Pox viral oncolytic vectors

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, virology and medicine. The method provides administering a pox virus containing the defect F2L gene into a host body or a cell. What is also described is using this pox virus for producing a drug preparation for treating proliferative diseases or diseases accompanied by osteoclast hyperactivity. The invention can be used in medicine.

EFFECT: what is presented is the method of treating proliferative diseases or diseases accompanied by osteoclast hyperactivity.

28 cl, 10 dwg, 3 tbl

 

Area of technology

Oncolytic viruses represent a new class of therapeutic agents used to treat cancer, has the unique ability to tumor-dependent sustainment (HERMISTON. A demand for next-generation oncolytic adenoviruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.322-30). Oncolytic viruses capable of selective replication in malignant cells and therefore offer levels of potency and specificity, which are potentially much larger than standard anti-cancer therapy (FISHER. Striking out at disseminated metastases: the systemic delivery of oncolytic viruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.301-13). The advantage of using these viruses is that when they replicate lisarow their host cells. Cancer cells are the ideal hosts for many viruses, because they inactivated antiviral interferon pathway or motroway genes-tumor suppressors that allow easily occur viral replication (CHERNAJOVSKY et al. Fighting cancer with oncolytic viruses. The British medical journal. 2006, vol.332, no.7534, p.170-2).

Some viruses are by nature able to selectively replicate in tumor cells, oncolytic viruses but can also be obtained by modification of natural viruses. With this goal, the main strategies currently used for modification of viruses include: functional deletions in su�significant viral genes; the tumor - or tissue-specific promoter used to control the expression of these viral genes; and the modification of the tropism to redirect adenovirus to the surface of the cancer cell. In the near future it is necessary to optimize oncolytic adenoviruses, in order to fully realize their potential as a critical anti-cancer tools and thus improve the prognosis for patients with malignant gliomas (JIANG, et al. Oncolytic adenoviruses as antiglioma agents. Expert review of anticancer therapy. 2006, vol.6, No. 5, p.697-708).

For example, ONYX-015, a selectively modified adenovirus to replicate and destroy cells that have p53 mutations is developed by company Onyx Pharmaceuticals for potential treatment of various solid tumors, including tumors of the head and neck, gastrointestinal and pancreatic tumors. He is a recombinant adenovirus that carries the mutation "loss of function" in the E1B locus, the product of which is a 55 kDa protein that binds to p53 tumor protein suppressor and inactivates it. Thus, the adenovirus ONYX-015, as expected, affects normal cells. Mutations in the p53 gene is a tumor suppressor gene are the most common type of genetic pathology in cancer, which occurs in more than half of all major types of cancer. Thus, these cell� susceptible to the virus which will be easy to replicate and cause cell death. Research continues into phase III of the application of ONYX-015 for treatment of recurrent head and neck cancer, phase II study - the treatment of colorectal tumors, tumors of the ovary, pancreas and oral cavity, and studies phases I - concerning diseases of the digestive system, tumors of the esophagus and liver (COHEN, et al. ONYX-015. Onyx Pharmaceuticals. Current opinion in investigational drugs. 2001, vol.2, no.12, p.1770-5).

Natural oncolytic viruses are replication-competent viruses possessing an innate ability to selectively infect and destroy tumor cells. Despite the fact that they were used in the original attempts at curing cancer live virus five decades ago, interest in natural oncolytic viruses has lagged behind support genetic engineering of adenovirus and herpes viruses as cancer therapy. However, recently there has been renewed interest in high potency and selectivity of these natural agents (ROBERTS, et al. Naturally oncolytic viruses. Current opinion in molecular therapeutics. 2006, vol.8, no.4, p.314-21).

Among natural oncolytic viruses, the vaccinia virus (Poxviridae) possess many of the key features necessary for a perfect viral frame for use in oncolytic virotherapy. They include the short period polywave�t, with the rapid intercellular spread, strong analytical ability, strong ability to precise molecular cloning and biology. In addition, although they are capable of replication in human cells, they are not considered a natural health problem and is especially well characterized by the fact that they were introduced to millions of people during the campaign for the destruction of smallpox. Early clinical results using or vaccine strains, or genetically modified strains of vaccinia have demonstrated antitumor effects (THORNE, et al. Vaccinia virus and oncolytic virotherapy of cancer. Current opinion in molecular therapeutics. 2005, vol.7, No. 4, p.359-65).

On the contrary, poxvirus myxoma is a new oncolytic candidate who has no history of direct use in humans because it has a distinctive and absolute tropism for referring the owner to Lagomorpha (rabbits). The myxoma virus, as recently shown, may also selectively infect and destroy human tumor cells, a unique tropism associated with deregulirovannym intracellular signalling pathways found in most human cancers. This review outlines the existing data regarding the tropism of myxoma virus to human cancer cells, as well as preclinical data, pokazivaushee� its ability to infect and destroy tumors in animal models of cancer (STANFORD, et al. Myxoma virus and oncolytic virotherapy: a new biologic weapon in the war against cancer. Expert opinion on biological therapy. 2007, vol.7, No. 9, p.1415-25).

Technical problem

Injection of high doses of poxviruses required to achieve antitumor effect have caused problems related to toxicity. Most adverse events are minor adverse reactions, which are usually associated with vaccinia virus are limited and include fever, headache, fatigue, myalgia, chills, local skin reactions, nonspecific rash, erythema multiform, swollen lymph nodes and pain at the site of inoculation. Other reactions might require additional therapies (e.g., VIG, first-line therapy and cidofovir, second line therapy). Adverse reactions that might require further evaluation or therapy, include inadvertent inoculation, generalized vaccinia (smallpox) (GV), eczema after vaccination (EV), progressive vaccinia (smallpox (PV), post-vaccinal disease of the Central nervous system and fetal vaccinia (smallpox) (CONO et al. Smallpox vaccination and adverse reactions. Guidance for clinicians. MMWR. Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports / Centers for Disease Control. 2003, vol.52, No. RR-4, p.1-28).

Thus, there is a need for safer poxviruses with the same oncolytic activity, just as their finely�x copies.

Background of the invention

In US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15/11/1994 describe a modified recombinant poxvirus, particularly a vaccinia virus having inactivated nonessential encoded by the virus genetic functions so that the recombinant poxvirus had reduced toxicity and enhanced security. In particular, genetic functions have been inactivated by deletion of the open reading frame encoding a virulence factor, or insertional inactivation of the open reading frame encoding a virulence factor. In more detail, this patent describes a vaccinia virus in which the open reading frame for J2R, B13R+B14R, A26L, A56R, C7L - K1L, and I4L had been inactivated. This virus (NYVAC) can be designed as a vector for alien nucleic acid and be used as a vaccine for inducing an immunological response in the animal host. However, NYVAC unable to effectively replicate in most mammalian cells and cannot be used as an oncolytic virus (XIANGZHI, et al. Vaccinia virus K1L protein supports viral replication in human and rabbit cells through a cell-type-specific set of its ankyrin repeat residues that are distinct from its binding site for ACAP2. Journal of virology. 2006, vol.353, No. 1, p.220-233.).

WO 2004/014314 (DAVID KIRN (US)) 19/02/2004 describes a modified vaccinia virus, which comprises one or more mu�tions in the viral genome. Described mutations are in one or more of the following classes of polypeptides: 1) interferon-modulating polypeptide; 2) a complement control polypeptide; 3) TNF or chemokine-modulating polypeptide; 4) serine protease inhibitor; 5) IL-1p-modulating polypeptide; 6) non-infectious EEV form polypeptide; and 7) a viral polypeptide that acts to inhibit release of infectious virus from cells (anti-infectious virus form polypeptide). In addition, also disclosed mutations in the vaccinia virus A41L or C11R.

Portions of the vaccinia genome, such as A34R, A41L, A53R, B5R, B7R, B8R, B13R, B15R, B18R, B22R, B28R, B29R, CUR, E3L, K2L, N1L, vC12L, vCKBP described in more detail in this application. The methods of the invention involve the use of any of poxviruses discussed by the authors. The inventors also disclose methods for treating cancer by introducing into the cancer cell or patient an effective amount of the modified vaccinia virus.

Disclosure of the invention

The inventors surprisingly discovered that poxviruses comprising a defective F2L gene, have an improved safety profile, but you keep the equivalent of oncolytic activity (compared to their natural copy).

The present invention relates to poxvirus comprising a defective F2L gene.

As used throughout the application the terms in the only� the number used in the sense of what do they mean "at least one", "first", "one or more" or "many" components, or steps referenced, unless the context clearly indicates otherwise. For example, the term "cell" includes a variety of cells, including mixtures thereof.

The term "and/or" used by the authors, includes the meaning "and", "or" and "all or any other combination of items associated with the specified term.

The term "about" or "approximately" used by the authors, means within 20%, preferably within 10% and more preferably within 5% of a given value or range.

Authors use the terms "including" and "include" mean that the products, compositions and methods include components or stages that are referenced, but exclude others. "Consisting essentially of" when used to define products, compositions and methods, shall mean excluding other components or steps of any essential significance. Thus, a composition consisting essentially of these components, does not exclude the pollutants in trace amounts and pharmaceutically acceptable carriers. "Consisting of" shall mean excluding more than the elements in trace amounts of other components or stages.

Authors use the term "poxvirus, including a defective gene" �tositsa to poxvirus, comprising deletions, substitutions or additions of one or more nucleic acids of the defective gene, or any combination of these possibilities, and these modifications lead to the inability for the virus to produce a protein having the activity of a protein produced by the unmodified genome. In a preferred embodiment, poxvirus, including a defective gene, refers to poxvirus, which was removed entire gene sequence. Mutation can be implemented in many ways, known qualified specialists in the art using recombinant methods. Methods for modifying the genome of poxvirus available in this area. For example, methods disclosed in MCCART, et al. Systemic cancer therapy with a tumor selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res. 2001, no.61, p.8751-57., KIM, et al. Systemic armed oncolytic ans immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF. Molecular Therapeutic. 2006, no.14, p.361-70, WO 2004/014314 (DAVID KIRN (US)) 19/02/2004 and US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15/11/1994 can be used to produce poxvirus of the invention. The methods disclosed in the example of this application, in particular to obtain poxvirus according to the invention. The sequence of the genome of different poxviruses are available in this area, for example, the genomes of vaccinia virus, vaccinia virus, Canarypox virus, Ectromelia virus, VI�USA myxoma, available in Genbank (accession number NC_006998, NC_003663, NC_005309, NC_004105, NC_001132, respectively).

Authors use the term "poxvirus" refers to a virus belonging to the family Poxviridae. According to a preferred embodiment of the poxvirus according to the invention belongs to the subfamily Chordopoxvirinae, more preferably an orthopoxvirus genus and even more preferably to the species Vaccinia virus.

For example, can be used strains of vaccinia virus Dairen I, IHD-J, L-IPV, LC16M8, LC16MO, Lister, LIVP, Tashkent, WR 65-16, Wyeth, Ankara, Copenhagen, Tian Tan and WR. According to a particularly preferred embodiment of the poxvirus according to the invention represents the strains of vaccinia virus Copenhagen. Poxvirus vaccinia contains a large double genome DNA (187 pairs of cilaisnowbuy) and is the only known member of a family of DNA viruses that replicate in the cytoplasm of infected cells. Because infected cell must supply the precursors of DNA in the cytoplasmic sites of replication, the virus encodes and expresses a lot of the enzymatic activities required for the metabolism and synthesis of DNA, including deoxyuridine 5'-triphosphate-nucleotidases (dUTPase).

Deoxyuridine 5'-triphosphorylated (dUTPase, EC 3.6.1.23) catalyzes hydrolysis of dUTP to dUMP and pyrophosphate ions in the presence of Mg (2+). dUTP�, when you delete from a pool of dUTP dNTP and production dUMP, involved in maintaining the devotion of DNA replication and in the provision of the predecessor for the production of the TSR timidilatsintazei. dUTP vaccinia is a 15-kDa protein, encoded by the genome F2L (MCGEOGH. Nucleic Acids Research. 1990, no.18, p.4105-10; BROYLES. Virology. 1993, no.195, p.863-5). The sequence F2L gene of vaccinia virus is available in gene Bank by stock number M, sequence and location F2L gene in different genomes of poxviruses also available in gene Bank, for example, by stock number NC_006998, DQ121394, NC_001611, AY689436, AY689437, NC_008291, DQ437594, DQ437593, AY313847, AY313848, NC_006966, NC_005309, NC_003391, NC_003389, NC_001132, NC_003310, NC_002188, M35027, AY243312, AF170726, DQ011157, DQ011156, DQ011155, DQ011154, DQ011153, X94355, Y16780, AY318871, U94848, AF198100 and M34368.

Gene nomenclature used in this description, is the nomenclature of Copenhagen strain vaccinia and is also used for homologous genes of other poxviridae, unless specified otherwise. However, gene nomenclature may differ depending on the strain of smallpox. For information, the correspondence between genes Copenhagen and MVA, see Table I ANTOINE. Virology. 1998, no.244, p. 365-396.

According to a preferred embodiment of the poxvirus of this invention additionally includes a defective J2R gene.

J2R gene encodes timedancing (TC), which are part of the "reutilization" route for the synthesis of d�of oxyribonucleotides pyrimidine. The reaction catalyzed by TC, involves the transfer of the γ - phosphorylated from APR 2 desoxy - thymidine (dThd) to produce thymidine 5'-monophosphate (dTMP). TC of vaccinia virus is the type 2. TC type 2 have a smaller polypeptide chain compared to type 1, ~25 kDa, but the form homotetramer. They are sensitive to inhibitors of feedback or dTDP dTTP, which are generated at the end of a metabolic pathway. The TC type 2 is more narrow specificity to the substrate compared to the TC type 1 and only phosphorylate 2 deoxyuridine (dU) and/or dThd (EL OMARI et al. Structure of vaccinia virus thymidine kinase in complex with dTTP: insights for drug design. BMC structural biology. 2006, no.6, p.22).

The poxviruses, defective at the site J2R, and methods for their preparation are available in the art. For example, management MCCART, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. cancer research. 2001, vol.61, No. 24, p.8751-7, PUHLMANN et al. Vaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutant. Cancer gene therapy. 2000, vol.7, No. 1, p.66-73, GNANT, et al. Systemic administration of a recombinant vaccinia virus expressing the cytosine deaminase gene and subsequent treatment with 5-fluorocytosine leads to tumor-specific gene expression and prolongation of survival in mice. Cancer Research. 1999, vol.59, No. 14, p.3396-403 can be used to obtain poxviruses with the deletion of section J2R.

According to a preferred embodiment of poxvirus according to the invention additionally includes a target nucleic acid.

In a preferred embodiment the present�in termination of the target nucleic acid contains at least one target sequence, encoding a gene product that is a therapeutic molecule (i.e., a therapeutic gene).

"Therapeutic molecule" is a molecule with pharmacological or protective activity when properly administered to a patient, especially a patient suffering from the disease condition or disease or someone that should be protected from the disease or condition. Such pharmacological or protective activity represents the activity that is expected to be associated with favorable effects on stroke or a symptom of a specified disease or specified condition. When a qualified person chooses during the existing of the invention the gene encoding a therapeutic molecule, it generally binds your choice with the previously obtained results and can reasonably be expected, without undue experiment, except for carrying out the invention according to the formula, to get this pharmacological property. According to the invention, the target sequence may be homologous or heterologous Milenium cells, in which it is entered. Preferably, the target sequence encodes all or part of a polypeptide, especially a therapeutic or prophylactic polypeptide, which gives a therapeutic or prophylactic property. Polypeptide, as you know, is Liu�th translation product of a polynucleotide regardless of size, and regardless of glycosylation, and includes peptides and proteins. Therapeutic polypeptides include as a primary example, those polypeptides that can compensate for defective or imperfect proteins in the animal or human body, or those that act through toxic effects to limit or remove harmful cells from the body. They may also be giving immunet polypeptides that act as endogenous antigen to induce a humoral or cellular response, or both.

Examples of polypeptides encoded therapeutic gene include genes encoding a cytokine (alpha, beta or gamma interferon, interleukin, particularly IL-2, IL-6, IL-10 or IL-12, tumor necrosis factor (TNF), the normal stimulating factor GM-CSF, C-CSF, M-CSF...), immunostimulatory polypeptide (V7.1, V7.2, etc.), the coagulation factor (FVIII, FIX...), growth factors (transforming growth factor TGF, fibroblast growth factor FGF, etc.), an enzyme (urease, renin, thrombin, metalloproteinase, synthase of nitric oxide NOS, SOD, catalase...), enzyme inhibitor (alpha 1-antitrypsin, antithrombin III, viral protease inhibitor, an inhibitor of plasminogen activator PAI-1), CFTR (regulator transmembrane conductance cystic fibrosis) protein, insulin, dystrophy, antigen MHC class I or II, polypeptide, which can modulate/regulation�ü the expression of cellular genes, polypeptide capable of inhibiting a bacterial, parasitic or viral infection or its development (antigenic polypeptides, antigenic epitopes, transdominant variants inhibiting the action of a native protein by competition....), an inducer or inhibitor of apoptosis (Bax, Bc12, BclX...), cytotoxic agent (P21, P16, Rb...), an apolipoprotein (ApoAI, ApoAIV, APOE genes...), an inhibitor of angiogenesis (angiostatin, endostatin...), angiogenic polypeptide (family of vascular endothelial growth factors VEGV, the FGF family, the family of CCN, including CTGF, Cyr61 and Nov), a scavenger of oxygen radicals, the polypeptide having an antitumor effect, an antibody, a toxin, immunotoxin and marker (beta-galactosidase, luciferase....) or any other target genes recognized in the art as useful for the treatment or prevention of a clinical condition.

Suitable antitumor genes include, among others, genes that encode genes-tumor suppressors (e.g., Rb, p53, DCC, NF-1, Wilms tumor, NM23, BRUSH-1, p16, p21, p56, p73 and their respective mutants), the products of suicidal gene, antibodies, polypeptide, inhibiting cell division or signal transduction.

According to a particularly preferred embodiment of poxvirus present invention further comprises a suicide gene.

Suicidal gene refers to a gene, Cody�following protein able to convert a precursor of a drug into a cytotoxic compound.

Suicide genes include, among others, genes encoding a protein having a cytosine-desaminase activity, thymidine kinase activity, uracil-phosphoribosyl transfersno activity, polynucleotide-phosphorylase activity and/or thymidylate-kinase activity.

Examples of suicide genes and the respective predecessors of the medicinal product, including one the rest of nucleobase, are disclosed in the following table:

Table 1
Suicidal geneA prodrug
TimedancingGanciclovir;
ester of ganciclovir Sadovoy acid;
penciclovir;
Suicidal geneA prodrug
acyclovir;
valacyclovir;
(E)-5-(2-bromovinyl)-2'-deoxyuridine;
zidovudine;
2'-Exo-metanarrative
Sitoindosides5-Fertilizin
Purine nucleoside phosphorylase6-methylpyridoxine;
fludarabine
Braceletsbracelets5-Fertilizin;
5-florouracil
TimetrackingAzidothymidine

According to a preferred embodiment of the invention, the suicide gene encodes a protein that has at least the activity CD. CD involved in pyrimidine metabolic pathway by which exogenous cytosine is converted to uracil by hydrolytic deamination. While activity D have been demonstrated in prokaryotes and lower eukaryotes (JUND, et al.. Journal of Bacteriology. 1970, no.102, p.607-15; BECK, et al. Journal of Bacteriology. 1972, no.110, p.219-28; HOEPRICH, et al. Journal of Infectious Diseases. 1974, no.130, p.112-18; ESDERS, et al. J. biol. chem. 1985, no.260, p.3915-22), they are not present in mammals (KOECHLIN, et al. Biochemical pharmacology. 1966, no.15, p.435-46; POLAK, et al. Chemotherapy. 1976, no.22, p.137-53).

CD will also desaminase analogue of cytosine, i.e. 5-fertilizin (5-FC), thus forming 5-florouracil (5-FU), which is a compound, which is very cytotoxic �ri its conversion to 5-fluoro-UMP (5-FUMP). Cells that are not actively CD, or due to a mutation that inactivates the gene encoding the enzyme, or because they have no natural this enzyme, like mammalian cells, are resistant to 5-FC (JUND et al. Journal of Bacteriology. 1970, no.102, p.607-15; KILLSTRUP, et al. Journal of Bacteriology. 1989, no.171, p.2124-2127). Unlike mammalian cells, which have migrated sequence encoding activity D, became sensitive to 5-FC (HUBER, et al. Cancer Research. 1993, no.53, p.4619-4626; MULLEN, et al. Proceedings of the National Academy of Sciences of the United States of America. 1992, no.89, p.33-37; WO 93/01281 (US HEALTH)). In addition, neighboring untransformed cells also become sensitive to 5-FC (HUBER, et al. Proceedings of the National Academy of Sciences of the United States of America. 1994, no.91, p.8302-6). This phenomenon is called the effect of "witness", is due to cells that Express the activity CD secreting 5-FU, which intoxicated neighboring cells by direct diffusion through the plasma membrane. This property is 5-FU relative to passive diffusion is the advantage compared to tk/GCV reference system in which the effect of the "witness" requires contact with cells that Express tk (MESNIL et al. Proceedings of the National Academy of Sciences of the United States of America. 1996, no.93, p.1831-35). All the benefits CD offers within the context of gene therapy, particularly anti-cancer gene therapy, m�gut so to be easily understood.

Genes codA Saccharomyces cerevisiae (S. cerevisiae) FCY1, FCA1 of Candida Albicans and E. coli, which respectively encode CD these two organisms, are known and their sequences have been published (SEQ ID N°: 4; SEQ ID N°: 5; SEQ ID N°: 6, respectively).

In this respect, according to a more preferred variant of embodiment of the present invention the gene encoding the protein having the activity CD is FCY1, FCA1 or CodA or their equivalent. Analogs of these genes affect gene having a nucleic acid sequence, which has a degree of identity of at least more than 70%, preferably more than 80%, preferably more than 90% and most preferably more than 95% with the nucleic acid sequence of the parent gene.

Patent WO 2005/007857 describes the gene encoding the protein having improved activity CD. These polypeptides obtained from native CD by supplementing the amino acid sequence. According to another preferred variant of embodiment of the present invention is a protein having the activity CD, is a polypeptide, disclosed in WO 2005/007857, and more preferably, the polypeptide FCU1-8, presented in the sequence identifier SEQ ID N°: 2, and analogs thereof.

In prokaryotes and lower eukaryotes uracil is converted to UMP under the action brazilhorselovers (UPR). This enzyme is excellent�aweet 5-FU into 5-FUMP. According to another preferred variant of embodiment of the present invention, the suicide gene encodes a protein having the activity UPR.

Consider UPRT may be of any origin, in particular prokaryotic origin, fungal or yeast origin. By way of illustration of the sequence of nucleic acids that encode, UPRT from E. coli (ANDERSEN, et al. Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12. European Journal of Biochemistry. 1992, no.204, p.51-56), Lactococcus lactis (MARTINUSSEN, et al. Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis. Journal of Bacteriology. 1994, vol.176, no.21, p.6457-63), Mycobacterium bovis (KIM, et al. Complete sequence of the UPP gene encoding uracil phosphoribosyltransferase from Mycobacterium bovis BCG. Biochemistry and molecular biology international. 1997, vol.41, no.6, p.1117-24) and from Bacillus subtilis (MARTINUSSEN, et al. Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis. Journal of Bacteriology. 1995, vol.177, no.1, p.271-4) can be used in the context of the invention. However, it is particularly preferable to use yeast UPRT and especially coded S. cerevisiae FUR1 gene, the sequence of which is disclosed in KERN et al. The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene. 1990, vol.88, no.2, p.149-57, which is introduced by the authors by reference. As a guide sequence genes and sequences corresponding UPRT can be found in the literature and data banks Spa�of alistov (SWISSPROT, EMBL, Genbank, Medline, etc.).

Application EP 0998568 A describes the FUR1 gene, having 105 nucleotides in the 5' coding portion, allowing the synthesis UPRT from which have been removed first 35 residues in the N-terminal position and beginning with methionine at position 36 in the native protein. The expression product of the mutant gene called FUR1Δ105 able to complementarian fur1 mutant of S. cerevisiae. In addition, a truncated mutant shows higher activity UPRT than that of native enzyme. Thus, according to particularly preferred variant of embodiment of the present invention, the suicide gene encodes a deletion mutant of the native UPRT. The deletion is preferably located at the N-terminal region of the original UPRT. It may be complete (involving all remnants of the indicated N-terminal region) or partial (affecting one or more continuous or discontinuous residues in the primary structure). Generally, the polypeptide consists of the N-terminal, Central and C-terminal parts, each of which represents approximately one-third of the molecule. For example, because UPRT S. cerevisiae has 251 amino acid, its N-terminal part consists of the first 83 residues, starting with the so-called initiator methionine, which is located in the first position of the native form. As for UPR-Basins E. coli, its N-terminal part of�vative provisions 1-69.

Preferred is a protein having the activity UPRT, includes an amino acid sequence essentially as presented in the sequence identifier SEQ ID N°: 1 from EP 0998568 A, starting with the Met residue in position 1 and ending at the Val residue in position 216. The term "substantially" refers to a degree of identity to the specified sequence SEQ ID N°: 1 EP 0998568 more than 70%, preferably more than 80%, preferably more than 90% and most preferably more than 95%. Still more preferably, it comprises the amino acid sequence represented in the sequence identifier SEQ ID N°: 1 EP 0998568 A. As mentioned above, it may contain additional mutations. In particular, may be mentioned the substitution of the serine residue in position 2 (position 37 in native UPRT) on alanine residue.

According to another preferred variant of embodiment of the present invention, the suicide gene encodes a protein that has at least one activity CD and one activity UPR-Basins. Patent application WO 96/16183 and EP 0998568 A describe the use of fused protein encoding an enzyme with two domains having activity CD and UPRT, and demonstrate that the transfer of a hybrid gene codA::upp or FCY1::FUR1 or FCY1::FUR1Δ105 (i.e. FCU1), which carries a plasmid expression increases sensitivity� transfected B16 cells to 5-FC. According to a more preferred variant of embodiment of the present invention, the suicide gene encodes a polypeptide comprising the amino acid sequence, essentially as presented in the sequence identifier SEQ ID N°: 3 (coda::upp), SEQ ID N°: 1 (FCU1) or FCY1::FUR1. The term "substantially" refers to a degree of identity to the specified sequence, more than 70%, preferably more than 80%, preferably more than 90% and most preferably more than 95%. Still more preferably, it includes the amino acid sequence, essentially as presented in the sequence identifier SEQ ID N°: 3 (coda::upp), SEQ ID N°: 1 (FCU1) or FCY1::FUR1. As mentioned above, it may include additional mutations.

Nukleinovokisly sequences can be easily obtained by cloning, by PCR or by chemical synthesis according to conventional methods used. They can be native genes or genes obtained from them by mutations, deletions, substitutions and/or additions of one or several nucleotides. In addition, their sequences are described in the literature, can be accessed professionals, skilled in the art. Professionals, skilled in the art, is able to clone the sequence CD or UPRT on about�the basis of published data and perform possible mutations to test the enzymatic activity of the mutant forms in an acellular or cellular system according to the technology in the prior art or based on the Protocol identified in the application EP 0998568 A, and fusing, especially in the phase, the polypeptides with the activity of CD and UPRT, and therefore, with all or part of the relevant genes.

According to a more preferred embodiment of poxvirus of the invention additionally includes nukleinovokisly sequence comprising the gene encoding permease.

Permease refers to a transmembrane protein involved in the transfer of a medicinal product, including one the rest of nucleobase, or its precursors across the cell membrane.

Permease includes, but is not limited to the mentioned above, puerperas, cytokinemia and nucleoside transporters.

According to a preferred embodiment of an embodiment of this invention permease is a purine or cytokinemia S. Cerevisiae. The conveyors nucleobase S. cerevisiae consist of a purine-cytosine, permease known as FCY2, and uretilirmis known as FUR4. Purine-cytosine, permease FCY2 mediates symport of protons and adenine, guanine, hypoxanthine and cytosine across the plasma membrane of yeast (Grenson 1969, Jund and Lacroute 1970, Polak and Grenson 1973, Chevallier et al. 1975, Hopkins et al. 1988). FCY2 protein OPOS�should also transport 5-fertilizin, analogue of cytosine (Grenson 1969, Jund and Lacroute 1970). FCY2 gene encodes a protein of 533 amino acids (58 kDa), as initially expected, has 10-12 transmembrane rotating domains (Weber et al. 1990), nine of whom on the date approved by (Ferreira et al. 1999). FCY2 shows a similar affinity for purine nucleobase and cytosine (Brethes et al. 1992). The capture of uracil in S. cerevisiae is mediated by precipitously, FUR4 (Jund and Lacroute 1970, Jund et al. 1977). FUR4 is a uracil-proton symporter (Hopkins et al. 1988), which, apparently, is a protein of 633 amino acids (of 71.7 kDa) with 10 transmembrane domains and a long cytoplasmic hydrophilic N - and C-terminal tails (Jund et al. 1988, Garnier et al. 1996). Protein FUR4 may also mediate the transport of 5-florouracil, the analogue of uracil (Jund and Lacroute 1970).

Amino acid sequence of the FCY2 and Fur4 especially available in swissprot database (accession number R and R respectively). Preferably, permease has an amino acid sequence selected from the group comprising the amino acid sequence of SEQ ID N0: 1 and SEQ ID N0: 2, as disclosed in the patent application WO 2006/048768.

In this respect, according to a preferred embodiment of an embodiment of this invention permease is selected from the group including FCY2 and Fur4 and their analogues. Analogues of Fur4 and FCY2 related polypeptide having the amino acid sequentially�th which has a degree of identity of at least more than 70%, preferably more than 80%, preferably more than 90% and most preferably more than 95%, with amino acid sequence of the parent protein, as described by the authors above, and which retain the ability to transport the drug, including one the rest of nucleobase through the cell membrane.

Specialist, skilled in the art can choose permease to be associated with the drug or a precursor of a drug comprising one remaining nucleobase. For example, FCY2 and Fur4 preferably contacted with 5-fortitudinem (5-FC).

In accordance with preferred option embodiment of poxvirus of the invention may further comprise elements necessary for expression of the target nucleic acid.

In accordance with preferred option embodiment of poxvirus of the invention may further include elements required for expression nukleinovokisly sequence comprising the gene encoding permease. These elements are required for expression of the target nucleic acid and/or nukleinovokisly sequence comprising the gene encoding permease included the elements required for transcription of the indicated DNA in mRNA �, if necessary, for translation of mRNA into a polypeptide. Transcriptional promoter suitable for use in various vertebrate systems, widely described in the literature. For example, among the suitable viral promoters promoter, such as RSV, MPSV, SV40, CMV or 7.5 k vaccinia promoter, the inducible promoter, etc. Preferred promoter isolated from poxviruses, such as 7.5 K, H5R, TK, P28, p11 or of vaccinia virus K1L. Alternatively, you can use synthetic promoter, such as described in CHAKRABARTI. Biotechniques. 1997, no.23, R. 1094-97, HAMMOND, et al. Journal of Virological Methods. 1997, no.66, p.135-38. and KUMAR. Virology. 1990, no.179, p.151-8, as well as the chimeric promoter between early and late conserved poxvirus by promoteri.

Target nukleinovokisly sequence and nukleinovokisly sequence comprising the gene encoding permease may optionally include additional functional elements, such as sequences of intron guiding sequences, transport sequences, secretion signal, nuclear localization signal, IRES, poly And the sequence termination of transcription, tripartite leader sequences, sequences involved in replication or integration. About these sequences reported in the literature, and they can easily be obtained by qualified� experts in this field of technology.

The invention also relates to a method for obtaining poxvirus in accordance with the invention, in which method:

(i) poxvirus according to the invention is introduced into the cell,

(ii) the specified cell is cultivated under conditions that are relevant in order to allow the products specified poxvirus, and

(iii) the specified poxvirus isolated from cell culture.

While poxvirus can certainly be recovered from the culture supernatant, it can also be isolated from cells. One of the commonly used methods is the decomposition of cells through successive cycles of freezing/thawing to assemble virions in litany supernatant. Then these virions can be amplified and purified using the methods of the art (chromatographic method, ultracentrifugation, in particular using a gradient of cesium chloride, etc.).

The present invention also relates to compositions that includes poxvirus in accordance with the invention in combination with pharmaceutically acceptable auxiliary substance.

The composition in accordance with the invention is more particularly intended for the preventive or curative treatment of diseases by gene therapy, and the more specific aims of proliferative diseases (cancers�e education, tumors, restenosis, etc.) or targeted at the disease associated with increased activity of osteoclasts (for example, rheumatoid arthritis, osteoporosis).

The composition in accordance with the invention can be made traditionally in order of its introduction locally, parenterally or by digestive. In particular, a therapeutically effective amount of the recombinant vector or poxvirus of this invention combined with a pharmaceutically acceptable auxiliary substance. It is possible to provide a large number of routes of administration. Examples that may be mentioned include intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumoral, intranasal, intrapulmonary and intratracheal route. In the case of these last three embodiments it is preferable that the introduction was by aerosol, or by using the installation. The introduction may occur as a single dose or a dose that is repeated in one or more cases, after a certain time interval. The appropriate route of administration and dosage varies depending on many parameters, such as the patient, the disease to be treated, or the target gene(s) that(s) is(are) replaced by(s). Drugs based on viral particles with�line with the invention, can be formulated in the form of doses comprising from 104up to 1014pfu (blackebrry unit), preferably between 105up to 1013pfu, preferably from 106up to 1012pfu, more preferably from 106up to 107.

The composition can also include a solvent, adjuvant or excipient which is acceptable from a pharmaceutical point of view, as well as solubilisers, a stabilizing agent and a preservative. In the case of injections of preference is given to compositions in aqueous or non-aqueous isotonic. It can be presented in a single dose or multidose, in liquid or dry form (powder, lyophilisate, etc.), which can be prepared while using the appropriate solvent.

The present invention also relates to the use of poxvirus or compositions according to the invention for the preparation of medicines intended for the treatment of human or animal gene therapy. This medicine can be injected directly in vivo (for example, intravenous injection, in an accessible tumor, into the lungs by aerosol, into the vascular system using a catheter, etc.). The preferred use is in the treatment or prevention �ekovich entities tumors and diseases that result from undesirable cell proliferation. Imaginable use which can be mentioned include cancers of the breast, uterus (especially caused by papilloma viruses), prostate, lung, bladder, liver, intestines, pancreas, stomach, esophagus, larynx, Central nervous system (e.g., glioblastoma) and blood (lymphomas, leukemia, etc.). Another preferred use is in the treatment or prevention of rheumatoid arthritis, osteoporosis and other diseases associated with increased activity of osteoclasts. It can also be used in the context of cardiovascular disease, e.g., inhibiting or delaying cell proliferation smooth muscle in the walls of blood vessel (restenosis). Finally, in the case of infectious diseases, it is possible to apply a remedy for AIDS.

When poxvirus, composition or method of the invention are used to treat cancer, the preferred route of administration is a systematic way, as well as poxvirus according to the invention is able to target specific tumor cells.

The invention also covers a method for treating a disease, characterized by the fact that poxvirus, the composition in accordance with the invention is administered in organizatin or cage desperately in need of such treatment.

According to an advantageous variant of an embodiment therapeutic use or method of treatment also comprises the additional step, in which the pharmaceutically acceptable quantities of a prodrug, mainly analogue of cytosine, in particular 5-FC, is introduced into a host organism or cell. By way of illustration it is possible to use a dose of from 50 to 500 mg/kg/day, with a preferred dose of 200 mg/kg/ day or 100 mg/kg/day. Within the context of the present invention, the prodrug is administered in accordance with standard practice (for example, orally, systemically).

Preferably the introduction, occurring after the introduction of poxvirus or compositions in accordance with the invention, preferably at least 3 days, more preferably at least 4 days and even more preferably at least 5 days after administration of poxvirus or compositions in accordance with the invention. In accordance with an even more preferred embodiment of this invention the introduction of the prodrug occurs after 7 days after administration of therapeutic agent. Preferred oral path. It is possible to enter a single dose of prodrug or doses are repeated for some time, sufficient to allow the production of toxic body�ITA within the host organism or cell.

Furthermore, the composition or method according to the invention can be combined with one or more substances which patentiert the cytotoxic effect of 5-FU. In particular we can mention drugs that inhibit the enzymes of the pathway for de novo biosynthesis of pyrimidines (for example, those mentioned below), drugs such as leucovorin (Waxman et al., 1982, Eur. J. Cancer Clin. Oncol. 18, 685-692), which, in the presence of the product of the metabolism of 5-FU (5-FdUMP), increases the inhibition timedilation, resulting in a reduction of the pool of dTMP required for replication, and finally, drugs such as methotrexate (Cadman et al., 1979, Science 250, 1135-1137), which inhibits digidrofolatreduktazu and increasing the pool of PRPP (phosphoribosylpyrophosphate), causes an increase in the introduction of 5-FU in cellular RNA. According to the present invention drugs that inhibit the enzymes of the pathway for de novo biosynthesis of pyrimidines, preferably selected from the group consisting of PALA (N-(phosphonacetyl)-L-aspartate; Moore et al., 1982, Biochem. Pharmacol. 31, 3317-3321), Leflunomide, A771726 (the active metabolite of Leflunomide; Davis et al., 1996, Biochem. 35, 1270-1273) and brequinar (Chen et al., 1992, Cancer Res. 52, 3251-3257).

The composition or method in accordance with the invention can be combined with one or more substances that are effective anti-cancer therapy. Among headlights�aceticacid substances effective in anti-cancer therapy that can be used in Association or in combination with the compositions according to the invention, may be mentioned alkylating agents, such as, for example, mitomycin C, cyclophosphamide, busulfan, ifosfamide, isospeed, melphalan, hexamethylmelamine, thiotepa, chlorambucil or dacarbazine; antimetabolites, such as gemcitabine, capecitabine, 5-florouracil, cytarabine, 2-fordetection, methotrexate, edatrexate, tomudex or trimetrexate; inhibitors of topoisomerase II, such as, for example, doxorubicin, epirubicin, etoposide, teniposide or mitoxantrone; inhibitors of topoisomerase I, such as, for example, irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) or topotecan; antimitotic drugs such as paclitaxel, docetaxel, vinblastine, vincristine or vinorelbine; and platinum derivatives, such as, for example, cisplatin, oxaliplatin, spirometer or carboplatin. Compositions or methods in accordance with the invention can also be used in combination with radiation therapy.

Compositions or methods in accordance with the invention can also be used in combination with one or more other agents, including, among others, immune modulating agents, such as, for example, alpha, beta or gamma interferon, interleukin (in person�ness, IL-2, IL-6, IL-10 or IL-12) or tumor necrosis factor; agents that affect the regulation of cell surface receptors, such as, for example, inhibitors of the receptor of epidermal growth factor (in particular, cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, gefitinib, erlotinib or lapatinib) or inhibitors of the receptor for human epidermal growth factor 2 (in particular, trastuzumab); and agents that affect angiogenesis, such as, for example, an inhibitor of vascular endothelial growth factor (especially bevacizumab or ranibizumab).

Brief description of Figures in drawings

Figure 1. The in vitro sensitivity to 5-FC from infected with vaccinia virus human cells colorectal tumors (LoVo). The LoVo cells infected at MOI of 0.0001 specified viruses (example () VVTK-/FCU1 () or VVTK-F2L-/FCU1 (), subjected to different concentrations of 5-FC. Survival of cells is determined after 5 days after infection. The results are expressed in percentage of cell viability in the presence or in the absence of drugs. The values represent as mean values ± SD of three individual definitions of immortality cells because of the replication of viruses.

Figure 2. The replication efficiency of the viruses i in vitro LoVo, infected at MOI of 0.0001 indicated virus on day 5 after infection. The values represent as mean values ± SD of three individual determinations.

Figure 3. Mean tumor volume ± SEM n/a LoVo in Swiss Nude mice after intravenous injection of the virus. 7 days after inoculation of the tumor (palpable tumor), mice treated with 107pfu buffer + salt solution (), buffer + 5-FC (), VVTK-F2L-/FCU1 + saline () or VVTK-F2L-/FCU1 + 5-FC (). Animals treated with saline or 5-FC 100 mg/kg/j twice a day Christendom feeding, 7 days after viral injection for 3 weeks. Twice a week determine tumor volume.

Figure 4. Mean tumor volume ± SEM n/a HepG2 tumors in Swiss Nude mice after intravenous injection of the virus. 14 days after inoculation of tumor (palpable tumor), mice treated with 107pfu buffer + water (), or buffer + 5-FC (), or 106pfu VVTK-/FCU1 + water (), or 106pfu VVTK-/FCU1 + 5-FC () (A); or buffer + water (), or buffer + 5-FC () or 106pfu VVTK-F2L-/FCU1 + water (), or 106pfu VVTK-F2L-/FCU1 + 5-FC ( ) (). Animals treated with 5-FC at 100 mg/kg twice a day Christendom feeding, 7 days after viral injection and for 3 weeks. Twice a week determine tumor volume.

Figure 5. The ratio of the yield of virus in dividing cells compared to confluent cells. Cells PANC1 (pancreatic cancer), H1299 (human lung tumor) or U118MG (tumor glioma man) infect with 100 pfu () VVTK-/FCU1 or () VVTK-F2L-/FCU1. 48 hours after infection to determine viral titers. Values represent the ratio between the outputs of the virus in dividing cells compared to confluent cells.

Figure 6. Viral titers (pfu/mg tissue) in organs or tumors on day 6 and day 21 after infection in Swiss Nude mice bearing subcutaneous human tumors with 1×106PFU VVTK-/FCU1 () or VVTK-F2L-/FCU1 ().

Figure 7. Survival Swiss bare mice after treatment with 1×108pfu VVTK-/FCU1 () or VVTK-F2L-/FCU1 () by intravenous injection.

Figure 8. Survival of immunocompetent B6D2 mice after treatment with 1×107pfu (A) or 1×108pfu (B) VVTK-/FCU1 () or VVTK-F2L-/FCU1 (by/in the injection.

Figure 9. The average number of craters on the tails after intravenous injection of 1×106pfu VVTK-/FCU1 or VVTK-F2L-/FCU1 in Swiss Nude mice at day 13 after infection and at day 34 after infection.

Figure 10. The average number of craters on the tails after intravenous injection of 1×107pfu VVTK-/FCU1 or VVTK-F2L-/FCU1 in Swiss Nude mice at day 15 after infection and at day 31 after infection.

Method(s) for carrying out the invention

Examples

Design vector plasmids

Build a Shuttle plasmid for deletion F2L using DNA of the Copenhagen strain of vaccinia virus (inventory number M). Flanking DNA regions F2L amplificateur by PCR. Primers rising flanking plot F2L represent 5' - CGCGGA TCCGAA AGC GAA GAT HUNDRED AAT GTT C - 3' (SEQ ID N°: 7; BamHI site underlined), and

5' - TCCCCC GGGGTT TTC AGT CTT AAC AAA TST AAC - 3' (SEQ ID N°: 8; the SmaI site is underlined). Primers descending part represent 5' - GCC CCA TGG ACA AAT AGA GGA GAT CAA GGG T - 3' (SEQ ID N°: 9; plot MscI underlined) and 5' - GCCCAG CTGACC ACT ACA TCA ATT TTA CAA AAG - 3' (SEQ ID N°: 10; PvuII site is underlined). Amplificatory the DNA fragment cleaved by the restriction enzyme SmaI/BamHI or MscI/PvuII and be ligated into the corresponding sites in the plasmid PpolyIII. Re-plot the downward flanking plot F2L amplificateur by PCR using primer 5' - GCCGA TGC TCC AGA ATT GAT CAT AGT GGA TA - 3' (SEQ ID N°: 11; SphI site underlined) and 5' - GCT HUNDRED GAG TTA GTT TCC TTA ACA AAT HUNDRED AC - 3' (SEQ ID N°: 12; XbaI site underlined) and inserted into the plasmid PpolyIII. Re-plot is used for the removal of the cassette selection during the production of deleted viruses. The cassette selection, the corresponding fused GFP gene/GPT in the control pH5R of vaccinia promoter, inserted into the SmaI site/SphI plasmid PpolyIII. The obtained plasmid is a recombinant Shuttle plasmid called pΔF2L to remove F2L gene.

The preparation of recombinant vaccinia viruses.

Cells infect CEF strain Copenhagen VVTK-FCU1 (Vaccinia virus, a defective J2R gene expressing gene FCUL under the control of the synthetic promoter p11k7.5) at MOI of 0.1 and incubated at 37°C for 2 h, then transferout with coprecipitation CaCl2recombinant Shuttle plasmid (0.2 mg). Cells were incubated for 48 h at 37°C. the resulting dilution of the virus used to infect CEF cells in a selective medium containing hypoxanthine at a final concentration of 15 μg/ml, xanthine at a final concentration of 250 μg/ml, and mycophenolic acid at a final concentration of 250 μg/ml Fluorescence (GFP) and positive (GPT breeding) plaques are isolated and selected for multiple rounds of selection in CEF cells in the presence of selective medium GPT. The presence of Il� the lack of VVTk-FCU1 determine 40 cycles of PCR with primers in the site deletion. After elimination of the parent virus used double remote virus to infect CEF without selective medium GPT to remove the selection cassette. Afluorescent plaques are isolated and selected for 2 cycles in the CEF. The final recombinant virus VV amplificateur in CEF, viral cleanse and original solutions titrated on CEF with a test for plaque.

Cellular in vitro sensitivity to 5-FC

Human tumor cells convert the respective recombinant VV at a MOI of 0.0001. A total of 3×105cells/well were placed on 6-hole Cup for culture in 2 ml of medium containing various concentrations of 5-FC. Then the cells are cultured at 37°C for 5 days and viable cells are counted by exception by tripan blue. The results depicted in figures 1, 2 show that the activity FCU1 equivalent in viruses, defective J2R gene than that of the virus, a defective J2R gene and F2L.

In vitro replication in cultured cells

Dividing cells or confluent infect cells in 6-of linkovich disks with 100 PFU of virus (MOI almost 0,0005). Add 2 ml of medium with 10% FCS for dividing cells and without additives for merging cells. Cells are harvested 48 hours after infection. Cells stored at -20°C and treated with ultrasound to release the virus, the virus will also determine quantitatively t�trevanian drives on CEF cells. The ratio of replication in dividing cells and merged cells like all cells. Both viruses VVTK-/FCU1 and VVTK-F2L-/FCU1 replicated more in dividing cells than in confluent cells.

As an indirect value for the quantitative determination of viral specificity to replicate, determine the yield of virus obtained in dividing cells compared to confluent tumor cells (human pancreatic cancer PANC1; human H1299 lung tumor; human tumor U118MG glioma). Confluent cells were placed on the plates with 1×106cells/well and cultured in complete media for 7 days, then 1 day before infection the cells were washed and cultured in medium without serum. Dividing cells are placed on the tablet, with 3×105cells/well one day before infection. To assess the level of cell division, the number of titrated thymidine included in the nucleic acid, determined after 5 hours, 24 hours and 48 hours of culturing cells on a tablet. During this period, the incorporation of thymidine is relatively constant in confluent cells, whereas in dividing cells, the increase in the inclusion notice. Then infect cells with 100 pfu of virus, and 48 hours after infection, the ratio between output Viru�and, produced in dividing tumor cells and in confluent tumor cells, determined by titration disks on CEF. The results depicted in figure 5, show that both viruses VVTK-/FCU1 and VVTK-F2L-/FCU1 more replicates in dividing cells than in confluent cells.

Subcutaneous tumor model

Female Swiss Nude mice are obtained from Charles River Laboratories. The animals used in the studies are homogeneous in age (6 weeks) and body weight ranged from 20 to 23 in Swiss Nude mice injected subcutaneously (s/to) in the side 5×106of LoVo cells/100 μl. When tumors reached a diameter of 50-70 mm3, mice randomizer arbitrary manner and treated with the indicated vectors for in vivo experiments.

Bioespeleo virus.

The presence of VV-FCU1 and VVTK-F2L-/FCU1 assess viral titration in samples of organs and tumors. 1×106virus is injected intravenous (IV) injection in the tail vein of Nude mice with specified n/a LoVo tumors. Mice are being killed on day 14 after infection and tumors and other organs were collected and weighed. Tumors and organs was homogenized in PBS and titers determined on CEF as described previously. Viral titers to standardize milligram of tissue. Viral titers to standardize milligram of tissue. The results depicted in Table 2 and 3 (range of virus titres are presented in pfu fabric) show that after 14 days, the virus according to the invention is mainly detected in the tumor. In the second set of experiments, under the same conditions as described above, mice are being killed on day 6 and 21 days after infection. The results depicted in figure 6, show that both viruses VVTK-/FCU1 and VVTK-F2L-/FCU1 target the tumor approximately 1000-10000 times more virus in the tumor than in the other organs analyzed except for the tail in case of VVTK-/FCU1. A small amount of VVTK-/FCU1 detected in lung, spleen, kidney and lymph nodes (less than 10 pfu/mg) and more in the skin, the tail and the bone marrow on day 6, and the skin and tail on day 21. On the contrary, VVTK-F2L-/FCU1 has a higher specificity to the tumor with only a small amount in the lungs, spleen, kidney, lymph nodes and skin on day 6, and in the skin and tail on day 21.

Table 2
TumorLightSpleenKidneyHeartLymph nodes
VVTK-/FCU1(0,2-3,3)×1050,1-20-2,2 0-1,80-0,30-61
VVTK-F2L/FCU10-8,1×1040-5,70-9,30,2-30,20-83

Table 3
OvariesLeatherTailBone marrowBrainMuscleHeart
VVTK-/FCU12,2-740,1-2413,5-7,1040-8000-1,80-220-0,3
VVTK-F2L/FCU10-1080-880,8-16,3n/R0-0,20-1,80,2

Antitumor activity of poxvirus invention on the n/a model tumor

Naked mouse with a mouth�target set n/a LoVo tumors (50-70 mm 3) receive treatment once nutripure or twice intravenously (tail vein) these vectors at a dose of 1.104PFU, 1.106PFU ABO 1.107PFU, respectively. Starting from day 7 after virus injection, 5-FC is introduced orally by gavage at 100 mg/kg (0.5 ml of 5-FC of 0.5% in water) twice a day for 3 weeks. The size of the tumor is determined twice a week using calipers. Tumor volume is calculated in mm3by using the formula (p/6) (length × width2). The results depicted in figure 3, show that 2 viruses have similar efficacy with oncolytic activity (p<0,05), capable of controlling tumor growth, and the combined activity of oncolytic virus and a therapeutic gene FCU1) with the introduction of 5-FC, which may further improve the control of tumor growth (p<0.01). Naked mice with established p/HepG2 tumors (cells hepatocellular liver carcinoma human) receive treatment intravenously (tail vein) these vectors at a dose of 1.106PFU in accordance with the following scheme: 14 days after inoculation with tumor (palpable tumor) mice treated with buffer + water, or buffer + 5-FC, or 106pfu VVTK-/FCU1 + water, or 106pfu VVTK-/FCU1 + 5-FC, or 106pfu VVTK-F2L-/FCU1 + water, or 106pfu VVTK-F2L-/FCU1 + 5-FC. Animals treated with 5-FC at 100 mg/kg twice a day of peror�flax through the probe, 7 days after viral injection and for 3 weeks. The size of the tumor is determined twice a week using calipers. Tumor volume is calculated in mm3by using the formula (π/6) (length × width2). The results depicted in figure 4. show control of tumor development after injection of VVTK-/FCU1 and VVTK-F2L-/FCU1 (p<0,0001) relative to the buffer. The activity does not increase after the administration of 5-FC. Independent oncolytic activity of the virus is already very strong when injected virus in a dose of 1×106PFU.

Viral pathogenicity

Viral pathogenicity assess the survival studies, which are conducted on Swiss bare mice (figure 7) and in immunocompetent B6D2 mice (6 weeks from Charles Rivers) (figure 8)). Mice/injected 1.107or 1.108PFU of all viruses in 100 ál of buffer to the mouse. The mice were observed daily during the experiment. Swiss bare mice (figure 7) injection of 1×108PFU VVTK-/FCU1 leads to the death of 40% of the animals after 3 days after infection. The remaining mice die between day 50 and day 80 after infection. Introduction VVTK-F2L-/FCU1 is less pathogenic, most of the animals die between day 65 and 115 (p<0,05). Not observed no evidence of toxicity with both viruses, with 107pfu. All mice die after intravenous injection of 108pfu VVTK-/FCU1. The group �the Directors VVTK-F2L-/FCU1 has a significantly longer survival to 92% (p< 0,00005) compared with VVTK-/FCU1 infected mice. Therefore, this result demonstrates the decrease in toxicity with double remote virus VVTK-F2L-/FCU1.

Model the defeat of the tail pockmarks

Swiss bare mice/injected 1.106(Figure 9) or 1.107(Figure 10) PFU of each virus. The defeat of the tail is calculated once a week. In mice, which is administered 1.106PFU VVTK-F2L-/FCU1, it is noted in less than 1 Ospina/mouse compared to mice injected with VVTK-/FCU1 with an average number equal to 8 pocks on the mouse at day 13 after infection (p<0,001), as shown in figure 9(A). The results are similar to day 34 after infection with an average number equal to 4 craters with VVTK-/FCU1 compared to almost 1 for VVTK-F2L-/FCU1 (p<0,0001), as shown in figure 9(B). In mice, which is administered 1.107PFU VVTK-F2L-/FCU1, marked respectively the average number, equal to 3.5 pocks/2 mouse and pockmarks/mouse compared to mice injected 1.107PFU VVTK-/FCU1, with an average of 10 pocks/mice at day 15 after infection (figure 10(A)). On day 31 after infection in mice, which is administered VVTK-F2L-/FCU1, celebrated respectively on average 3.5 pocks/mice compared with mice injected with VVTK-/FCU1, with an average of 7 pocks/mice (figure 10(B)). The difference in the number of craters between VVTK-/FCU1 and VVTK-F2L-/FCU1 is statistically significant (p<0,05). The formation of craters is correlated with the replica�tion of the virus in the tail, and with the virulence and toxicity. Injection/VVTK-F2L-/FCU1 is less toxic than single-remote virus TC.

Statistical analysis

Conduct statistical studies, using non-parametric U test of Mann-Whitney and the software STATISTICA 7.1 (StatSoft, Inc). P<0.05 are considered statistically significant.

Links

- US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15.11.1994

- WO 2004/014314 (DAVID KIRN (US)) 19.02.2004

- WO 2004/014314 (DAVID KIRN (US)) 19.02.2004

- US 5364773 (VIROGENETICS CORPORATION (TROY, NY)) 15.11.1994

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- EP 0998568 A

- EP 0998568 A

- EP 0998568 A

- WO 96/16183

- EP 0998568 A

- EP 0998568 A

- WO 2006/048768

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1. A method of treating proliferative diseases or diseases with increased activity of osteoclasts, characterized in that poxvirus comprising a defective F2L gene is introduced into the organism-owner or KL�weave, desperately in need of such treatment, an effective amount.

2. A method according to claim 1, where poxvirus additionally includes a defective gene F2R.

3. A method according to claim 1 or 2, where the proliferative disease is cancer or restenosis.

4. A method according to claim 1 or 2, where the disease associated with increased activity of osteoclasts, is rheumatoid arthritis or osteoporosis.

5. A method according to claim 1 or 2, where the specified poxvirus belongs to the subfamily Chordopoxvirinae.

6. A method according to claim 5, where the specified poxvirus belongs to the species Vaccinia virus.

7. A method according to claim 6, where the specified poxvirus belongs to the Copenhagen strain Vaccinia virus.

8. A method according to claim 6, where the specified poxvirus strain belongs to the WR Vaccinia virus.

9. A method according to claim 1, where the specified poxvirus additionally includes suicidally gene.

10. A method according to claim 9, wherein said suicide gene encodes a protein that has at least the activity sitoindosides.

11. A method according to claim 9, wherein said suicide gene encodes a protein that has at least the activity brazilhorselovers.

12. A method according to claim 10, wherein said suicide gene is a FCY1, FCA1 or CodA or their equivalent.

13. A method according to claim 10, wherein said protein having at least the activity sitoindosides, is a polypeptide FCU1-8, presented in the identifier sequence�the sequences SEQ ID N°:2, and its analogs.

14. A method according to claim 10 or 11, wherein said suicide gene encodes a protein that has at least one activity sitoindosides and one brazilhorselovers.

15. A method according to claim 14, wherein said suicide gene encodes a polypeptide comprising the amino acid sequence is almost the same as presented in the sequence identifier SEQ ID N°:3 (coda::upp), SEQ ID N°:1 (FCU1), or the amino acid sequence of FCY1::FUR1.

16. A method according to claim 1, wherein said poxvirus additionally includes nukleinovokisly sequence comprising the gene encoding permease.

17. A method according to claim 16, in which permeate is a purine or chitosanpharmacia S. Cerevisiae.

18. A method according to claim 17, in which permease selected from the group including FCY2 and Fur4 and their analogues.

19. A method according to claims. 9-13 and 15, wherein said poxvirus additionally includes the elements necessary for expression of the suicide gene.

20. A method according to claims. 16-18, in which the specified poxvirus additionally includes the elements necessary for expression of permease.

21. A method according to any one of claims. 1, 2, 6-13, and 15-18, wherein said poxvirus or composition administered system path.

22. A method according to any one of claims. 1, 2, 6-13, and 15-18, comprising the additional step, in which the pharmaceutically acceptable amount of policestate specified in master-organism or cell.

23. A method according to claim 22, in which the introduction of this prodrug is carried out preferably after at least 3 days after administration of poxvirus.

24. A method according to claim 23, in which the introduction of this prodrug is carried out 7 days after the imposition of poxvirus.

25. A method according to any one of claims. 1, 2, 6-13, and 15-18, where poxvirus administered in combination with one or more substances which patentiert cytotoxic effect of 5-fertilizin.

26. A method according to claim 25, in which the abovementioned substances, potentiating the cytotoxic effect of 5-fertilizin, are drugs that inhibit the enzymes of the pathway for de novo biosynthesis of pyrimidines, preferably selected from the group consisting of PALA, Leflunomide and And 771726.

27. A method according to claim 25 in which the specified substance, potentiating the cytotoxic effect of 5-fertilizin, is an methotrexate.

28. The use of poxvirus, as specified in any one of claims. 1-21, for the preparation of a medicine for the treatment of proliferative diseases or diseases with increased activity of osteoclasts.



 

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FIELD: medicine, pharmaceutics.

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6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: medicine, pharmaceutics.

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28 cl, 14 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention represents an agent for neutralising smallpox virus representing an artificial single-chain human 1A antibody having an amino acid sequence presented in the claim material, and exposed on a surface of the filamentous phage M13.

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7 dwg, 4 ex

FIELD: chemistry.

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21 cl, 2 dwg, 6 ex

FIELD: biotechnologies.

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31 cl, 12 dwg, 3 tbl

FIELD: medicine, pharmaceutics.

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29 cl, 20 ex

FIELD: chemistry.

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5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: chemistry.

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28 cl, 18 dwg, 3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.

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8 cl, 16 dwg, 1 tbl, 8 ex

FIELD: chemistry.

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EFFECT: novel polymorphs of (-)trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolon-I-yl)-4-(1H-indol-3-yl)pyrrolidin-2,5-dione are obtained, useful in cancer treatment.

23 cl, 26 dwg, 2 tbl, 27 ex

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22 cl, 7 dwg, 2 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely, to novel heterocyclic compounds of the general formula or to their pharmaceutically acceptable salts, where R1 stands for cyano, nitro, amino, -NHCOOR4 or -NHCOR4; R2 stands for a halogen, C1-alkyl, halogenC1-alkyl or C1-alkoxy; R3 stands for C1-alkyl; or both radicals R3 form a cycloalkyl, containing 3 members, together with carbon atom, which they are bound to; X stands for either an alkylene chain of 4-7 carbon atoms, linear or branched, and the said chain can contain one or several similar or different additional units, selected from -O-, -N(R5)-; either a group where n1 and p1 stand for two integer numbers, the sum of which n1+p1 is an integer number, selected from 2; R6 and R7 together form a covalent bond or R6 and R7 together with carbon atoms, which they are bound to, form a cycle or a cycloalkyl, containing 3 members; R4 stands for C1-alkyl; R5 stands for C1-alkyl. The invention also relates to particular compounds, a pharmaceutical composition based on formula (I), application of the formula (I) compound.

EFFECT: obtained are the novel heterocyclic compounds, useful in treating cancer.

23 cl, 10 dwg, 23 ex

FIELD: medicine, pharmaceutics.

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EFFECT: invention provides extending the range of products applicable in the therapeutic or preventive treatment of hypercalcemia.

19 cl, 8 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to medicine and deals with an immunobiological agent for treating urinary bladder cancer, based on BCG vaccine, including a peptidoglycan fragment, which contains diaminopimelic acid as a part of its structure. The group of inventions also deals with a method of applying the claimed immunobiological agent for the treatment of urinary bladder cancer.

EFFECT: group of inventions provides an increased anti-tumour activity.

5 cl, 5 ex, 5 dwg, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and aims at preventing, treating or managing cancer, preferentially metastatic cancer, in a patient. The method involves administering an effective amount of Axl inhibitor in a combination with an effective amount of one or more chemotherapeutic substance. The Axl inhibitor represents 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N3-(7-(pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)-1H-1,2,4-triazole-3,5-diamine in the form of a recovered stereoisomer or a mixture thereof, or in the form of a tautomer or a mixture thereof, or their pharmaceutically acceptable salt or N-oxide. The chemotherapeutic substance is specified in a group consisting of: cisplatin, lapatinib, erlotinib, gemcitabine, doxorubicine, paclitaxel, cytarabine and docetaxel.

EFFECT: method enables increasing the clinical effectiveness in cancer, reducing side effects.

5 cl, 11 ex, 16 dwg

FIELD: medicine.

SUBSTANCE: method involves intravenously administering 0.1-1% aqueous solution of khlorin, selected from group containing photolon, radachlorine or photoditazine at a dose of 0.2-0.5 mg/kg or 0.2-1% aqueous solution of porphyrin like photogem at a dose of 0.2-1 mg/kg. Laser irradiation of blood is carried out 5-15 min later after beginning photosensitizer injection into cubital vein of one arm via laser light guide set in advance in the cubital vein of the other arm during 10-40 min at wavelength of 661-666 nm and power of 20-50 mW one session per day during 3-10 days with the aqueous solution of khlorin used as the photosensitizer, or laser irradiation of blood with wavelength equal to 630-633 nm during 10-45 min with power of 20-50 mW one session per day with the aqueous solution of porphyrin used as the photosensitizer. Repeated intravenous administration of photosensitizer is carried out 1-3 months later combined with repeated laser irradiation of blood.

EFFECT: reduced risk of tumor cells dissemination and metastasis development.

2 cl

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