH 2-OH, -CO-CH 3 , CN, -NH 2 , -N(CH 3 ) 2 , pyrrolidine, piperidine derivatives,

perhydration, -OH-OH-CO-15 C H 31, etc. Preferred inhibitors ChoK, having defined above formula, are compounds 1-6 described Conejo-Garcia at al. (J. Med. Chem., 2003, 46:3754-3757) and has the following structure:

where R is H or

The connection defined above General formula, selected from a group of compounds GRQF-JCR795b, GRQF-MN94b and GRQF-MN58b with patterns:

Connections are described in the international patent application WO 9805644 with common structural formula:

where n is 0, 1, 2, 3, etc., X is a structural element that is selected from group A, B, C, D and E:

where Y is selected from the-H, -CH 3 , -CH 2-OH, -CO-CH 3 , CN, -NH 2 , -N(CH 3 ) 2 , pyrrolidine, piperidine derivatives, perhydration, -OH, -O-CO-15 C H 31 , and where R 1 , R 2 and R 3 are alkyl groups, such as-Me, -Et and similar, although in some cases R 2 and 3 may be more difficult groups such as-CH 2-CH(OMe) 2-CH 2-CH(OEt) 2 . Preferred connection with the above General structure, are GRQF-FK3 and GRQF-FK21 that has the following structure:

Connections are described in the international patent application WO 9805644 with common structural formula:

where X corresponds to the group selected from the group A, B, C and E:

where Y is a Deputy, such as-H-CH 3 , CH 2 OH-CN-NH 2 , -N(CH 3 ) 2 , pyrrolidyl, piperidinyl, perhydration, -OH-OH-CO-C 15 H 31 and the like; where Z denotes alkyl (Me, Et, and others), aryl, phenyl group or donor groups, such as-OMe, -NH 2 , -2 and NMe other Preferred connection with the above General structure, are GRQF-MN98b and GRQF-MN164b that has the following structure:

Connections are described in the international patent application WO 9805644 that has the following General structural formula:

where X corresponds to the group selected from the group A, B, C and E:

where Y is Deputy such as-H-CH 3 , CH 2 OH, -CO-CH 3 , CN, -NH 2 , -N(CH 3 ) 2 , where Z denotes alkyl (Me, Et, and others), aryl (phenyl and similar) group or donor groups, such as-OMe, -NH 2 , -2 and NMe other Preferred connection with the above structure, are GRQF-FK29 and GRQF-FK33 that has the following structure:

oxazoles)]propane, 2,2-bis[(5-trifluoromethyl-4-(4-pyridyl)-2-oxazole)]propane, 4,4'-bis[(5-trifluoromethyl-4-(1-methyl-4-pyridine)-2-oxazole)]biphenyl, 4,4'-bis[(5-pentafluoroethyl-4-(1-methyl-4-pyridine)-2-oxazole)]biphenyl, 4,4'-bis[(5-trifluoromethyl-4-(1-methyl-4-pyridine)-2-oxazole)]hexafluoroisopropylidene, 2,2-bis[(5-trifluoromethyl-4-(4-pyridyl)-2-thiazolyl)]propane and 4,4'-bis[(5-trifluoromethyl-4-(1-methyl-4-pyridine)-2-thiazolyl)]-1,1'-occipital.

Hemikalii-3 described Cuadrado at al. (Oncogene, 1993, 8: 2959-2968), Jimenez at al. (J.Cell Biochem., 57: 141-149) and HeRNAndez-Alcoceba at al. (Oncogene, 1997, 15: 2289-2301).

The connection described in the international patent application WO 2007077203 with the General structure of the formula:

where R 1 , R 2 , R 3 , R 4 R 5 R 6 R 7 R 8 , R 11 R 12 independently represent an atom of hydrogen, hydroxyl; halogen atom; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; N(R')(R)the amino group, where R' and R ' independently represent a hydrogen atom or C 1-C 12 alkyl group; the group OCOR, where R is (CH 2 ) 2-COOH or

(CH 2 ) 2 CO 2 CH 2 CH 3 ; or each couple can form a group (C=O) together with the carbon atom to which they relate; R 9 R 10 independently represent a hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; 6 C-C 10 aryl; group COR"' (where R"' means a hydrogen atom; hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; C 1-C 12 alkyl or N(R IV )(a R V )the amino group, where R IV and R V independently represent a hydrogen atom or C 1-C 12 alkyl group); (CH 2 ) n-OH carbinol group (where n is an integer from 1 to 10); or together form methylene group; communication ------ means of the double bond or simple communication; and where tricyclic structure:

selected from the following structures:

where R 13 R 14 , R 15 , R, 16 , R 21 , 22 and R

R 23 independently represent a hydrogen atom; hydroxyl; halogen atom; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; N(R VI )(R VII )the amino group, where R VI and R VII independently represent a hydrogen atom or C 3-C 12 alkyl group; the group OCOR VIII , where R VIII means (CH 2 ) 2 COOH or (CH 2 ) 2 CO 2 CH 2 CH 3 ; or each couple can form a group (C=O) together with the carbon atom to which they relate; R 17 means a hydrogen atom or methyl; R 18 R 18 ' independently represent a hydrogen atom; hydroxyl; halogen atom; C 1-C 12 alkyl; 6 C-C 10 aryl; COR IX (where R IX means atom of hydrogen, hydroxyl; C 1-C 12 alkyl; N(R, X )(XI R )the amino group, where R X and XI R independently represent a hydrogen atom or C 1-C 12 alkyl group; or C 1-C 12 alkoxyl); or (trifluoromethyl; R 19 , R 19 ', R 20 and R 20 ' independently represent a hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; group COR XII (where R XII means atom of hydrogen, hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; or N(R XIII )(R XIV )the amino group, where R XIII and R XIV independently represent a hydrogen atom or C 1-C 12 alkyl group); group [(C 1-C 12 )alkyl-O-C 1-C 12 )alkyl-] n (where n has values between 1 and 3); trifluoromethyl; or each pair 19-19' or 20-20' may form a group, C=O, together with the carbon atom to which they relate; R 24 and R 25 independently represent the atom

hydrogen, hydroxyl or halogen. The preferred connection, defined above structure, selected from the group comprising the following connections: 3,9-dihydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-7,8,8a,11,12,12a,12b,13,14,14a-decahydro-6bH,9H-pecen-2,10-dione; 9-hydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt ether of acetic acid; 9-hydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt ether propionic acid; 9-hydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt ether dodekanisou acid; 9-hydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt ether dimethylcarbinol acid; 9-hydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt ester of nicotinic acid; 4-bromo-(9-hydroxy-6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt) ether benzoic acid; 14-bromo-3,7,9-trihydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-7,8,8a,11,12,12a,12b,13,14,14a-decahydro-6bH,9H-pecen-2,10-dione; 12-bromo-9-hydroxy-6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt ether dimethylcarbinol acid; 4-bromo-(12-bromo-9-hydroxy-6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt) ether benzoic acid; 12-bromo-3,9-dihydroxy-6b,8a,11,12b,14a-HEXAMETHYL-7,8,8a,11,12,12a,12b,13,14,14a-decahydro-6bH,9H-pecen-2,10-dione; 3,9,10-trihydroxy-6b,8a,11,12b,14a-HEXAMETHYL-7,8,8a,9,10,11,12,12a,12b,13,14,14a-dodecahydro-6bH-pecen-2-it; mono(10-hydroxy-2,4a,6a,9,12b,14a-HEXAMETHYL-3,11-dioxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-terradicalization-4-sludge) ether succinic acid; ethyl ether 10-hydroxy-2,4a,6a,9,12b,14a-HEXAMETHYL-3,11-dioxo-1,2,3,4,4a,5,6,6a,11,12b,13,14,14a,14b-terradicalization-4-silt ether succinic acid.

The connection described in the international patent application WO 2007077203 with the General structure of the formula:

where R 1 , R 2 , R 3 , R 4 R 5 R 6 R 9 R 10 R 11 R 12 R 13 R 14 , R 15 , R, 16 , R 17 , R 18 , R 19 and R 20

independently represent the atom of hydrogen, hydroxyl; halogen atom; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; N(R XV )(R XVI )the amino group, where R XV and R XVI

independently represent a hydrogen atom or C 1-C 12 alkyl group; or each couple can form (C=O) carboxyl group together with the carbon atom to which they relate; R 7 and R 8 independently represent a hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; 6 C-C 10 aryl; group COR XVII (where R XVII means atom of hydrogen, hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; O-C 1-C 12 alkyl; or N(R XVIII )(R XIX )the amino group, where R XVIII and R XIX independently represent a hydrogen atom or C 1-C 12 alkyl group); (CH 2 ) n-OH carbinol group (where n is an integer from 1 to 10); or together form methylene group, R 21 R 24 independently represent a substituted or unsubstituted C 1-C 12 alkyl; group COR XX (R XX indicates the atom

of hydrogen, hydroxyl; or substituted

unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; or N(R XXI )(R XXII )the amino group, where R XXI

and R XXII independently represent a hydrogen atom or C 1-C 12 alkyl group); group [(C 1-C 12 )alkyl-O-C 1-C 12a )alkyl-] n (where n is a value from 1 to 3); or (trifluoromethyl; R 22 R 23 are: [1] the hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; group COR XXIII

(where R XXIII means the hydrogen atom; hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; or N(R XXIV )(R XXV )the amino group, where R XXIV and R XXV

independently represent a hydrogen atom or C 1-C 12 alkyl group); group [(C 1-C 12

) alkyl-O-C 1-C 12a )alkyl-] n (where n has a value from 1 to 3); or (trifluoromethyl, if R 24 is paraprotein regarding R 20 ; or [2] OR 22 ' and OR 23', respectively, where R 22 ' and R 23 ' independently mean the hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; group COR XXVI (where R XXVI

means a hydrogen atom; hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; or N(R XXVII )(R XVIII )the amino group, where R and XXVII

R XVIII independently represent a hydrogen atom or C 1-C 12 alkyl group); group [(C 1-C 12 )alkyl-O-C 1-C 12a )alkyl-] n (where n has a value from 1 to 3); or (trifluoromethyl, if R 24 is metaprogram regarding R 20 . The preferred connection, which are included in the above General structure, selected from the group: [3] 14-bromo-3-hydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-7,8,8A,11,12,12A,12b,13,14,14a-decahydro-6bH,9H-pecen-2,10-Dion; [4] 4,6b,8a,11,12,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt ester acetic acid; [5] 4,6b,8a,11,12b,14a-HEXAMETHYL-2,10-dioxo-2,6b,7,8,8a,9,10,11,12,12a,12b,13,14,14a-terradicalization-3 silt ether nicotine acid; [6] 3,10-dihydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-7,8,8a,9,10,11,12,12a,12b,13,14,14a-dodecahydro-6bH-pecen-2-it; [7] 3-hydroxy-4,6b,8a,11,12b,14a-HEXAMETHYL-7,8,8a,12a,12b,13,14,14a-octahydro-6bH,9H-pecen-2,10-Dion.

Connections are described in the international patent application WO 2007077203 with common structural formula:

where R 1 , R 2 , R 3 , R 4 R 5 R 6 R 7 R 8 , R 11 R 12 independently represent a hydrogen atom; hydroxyl; halogen atom; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; N(R')(R)the amino group, where R' and R ' independently represent a hydrogen atom or C 1-C 12 alkyl group; the group OCOR, where R is (CH 2 ) 2-COOH or (CH 2 ) 2 CO 2 CH 2 CH 3 ; or each couple can form a group (C=O) together with the carbon atom to which they relate; R 9 R 10 independently represent a hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; 6 C-C 10 aryl; group COR"' (where R"' means a hydrogen atom; hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; O-C 1-C 12 alkyl; or N(R IV )(a R V )the amino group, where R IV and R V independently represent a hydrogen atom or C 1-C 12 alkyl group); (CH 2 ) n-OH carbinol group (where n is an integer from 1 to 10); or together form methylene group; communication ----- means dual link or single link; and where tricyclic structure:

selected from the following structures:

where R 13 R 14 , R 15 , R 16 -, R-21 , R-22 and R 23 , independently represent a hydrogen atom; hydroxyl; halogen atom; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; N(R VI )(R VII )the amino group, where R VI and R VII independently represent a hydrogen atom or C 1-C 12 alkyl group; the group OCOR VIII , where R VIII is a (CH 2 ) 2 COOH or (CH 2 ) 2 CO 2 CH 2 CH 3 ; or each couple can form a group (C=O) together with the carbon atom to which they relate; R 17 means a hydrogen atom or methyl; R 18 R 18 ' independently represent a hydrogen atom; hydroxyl; halogen atom; C 1-C 12 alkyl; 6 C-C 10 aryl; COR IX (where R IX means atom of hydrogen, hydroxyl; C 1-C 12 alkyl; N(R, X )(XI R )the amino group, where R X and XI R independently represent a hydrogen atom or C 1-C 12 alkyl group; or C 1-C 12 alkoxyl) or trifluoromethyl; R 19 , R 19 ', R 20 and R 20 ' independently represent a hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; group COR XII (where R XII means atom of hydrogen, hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; or N(R XIII )(R XIV )the amino group, where R XIII and R XIV independently represent a hydrogen atom or C 1-C 12 alkyl group); group

[(C 1-C 12 )alkyl-O-C 1-C 12

) alkyl-] n (where n is a value from 1 to 3); trifluoromethyl; or each pair 19-19' or 20-20' may form a group, C=O, together with the carbon atom to which they relate; R 24 and R 25 independently represent a hydrogen atom, hydroxyl or halogen. The preferred compounds that have the above structure, selected from the group: [8] 7,10,11-trihydroxy-2,4a,6a,9,12b,14a-HEXAMETHYL-8-oxo-1,2,3,4,4a,5,6,6a,8,12b,13,14,14a,14b-terradicalization-2-methyl ester of carbonic acid; [9] 9-formyl-10,11-dihydroxy-2,4a,6a,12b,14a-pentamethyl-8-oxo-1,2,3,4,4a,5,6,6a,8,12b,13,14,14a,14b-terradicalization-2-methyl ester of carbonic acid; [10] 11-hydroxy-10-(2-methoxymethylethoxy)-2,4a,6a,9,12b,14a-HEXAMETHYL-8-oxo-1,2,3,4,4a,5,6,6a,8,12b,13,14,14a,14b-terradicalization-2-methyl ester of carbonic acid.

The connection described in the international patent application WO 2007077203 with the General structure of the formula:

where R 1 , R 2 , R 3 , R 4 R 5 R 6 R 9 R 10 R 11 R 12 R 13 R 14 , R 15 , R, 16 , R 17 , R 18 , R 19 and R 20 independently represent a hydrogen atom; hydroxyl; halogen atom; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; N(R XV )(R XVI )the amino group, where R XV and R XVI independently represent a hydrogen atom or C 1-C 12 alkyl group; or each couple can form a group (C=O) together with the carbon atom to which they relate; R 7 and R 8 independently represent a hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; 6 C-C 10 aryl; group COR XVII (where R XVII means atom of hydrogen, hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; O-C 1-C 12 alkyl; or N(R XVIII )(R XIX )the amino group, where R XVIII and R XIX independently represent a hydrogen atom or C 1-C 12 alkyl group); carbinol group (CH 2 ) n-OH (where n is an integer from 1 to 10); or together form methylene group, R 21 R 24 independently are replaced or unsubstituted C 1-C 12 alkyl; group COR XX (R XX indicates the atom

of hydrogen, hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; or

N(R XXI )(R XXII )the amino group, where R XXI and R XXII independently represent a hydrogen atom or C 1-C 12 alkyl group); group [(C 1-C 12 )alkyl-O-C 1-C 12 a)alkyl-] n (where n is a value from 1 to 3); or (trifluoromethyl; R 22 R 23

are: [11] a hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; group COR XXIII (where R XXIII means atom of hydrogen, hydroxyl; replaced or unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; or N(R XXIV )(R XXV )the amino group, where R XXIV and R XXV independently represent a hydrogen atom or C 1-C 12 alkyl group); group [(C 1-C 12 )alkyl-O-C 1-C 12a )alkyl-] n (where n is a value from 1 to 3); or trifluoromethyl, if R 24 is paraprotein regarding R 20 ; or [12] OR 22 ' and OR 23 ', respectively, where R 22 ' and R 23 '

independently mean the hydrogen atom; or substituted unsubstituted C 1-C 12 alkyl; group COR XXVI (where R XXVI means atom of hydrogen, hydroxyl; or substituted unsubstituted C 1-C 12 alkyl; or substituted unsubstituted 6 C-C 10 aryl; or N(R XXVII )(R XVIII )the amino group, where R and XXVII

R XVIII independently represent a hydrogen atom or C 1-C 12 alkyl group); group [(C 1-C 12 )alkyl-O-C 1-C 12a )alkyl-] n (where n is a value from 1 to 3); or trifluoromethyl, if R 24 is metaprogram regarding R 20 . The preferred connection, which are included in the above structure, selected from the group: [13] 10,11-dihydroxy-2,4a,6a,9,14a-pentamethyl-1,4,4a,5,6,6a,13,14,14a,14b-decahydro-2H-pecen-3-it; [14] 10,11-dihydroxy-2,4a,6a,9,14a-pentamethyl-4a,5,6,6a,13,14,14a,14b-octahydro-4H-pecen-3-it.

ATP equivalents, including digitalisnye ATP equivalents, such as AMP-PCH 2 P, adenosinetriphosphate (AMP-PNP), AMP-PSP and AMP, where oxygen connecting the second and third phosphate ATP-analogues, replaced by CH 2 S (for example, γS, β and αS) and NH, respectively, and also suicidal substrates, such as 5'-(p-forcorporate)adenosine (FSBA), N 6-diethyl-beta,gamma dibromoethylene-ATP, 2-methylthio-ATP (APM), alpha,beta-methylene-ATP, beta,gamma methylene-ATP, diadenosinepentaphosphate (Ap5A), 1-N 6-aminoacetonitrile, adenosine-1-oxitriptofana, 2',3'-O-(benzoyl-4-benzoyl)-ATP (B-Z), family ATP equivalents described in US 2004204420, the contents of which are included in this description by reference, 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), 1-N 6 -(methoxy)ATF, 7-N 6 -(pyrrolidin)ATF, 2-N 6 -(ataxy)ATF, 8-N 6 -(cyclopentyl)ATF, 3-N 6 -(acetyl)ATF, 9-N 6 -(cyclopentyloxy)ATF, 4-N 6 -(isopropoxy)ATF, 10-N 6 -(piperidine)ATF, 5-N 6 -(benzyl) ATF, 11-N 6 -(cyclohexyl) ATF and the like.

Inhibitors halinowego conveyor, such as analogues of N-n-alternatine, HC-3 hemikalii, decamethonium, suksametonia, D-tubokurarin, Tetramethylammonium, tetraethylammonium, gexametoni, N-alkyl analogues (N-atilgan, N-metilholin), mono-, di - and tricillin, N-hydroxyethylpyrrolidine (pyrrolin) and DL-alpha-metilholin described Barker, L.A. and Mittag, T.W. (J Pharmacol Exp Ther. 1975; 192: 86-94), ion dimethyl-n-pentyl(2-hydroxyethyl)ammonium, decamethonium, gexametoni, substituted analogues bis-catechol and decamerone described Cai at al. (Bioorganic & Medicinal Chemistry, 2007, 15: 7042-7047)with structure:

Inhibitory antibodies, can specifically bind and inhibit the activity Kalinkina and, in particular, monoclonal antibodies that recognize the catalytic domain or domain dimerization ChoKα and thereby inhibit the activity ChoKα. In a preferred embodiment, inhibitory antibodies are a monoclonal antibody defined in WO 2007138143. In another preferred embodiment inhibitory antibodies are antibodies AD3, AD8 and AD11 defined in WO 2007138143.

Inhibitors fasttimesatnau N-methyltransferase (PEMT or EC 2.1.1.17). The cell treatment inhibitors ChoKα causes increased expression of PEMT (Spanish patent application P200802007, taken together with the present). In addition, excessive expression ChoKβ in cells also causes increased expression of PEMT (Spanish patent application P200802007, read in conjunction with this, suggesting that the activation PEMT can represent the path used ChoKβ to compensate for lower levels of phosphatidylcholine in response to the inhibition ChoKα. PEMT suitable for use in the compositions of the present invention, includes 3-dealaadanse (DZA) (Vance at al., 1986, Biochem. Biophys. Acta, 875: 501-509), 3-diasaranaaron (Smith and Ledoux, Biochim. Biophys. Acta. 1990, 1047: 290-3), bezafibrat and clofibrate acid (Nishimaki-Mogami T at al., Biochim. Biophys. Acta, 1996, 1304: 11-20).

Antisense oligonucleotide specific in terms of consistency Kalinkina.

The enzyme DNA or the ribosome, specific in terms of consistency Kalinkina.

Interfering RNA, specific in terms of consistency Kalinkina, for example, short hairpin RNA (sh), as defined in SEQ ID NO:3, or siRNAs defined Glunde at al. (Cancer Res., 2005, 65: 11034-11043).

In a preferred embodiment, the method personalized therapy for the invention carried out on patients with cancer, where the cancer is selected from the group: lung cancer, breast cancer, bladder or colorectal cancer.

Treatments for cancer, based on stimulation of activity ChoKβ

The authors of the present invention suddenly discovered that the expression ChoKβ in tumor cells results in a decrease in the rate of proliferation of these cells. Thus, in example 1.4 of the present invention is shown as due to excessive expression ChoKβ and ChoKα in cells is the formation of tumors as compared with the case where the tumor is the result of the expression ChoKα. Similarly, observed that the implantation Nude mice tumor cells, overly expressing ChoKβ and ChoKα, gives the tumor, the volume of which 73% less than in tumors arising from the implantation of cells expressing only ChoKα.

Thus, in the first aspect of the invention refers to the agent, inducing activity ChoKβ, for its use in the treatment of cancer. Alternatively, the invention relates to the use of an agent inducing activity ChoKβ, to obtain drugs for cancer treatment. Alternatively, an invention refers to the treatment of cancer in the subject, including the introduction of a specified individual agent inducing activity ChoKβ.

In a preferred embodiment, the agent inducing activity ChoKβ selected from the group:

(i) ChoKβ or functionally equivalent option ChoKβ,

(ii) polynucleotide encoding ChoKβ or its functional equivalent option,

(iii) the vector, including polynucleotide according to (ii),

(iv) cells can secrete ChoKβ or its functional equivalent option in the environment.

the ability to inhibit the proliferation of tumor cells, overly expressing ChoKα, for which you can apply the methods described in example 1.4 of the present invention;

the ability to promote increased levels of phosphoethanolamine (PEtn), if it is expressed in the cell in no ChoKα, or cause an increase levels PEtn, if it is expressed with ChoKα stronger than observed in the expression of only ChoKα, for which you can apply the methods described in example 1.4 of the present invention.

Used in the present invention, the term "polynucleotide" refers to the nucleotide polymeric form of any length, formed ribonucleotides and/or deoxyribonucleotide. The term includes both single-stranded and double polynucleotide and modified polynucleotide (methylated, protected polynucleotide and similar).

Polynucleotide, suitable for use as agents capable of inducing activity ChoKβ include, but without limitation, polynucleotide, the sequence of which correspond mRNA ChoKβ person (inventory number NM_005198 in NCBI according to the version of June 28, 2009), mouse mRNA ChoKβ (inventory number NM_007692 in NCBI version of 24 October 2008), rat mRNA ChoKβ (inventory number NM_017177 in NCBI version from 24 October 2008), mRNA ChoKβ striped Danio (inventory number NM_001100012 in NCBI version of March 22, 2009).

Alternatively, the agents capable of inducing activity ChoKβ include functionally equivalent options polynucleotides, pre-defined by their specific sequences. In the context of the present invention, the phrase "functionally equivalent polynucleotide" understand all such polynucleotide able to encode polypeptide with activity ChoKβ, which is pre-determined and which is obtained from the predefined polynucleotides by including, deletion or substitution of one or more nucleotide regarding pre certain sequences. Variant polynucleotide the present invention preferably are polynucleotide, the sequence of which allows them to gibridizatsiya in a very strict conditions with predefined polynucleotide. Specific rather strict conditions hybridization include incubation 6 x SSC (1 x SSC: 0,15M NaCl, 0,015M sodium citrate), and 40% formamide at 42 degrees C for 14 hours, followed by one or more cycles of washing using a 0.5 x SSC, the 0.1% SDS) at 60 C. Alternative, very strict conditions include hybridization at a temperature of about 50-55°C 6 x SSC and final washing at a temperature of 68°C 1-3 x SSC. Moderately strict conditions include hybridization at a temperature of about 50 C to approximately 65 C to 0.2 or 0,3M NaCl followed by washing at a temperature of about 50 C for about up to 55 C 0.2 x SSC, the 0.1% SDS (sodium dodecyl sulphate).

Preferably, if the agent is able to induce the activity ChoKβ, is polynucleotide, who promptly connection with the field of regulating expression. Regulatory sequences suitable for the present invention may constitute sequence nuclear promoters or, alternatively, enhancer sequences and/or other regulatory sequences that can increase the expression sequence heterologous nucleic acids. The promoter can be constitutive or induced. If desired constant expression of heterologous nucleic acid sequence, use the constitutive promoter. Examples of well-known constitutive promoters include predanniy the promoter of cytomegalovirus (CMV), a promoter of the rous sarcoma virus and such. A number of other examples constitutive promoters well known in this area and can be used in the practical application of the invention. If desired adjustable expression of heterologous nucleic acid sequence, you should use inducible promoter. In reingoldovna state inducible promoter "silent." The expression "silent" means that in the absence of the coil detects a small expression of heterologous nucleic acid sequence or no expression; however, in the presence of the inductor is the expression of heterologous nucleic acid sequence. Often the expression level can be adjusted by varying the concentration of the inductor. Regulate the expression, for example by varying the concentration of the inductor so that stronger or weaker stimulate inducible promoter, can affect the concentration of transcription product heterologous nucleic acid sequence. In the case of heterologous sequence of nucleic acid, gene encodes, you can adjust the amount of protein synthesized. Thus, it is possible to vary the concentration of therapeutic product. Examples of well-known inducible promoters are: estrogen-promoter or androgen-promoter, metallothioneins the promoter or a promoter that responds to Edison. In this area is well known for a number of other examples, and you can use them in practice the invention. In addition to the constitutive and inducible promoters (who usually work in widely diverse types of cells or tissues) can be used tissue-specific promoters to reach a specific expression of heterologous sequence of nucleic acid into cells or tissues. Well-known examples of tissue-specific promoters include some muscle-specific promoters, including: the promoter skeletal alpha-actin, the promoter heart actin, the promoter skeletal troponin C, the promoter heart/slow reduction troponin C, and the promoter/enhancer creatine kinase. There are a number of muscle-specific promoters, who are well known in this field and which can be used in the practical application of the invention (review the muscle-specific promoters, see Miller at al., (1993) Bioessays 15: 191-196).

In another embodiment, the agent inducing activity ChoKβ is a vector, including polynucleotide, which is described above, i.e. encoding ChoKβ or its functional equivalent option. Vectors suitable for such polynucleotides, are vectors obtained from the vectors of expression in prokaryotes, such as pUC18, pUC19, pBluescript and their derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phages and Shuttle vectors, such as pSA3 and pAT28, vectors expression in yeast, such as vectors of type 2-micron plasmids, integrative plasmids, YEP vectors, centromeric plasmids and such, vectors of expression in cells of insects, such as vectors series pAC and series pVL, vectors of expression in plants, such as vectors series pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE and the like, and the expression vectors in higher eukaryotic cells on the basis of viral vectors (adenoviruses, viruses associated with adenovirus, and retroviruses and, in particular, of lentiviruses)and non-viral vectors, such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.l/hyg pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDT1.

In another embodiment, the agent, inducing aktivnosti ChoKβ, is a cell that is able to secrete ChoKβ or its functional equivalent option in the environment. Cells suitable for expression ChoKβ or her functionally equivalent option, include, but are not limited to, cardiomyocytes, adipocytes, endothelial cells, epithelial cells, lymphocytes (B and T cells), Metacity, eosinophils, the cells of the inner lining of blood vessels, primary cell culture allocated from various bodies, preferably from cells isolated from the islets of Langerhans, hepatocytes, leukocytes, including menagerie, mesenchymal, umbilical cord or Mature cells (skin, lungs, kidneys, and liver), osteoclasts, chondrocytes and other cells of connective tissue. Suitable also are accepted cell lines, such as Jurkat T cells, NIH-3T3 cells, CHO, Cos, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, C2C12 the cultured myoblasts and cells W138.

The state of art in this area is known that some of the above polymers are not very stable and tend to lose the property gel, in addition to being a relatively porous, resulting antibody is able to enter into them and damage the cells. For these reasons, a particle of the invention may not necessarily be surrounded by a semipermeable membrane, letting the particle stability and forming a barrier is impervious to antibodies. Semi-permeable membrane believe membrane, which allows you to enter all dissolved substances that are necessary for the viability of the cells, and which allows having therapeutic proteins produced by cells contained within microparticles, but which is essentially impermeable to antibodies, thus, the cells are protected from immune response caused by living in the body of the microparticle. Materials suitable for the formation of a semi-permeable membrane, are materials not soluble in biological fluids, preferably polinikotinata, such as, for example, poly-L-lysine, poly-L-ornithine, poly-L-arginine, poly-L-asparagine, poly-L-aspartic acid, polyvinil-L-aspartate, poly-S-benzyl-L-cysteine, poly-gamma-benzyl-L-glutamate, poly-S-CBZ-L-cysteine, poly-e-CBZ-D-lysine, poly-Delta-CBZ-DL-ornithine, poly-O-CBZ-L-serine, poly-O-CBZ-D-tyrosine, poly(gamma-ethyl-L-glutamate), poly-D-glutamic acid, polyglucin, poly-g-N-hexyl-L-glutamate, poly-L-histidine, poly(alpha,beta-[N-(2-hydroxyethyl)-DL-aspartame]), poly-L-hydroxyproline, poly(alpha,beta-[N-(3-hydroxypropyl)-DL-aspartame]), poly-L-isoleucine, poly-L-leucine, poly-D-lysine, poly-L-phenylalanine, poly-L-Proline, poly-L-serine, poly-L-threonine, poly-DL-tryptophan, poly-D-tyrosine, or their combination.

In the context of the invention, the expression "treatment of cancer" means the combined introduction of the composition of the invention to prevent or delay the onset of symptoms, complications or biochemical signs of cancer or tumors to relieve symptoms or termination or inhibiting their development and progression, such as development of metastases. Processing can be preventive to delay the occurrence of the disease or prevent the symptoms of clinical or subclinical symptoms or therapeutic treatment for removing or alleviating symptoms after manifestations of the disease, or in conjunction with surgery or radiation.

Cancer treatment to be, in the context of the present invention can be any type of cancer or tumors. These tumors or cancer types include, but are not limited to, the hematologic cancers (such as leukemia or lymphoma), neurological tumours (for example, astrocytomas or glioblastoma), melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumours (for example, stomach cancer, pancreatic cancer or colorectal cancer), liver cancer (for example, hepatocellular carcinoma, renal cell cancer, urogenital tumours (for example, ovarian cancer, vaginal cancer, cervical cancer, bladder cancer, testicular cancer, prostate cancer), bone tumors and tumor vessels. Thus, in particular embodiment cancer, which is subject to treatment or prevention, is a cancer of the lung, breast, bladder or colorectal cancer.

In the context of the present invention under the "lung cancer" understand neoplastic disease of any type of lung tissue, including non-small cell lung cancer and NSCLC. In more concrete embodiment NSCLC selected from squamous cell carcinoma of the lung, both lung carcinoma and adenocarcinoma of the lung. In addition, this method is also applicable to a person who is suffering NSCLC at any stage (stage 0, IA, IB, IIA, IIB, IIIA, IIIB or IV).

In the context of the present invention, the term "breast cancer" is understood as any type of tumor lesions of the breast and he includes, but without restrictions duct carcinoma in situ (DCIS), infiltration or invasive carcinoma ductal, lobular carcinoma in situ (LCIS), infiltration or invasive lobular carcinoma and inflammatory carcinoma and tumor stage 0, I, II, IIIA, IIIB, IIIC and IV.

The expression "the cancer of a bladder bubble" refers to tumors of the urinary bladder and includes any subtype with histology, which usually occurs when cancer of the bladder, such as transitional cell carcinoma, squamous cell carcinoma and adenocarcinoma, any clinical subtypes, such as invasive cancer superficial muscles or metastatic disease and any TNM stage, including tumors T0-T4, N0-N4 and M0-M4.

Demonstrated that the composition of the invention is particularly effective in the treatment of tumors in which there are high levels of expression of ChoKα. Used in this specification, the expression "the high levels of expression of ChoKα" refers to the levels ChoKα above the levels observed for the reference sample. In particular, we can assume that the sample has high levels of expression of ChoKα, if the levels of expression exceed the levels specified reference sample of at least 1.1 times, 1.5 times, 5 times, 10 times, 20 times, 30 times 40 times, 50, 60, 70, 80 times, 90 times, 100 times, or even more.

The specified reference sample is usually obtained by combining an equal number of samples from the population of subjects. Typical reference samples are usually obtained from the entities that are clinically documented correctly and which do not have the disease. In these samples, you can define normal (reference) concentrations of biomarker, for example, receiving average concentration to control populations. When determining the reference concentration of the marker to take into account several factors. Such factors include the type of attracted sample (for example, fabric or CSF), age, weight, gender, General physical condition of the patient and kind. For example, an equal number in the group for at least 2 at least 10, at least, preferably more than 100 for 1,000 subjects, preferably classified by the above factors, for example, take as control group several age categories.

You can hold determination of the levels of expression of ChoKα as in the sample of the tumor, the treatment to be, and the reference sample, determining the levels of mRNA coded ChoKα, using well known techniques, such as RT-PCR analysis using protection RNA, Northern technique, in situ hybridization technique using Micronesia and the like, or by specifying the levels of protein ChoKα using for this purpose the well-known techniques like the Western blot turns or the Western blot, ELISA (adsorption enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme-linked immunosorbent assay), DAS-ELISA (sandwich method is a double antibody ELISA), immunocytochemical and immunohistochemical techniques, methods based on the use of biochips or micronorb proteins, including antibodies, or research, is based on the deposition of colloids, for example, in the format of strips.

Connection invention can be entered both in the form of sharp steps, and the forms of continuous action. Used in the present invention, the expression "permanent introduction" refers to the method of administration, in which connection is administered to the patient continuously for extended periods of time, to maintain therapeutic effect for a specified period. Form for continuous administration includes multiple dose compounds daily, twice a day, three times a day or less frequently. The continuous introduction can be done by several intravenous injection, administered periodically during the day. Alternative, permanent introduction includes an introduction bolus or through continuous transfusion that can be performed daily, every two days, every 3-15 days, every 10 days or more. Usually chronic introduction perform, at least, within 72 hours, at least 96 hours at least 120 hours, at least 144 hours, at least 1 week for at least 2 weeks, at least 3 weeks at least 4 weeks, at least 5 weeks at least 6 weeks at least 7 weeks at least 8 weeks at least 9 weeks at least 10 weeks at least 11 weeks at least 12 weeks at least 4 months, at least 5 months, at least 6 months at least 9 months, at least, years, at least 2 years or more.

Used in the present invention, the expression "acute introduction" refers to the method of administration, in which the patient is exposed to a single dose of connections or multiple doses, but for a reduced period of time, for example, 1, 2, 4, 6, 8, 12 or 24 hour or 2, 3 or 4 days.

The specialist in this area is clear that therapeutically effective amount and/or the drug is active connection is prepared depending on the type of introduction. Used in this specification, the expression "therapeutically effective amount" means the number of connections, which allows completely or partially to stop the tumor growth.

In case when it is desirable chronic introduction of connections according to the invention, you can enter it as a composition of a long-release, such as compositions, described in US 5672659, US 5595760, US 5821221, US 5916883 and WO 9938536. In contrast, if it is desirable acute introduction, it is preferable to use forms with the immediate-release. Regardless of the type of the introduction of the dosing amount and the interval can be adjusted individually, providing levels of compounds in plasma sufficient to maintain therapeutic effect. Specialist in this field are capable of optimizing a therapeutically effective local dose without undue experimentation.

Introduction compounds of the invention requires preparation in the form of pharmaceutical compositions, which constitute another aspect of the invention. Pharmaceutical compositions suitable for the practical application of the method according to the invention, contains a therapeutically efficient quantity of the active agent and a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable" means approved by the regulatory authority of the state or Federal government or included in the Pharmacopoeia the USA or other recognized Pharmacopoeia for use on animals, and more specifically on the people. The term "media" refers to the diluent, coadyuvante, excipiente or filler, which introduced a therapeutic compound. Such pharmaceutical media can be a sterile liquids such as water and oils, including mineral, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, saccharose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerin, propylene glycol, water, ethanol and such. If necessary, the composition can also contain a small amount of wetting agents or emulsifiers, or pH superyoshi agents. These songs can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, drugs slow release and the like. The composition can be prepared in the form of suppositories with traditional binders and substrates such as triglycerides. Oral drug can include standard media, such as mannitol, lactose, starch, magnesium stearate, Nachrichten, cellulose, magnesium carbonate pharmaceutical and other types Examples of appropriate pharmaceutical carriers described in "Remington's Pharmaceutical Sciences" by Martin E.W..

In the case when introduced nucleic acid (polynucleotide according to the invention, vectors or gene construct), the invention discusses pharmaceutical compositions, specially prepared for such a nucleic acids. The pharmaceutical composition may contain specified nucleic acids in unprotected form, i.e. in the absence of compounds that protect nucleic acids from their degradation under the action of nucleases of the body that includes the advantage of eliminating the toxicity associated with reagents used for transfection. Appropriate ways of introduction for unprotected connections include intravascular, intratumoral, intracranial, vnutribrjushinnye, vnutricletocny, intramuscular, subretinal, subcutaneous, through the mucous membrane, local and oral method (Templeton, 2002, DNA Cell Biol., 21:857-867). Alternative, nucleic acids can to enter as part of liposomes, associated with cholesterol or compounds capable promote translocation through cell membranes, such as Tat-peptide derived HIV-1 TAT protein, third spiral of homeodomain protein Antennapedia D.melanogaster, protein VP22 virus of a simple herpes (herpes simplex), oligomers arginine and peptides, such as peptides, described in WO07069090 (Lindgren, A. at al., 2000, Trends Pharmacol. Sci, 21: 99-103; Schwarze, S.R. at al., 2000, Trends Pharmacol. Sci., 21: 45-48; Lundberg, M at al., 2003, Mol. Therapy 8: 143-150 and Snyder, E.L. and Dowdy, S.F., 2004, Pharm. Res. 21: 389-393). Alternative, polynucleotide you can enter as part of a plasmid vector or viral vector, preferably vectors based adenoviruses, adeno-associated viruses and retroviruses, such as viruses on the basis of murine leukemia virus (MLV) or lentiviruses (HIV, FIV, EIAV).

The composition can be made up according to routine methods in the form of pharmaceutical compositions adapted for intravenous, subcutaneous or intramuscular person. If necessary, the composition can also include soljubilizatorami agent and a local anesthetic such as lidocaine, for the relief of pain at the injection. If the composition is injected through infiltration, it is possible to prepare and release with infiltration bottle, filled with water or physiological solution pharmaceutical quality. If the composition is administered through an injection, provide ampoules of water for injection or sterile saline, thus, the ingredients can be mixed immediately before the introduction.

Therapeutically effective dose for system introduction you can initially be determined from in vitro studies. For example, in animal models can be prepared dose to achieve blood levels in the range, including IC 50 which is defined in cell culture. Such information can be used for a more precise definition of the dose, applicable for people. The initial dose is also possible to determine from the data in vivo , for example, in animal models, applying techniques that are well known in this area. Specialist in this field can easily optimize introduction people on the basis of data for the animals.

Other aspects of the invention

Additional aspects of the invention refers to:

[1] the expression Vector enzyme ChoKβ for use in gene therapy method of inhibiting the enzyme ChoKα intended for the treatment of cancer.

[2] the expression Vector enzyme ChoKβ for use in gene therapy method of inhibiting the enzyme ChoKα intended for treatment of lung, breast, bladder or colorectal cancer.

[3] the expression Vector according to [1] or [2], wherein is the virus.

[4] the Use of the expression vector enzyme ChoKβ as agent that inhibits antioncogenes enzyme ChoKα.

[5] the Use of the expression vector enzyme ChoKβ when receiving songs for gene therapy, inhibiting the expression of enzymes ChoKα, intended for the treatment of cancer.

[6] the Application according to [5], wherein cancer is a cancer of the lung, breast, bladder or colorectal cancer.

[7] Application according to [4]-[6], wherein the vector is a virus.

[8] Song for gene therapy, wherein contains the expression vector enzyme ChoKβ able to inhibit the enzyme ChoKα.

[9] the Cell, transpirirovat expression vector enzyme ChoKβ, wherein has reduced the level of expression ChoKα and/or has the inability to grow or proliferate pathological way.

The invention is illustrated below by the following examples, which should be considered only as illustrative and in any case as limiting the amount of the invention.

EXAMPLES

Example 1

Application Chokβ as a tumor suppressor

1.1. Profile of expression genes ChoKα and ChoKβ in human cell lines

To determine whether there are differences in the expression of different isoforms ChoK in cancer, the present invention check the expression of endogenous mRNA ChoKα and ChoKβ in the set of cell lines derived from human breast tumors gland, urinary bladder, colorectal tumors and tumors of the lung, by quantitative PCR. Each type of tumor compare turns its relevant nontransgenic primary source lines as control.

The levels of mRNA ChoKα and ChoKβ different lines of small-cell and non-small cell lung cancer man is compared with the primary line of light (BEC). The results presented on figa show that in all tumor lines there is only the type of excessive expression ChoKα messenger compared with aging primary line. In addition, in the case isoforms ChoKβ see the opposite type, i.e. silencing the expression of this protein in tumor lines relative to a primary line. Similar results were obtained in tumor lines bladder, compared with normal line UROTsa (FIGU), in which levels ChoKα messenger overly expressed in tumor lines, while ChoKβ they keep silent.

Similar to the above mentioned cases in tumor lines derived from cancer of the breast of a man found elevated levels isoforms ChoKα relatively aging primary epithelial line (HMEC), while in the type of expression ChoKβ not found significant differences (figs).

These the results show that some tumor lines have a common characteristic increased expression of the gene ChoKα, while ChoKβ not affected, suggesting that high levels isoforms ChoKα essential for tumor process, but this is incorrect for levels ChoKβ that even downgraded some cases.

1.2. Profile of gene expression ChoKα and ChoK β in patient samples

In the same way as for the case of tumor lines person, determine the levels of expression of α and? isoforms ChoK in a series of 33 samples of patients diagnosed with lung cancer. For this purpose, define the levels ChoKα and ChoKβ by quantitative PCR, comparing them with commercial RNA normal lung tissue of a person as a reference.

The results of quantitative PCR reproduce the data previously obtained for cell lines, demonstrating the increasing more than twice the levels of expression of ChoKα in samples of tumors relative to normal tissue, corresponding to 39.4% (Figo). For the case isoforms ChoKβ (FIGU) receive lower levels of expression of tumor samples more than two times compared to normal tissue, corresponding to 66.7%, similar to the results found for cell lines.

The data obtained show that α and? isoforms ChoK have the opposite behavior in the conditions of transformation of cells, assuming different roles for two proteins in the carcinogenic process.

In addition, in the present invention assess the sensitivity ChoKβ to chemical inhibitor MN58b, making a conclusion that isoform ChoKα considerably more sensitive to the antiproliferative effect MN58b than isoform ChoKβ. As a result, the conditions under which the processing of that drug causes cell death, is concerned only isoform ChoKα. To verify whether or not this is a compensatory effect of ChoKα through ChoKβ, studying transcriptional response ChoKβ on pharmacological inhibition ChoKα through MN58b. To perform this study to take a set of human tumor cells of different origin, having effective in vitro response to antiproliferative effect processing MN58b, including Hek293T, Jurkat, H1299 and SW780. Cells treated with 20 microns MN58b (concentration at which inhibited ChoKα, but ChoKβ not affected to a significant extent) for 24 and 48 hours and control the effect of the medicinal product by immunodetection proteolysis PARP or degradation of caspase 3 as indicators cell death (figure 3). In addition, through quantitative PCR determine the levels ChoKβ person. As shown in figure 4, in all cases there is an increased levels ChoKβ in response to MN58b, although the maximum time of induction varies for each cell line.

These results suggest that deals with the regulation of both isoforms ChoK, with ChoKβ transcription is induced in response to pharmacological inhibition ChoKα.

1.4. The regulatory role ChoK β in transformation of mediated ChoKα

Excessive expression ChoKα, but not excessive expression ChoKβ, induces the transformation of the human cells Hek293T. In addition, various cell lines derived from human tumors and samples of patients with lung cancer have high levels of mRNA ChoKα and lowered levels of mRNA ChoKβ relative to normal controls. This implies different but related behavior both isoforms in the process of transformation of cells.

To study the possible combined regulation isoforms ChoK analyze the intracellular levels PCho and PEtn, generated by excessive expression of both isoforms together. To this end transferout cells Hek293T through empty vector pCDNA3b as control and vectors of expression, coding ChoKα, ChoKβ or both together, and mortality in vitro when the balance of 14 C-choline or 14 C-ethanolamine. The results, shown in figure 5, confirm that while ChoKα capable promote high levels PCho and PEtn, ChoKβ preferably involved in the induction levels PEtn. In case the total excessive expression of both isoforms there is an increase in the intracellular levels PEtn higher levels obtained with each of the two isoforms separately. However, the levels PCho fall relative to the levels obtained at excessive expression ChoKα, but still higher than the control. According to these results, as for the properties of dimerization of different isoforms ChoK forming α/alpha, beta/β homodimer or α/β heterodimer, previously demonstrated that are generated by different degrees of activity ChoK, with the most active dimers are dimers formed α/α molecules, and the least active are β/β dimers, heterodimer remain intermediate phenotype (Aoyama at al., 2004).

In addition, increased levels PCho plays an important role in mitogenesis, cell proliferation and carcinogenesis. Therefore, the reduction of intracellular levels PCho caused by excessive expression ChoKβ, may have an impact on the transformation of mediated ChoKα. To determine the importance of this effect cells Hek293T short transferout, as described above, and in each case check once exact excessive ectopic expression ChoK, administered through an injection 106 cells subcutaneously in each side Nude mice nu/nu- (n=10-12). Mice injected through the injection of cells Hek293T, transfetsirovannyh generated ChoKα tumor extent 25% similar degree previously obtained, taking into account that the mouse injection ChoKβ not produce tumors, being identical to the control (6). Suddenly, in the case of cells, together transfected with both isoforms is a General decline in the rate of occurrence of tumors.

In addition, tumors generated in immunocompromised mice at excessive expression ChoKα in similar conditions, surgically remove, then their cells ekspluatiruet, adapting them in culture. This cell line called ADJ has a constitutive activation ChoKα and causes neoplasms in the immunocompromised mice that recently described (Ramirez de Molina at al., 2008a). To confirm a negative effect ChoKβ on transformational ability ChoKα transferout cells ADJ expression vector ChoKβ or an empty vector pCDNA3b in quality control, after which they are administered through injections Nude mice, as described above, watching the growth of a tumor within 6.5 weeks. In the case of cells ADJ/ChoKβ generated tumor volume, that on 73% less than for the control cells ADJ, transfected with blank vector (Fig.7).

To study the effect ChoKβ on cells that have been transformed by ChoKα more investigated in vitro proliferative ability of cells ADJ, transfected ChoKβ or an empty vector pCDNA3b. Experiment with the proliferation conducted in time, staining cells crystal violet. According to the results obtained in vivo in Nude mice, the cells ADJ, transfetsirovannyh ChoKβ reflect a significant slowdown proliferation relative to the control cells after 96 hours maintenance of normal conditions of cultivation (Fig). Taken together, the results suggest that ChoKβ plays uncompressing role in the transformation of mediated ChoKα.

2. Application ChoK β and the ratio ChoKα/ChoK β as a marker of forecasting in patients with cancer

2.1. Materials and methods

Patients included in the study

Researched frozen samples of lung cancer tissue from 69 randomly selected patients who underwent surgical resection NSCLC in the University hospital of La Paz in Madrid (Spain) in the period from 2001 to 2007. Of these 69 39 samples are squamous cell carcinomas, 12 - adenocarcinoma and 17 - cancer of another type. Clinical characteristics of patients included in the study, summarized in table 1.

Statistical analysis

Expect to quantify gene expression (AQ) method 2-ΔCt (Applied Biosystems) and are as AQ x 106. Analysis of gene expression is done using the expression of an endogenous gene 18S for normalization.

Get the characteristic performance curves (ROC) to illustrate the relationship between sensitivity and false positive assessment at different thresholds expression ChoKβ for survival, relatively specific lung cancer and survival without relapse. The threshold value set by the best combination of sensitivity and false positive evaluations (1-specificity) on the basis of ROC curves.

To determine overall survival and survival without relapse apply the method of Kaplan-Meier. The study considered only death from lung cancer recurrence. Evaluate the effect of various factors on related to tumor recurrence and survival through the log-rank test for univariate analysis. To evaluate the effect of expression ChoKβ survival when adjusted for potentially interfering factors applied regression model of proportionality risk of Coke. From the regression model Coke calculated risk ratios (HR) and 95% confidence intervals (95% CI). All reported p values are two-sided. Statistical significance was defined as p<0.05. Statistical analysis performed using software SPSS (version 14.0).

Characteristics of patients included in the study

43-85 years (average 66)

61 (88,4%)

8 (11,6%)

Histology

Squamous cell carcinoma

39 (56,5%)

Adenocarcinoma

12 (17,4%)

17 (26,1%)

6 (8,7%)

32 (46,4%)

2 (2,9%)

11 (15,9%)

7 (10,1%)

48 (69,6%)

There are

17 (24,6%)

Not known

4 (5,8%)

2.2. Prognostic value of the expression ChoKβ in NSCLC

To study whether the expression ChoKβ with the clinical outcome of patients with NSCLC, analyze gene expression ChoKβ in 69 surgical NSCLC samples, using RT-PCR in real time. Analysis of gene expression shows that the expression of ChoKβ distributed in tumors in a differentiated way with the normalized values AQ mRNA copies in the range from 0.42 to 30,81 (Fig.9).

To install, how is the expression of ChoKβ in samples of tumors compared with healthy tissues, analyze the expression of ChoKβ in commercial RNA obtained from the lung tissue of a healthy person. Most of tumor samples demonstrate lower levels of expression in comparison with the levels for commercial normal tissue used as a reference (normal tissue has AQ expression 13,89).

Not found the ratio of expression ChoKβ available with clinicopathological parameters patients (stage, histological evaluation, age or gender). To analyze whether the reduced expression of ChoKβ observed in most cancers, with clinical outcome of patients, set an arbitrary threshold point 4,022 AQ (70% sensitivity, 54% specificity) according to the ROC methodology. In these conditions 35 of 69 (51%) of the samples tumors analyzed on the expression ChoK, are below this threshold point.

Patients with decreased expression of ChoKβ post poorer survival in lung cancer and survival without relapse rates than patients with higher concentrations of this enzyme, although these differences did not reach statistical significance (figure 10).

Taken together, the results suggest that the expression of ChoKβ closely connected with the survival without relapse and overall survival among patients with NSCLC. Multiple regression analysis Coke assumes that ChoKβ may be an independent factor of high risk bad survival for patients with decreased expression of ChoKβ (HR 0,38 [95% CI: 0,13-1,11], p=0.07). Thus, ChoKβ can be a new prognostic factor, which can be used to assist in the identification of patients with early stage NSCLC who may be at high risk of relapse, and to identify patients with a favorable prognosis, which could take a less aggressive treatment option or avoid additional system of treatment.

2.3. A combined analysis of gene ChoKα and ChoK β as a powerful tool for prognosis of NSCLC

In TCD Pharma previously been demonstrated that ChoKα plays a significant role in lung cancer, coming to the conclusion that over-expression of this enzyme is an independent predictive factor survival without relapse and survival, relatively specific lung cancer, in patients with early stage NSCLC (Ramirez de Molina, A. at al., Lancet Oncol 8, 889-97 (2007). It demonstrated the predictive value of the expression ChoKβ in tumor samples from patients with NSCLC. These results are strictly suggest that low expression ChoKβ associated with a worse clinical outcome for patients with early stage disease.

On the basis of these initial discoveries high expression ChoKα and low expression of ChoKβ defined as adverse factors associated with poor survival. Indeed, found that patients with low ChoKα and high ChoKβ levels of expression of exhibit more long survival, relatively specific lung cancer and survival without relapse, whereas patients with high levels ChoKα and simultaneously low levels ChoKβ demonstrate a shorter survival (p=0,19 for the overall survival p=0,099 for survival without relapse) (Fig).

The output from analysis Kaplan-Meier estimates for the other two groups combinations: for these two groups was observed that low levels of expression of ChoKα and simultaneously high levels of expression of ChoKβ and high levels of expression of ChoKα and simultaneously high levels of expression of ChoKβ demonstrate intermediate behavior and no difference in survival (Fig).

According multiple regression analysis Coke patients with high levels ChoKα and simultaneously low levels ChoKβ show a significant tendency for specific death from lung cancer (HR 7,8 (95% CI, 0,98-67,5); p=0,06) and with a cancer recurrence (HR 10,5 (95% CI, 1,22-90,0); p=0.03). These results are strictly show that the combined effect of expression of both isoforms ChoK may be the best prognostic factor than individually.

2.4. Conclusions

Determination of gene expression ChoKβ can predict clinical outcome in patients with NSCLC. This profile of expression may be useful for improving clinical care for patients with NSCLC. In addition, the results presented in this message, suggest that the combined effect of both isoforms ChoK provides a powerful tool for identifying patients at high risk of recurrence and death from lung cancer for patients with early stage NSCLC.

3. Application PEMT and/or ChoK β as a marker of response to treatment inhibitors ChoKα

3.1. Phosphatidylethanolamines (PEMT): functional relationship between ChoKα and ChoK β

In mammals, one of the points of the metabolic link between the two branches of the way Kennedy to generate PE and PC is the enzyme phosphatidylethanolamines (PEMT). This enzyme converts PE PC through two consecutive metilirovanie (Vance & Ridgway, 1988). Described that this enzyme has significant activity only in the liver cells, and its contribution is 30% of the total PC cells (DeLong at al., 1999; Li at al., 2005; Reo 35 at al., 2002; Sundler & Akesson, 1975). To determine involved whether this enzyme in cross-activity ChoKα and ChoKβ, determine the nature of gene expression PEMT in response to the processing MN58b in different cellular systems. To this end, the cells Hek293T, Jurkat, H1299 and SW780 treated with 20 microns MN58b and define the expression PEMT by quantitative PCR. The results are summarized in Fig where observe that in all cases there is an increased levels PEMT mRNA in response to specific inhibition ChoKα through MN58b.

In addition, as described above, processing through MN58b also induces excessive expression ChoKβ at the transcriptional level. To determine whether these effects, analyzed the levels of expression of PEMT by quantitative PCR in cells transfected expression vector ChoKβ, relatively cells transfected with blank vector control. As you can observe on Fig, in the cells, overly expressing ChoKβ, is inducing the expression PEMT, indicating that simple over-expression of this isoform is sufficient to cause the transcriptional induction PEMT.

1. The method for determining the prognosis for a patient suffering from cancer, which includes the determination of the levels of expression of ChoKβ in the sample from the patient, where lower levels ChoKβ relative to the levels in the reference sample indicate that the patient demonstrates bad forecast where the cancer is a NSCLC and where the forecast is determined by identifying the option selected from the group consisting of survival and survival without relapse.

2. The method according to claim 1, where cancer has high levels of expression of ChoKα.

3. The method according to claim 1 or 2, where the definition of the levels of expression of ChoKβ or levels of expression of ChoKα performed by determining the levels of mRNA encoding this protein.

4. The method for determining the prognosis for a patient suffering from cancer, which includes the determination of the levels of expression of ChoKα and ChoKβ in the sample from specified patient, where lower levels ChoKα and high levels ChoKβ relative to the levels of expression of these proteins in the reference sample indicate that the patient shows a good prognosis where the cancer is a NSCLC and where the forecast is determined by the parameter definition, selected from the group consisting of survival and survival without relapse.

5. The method according to claim 4, where cancer has high levels of expression of ChoKα.

6. The method according to claim 4, where the definition of the levels of expression of ChoKβ or levels of expression of ChoKα performed by determining the levels of mRNA encoding this protein.