Cell elimination by using viruses

FIELD: medicine, in particular uses of viruses for cell elimination.

SUBSTANCE: invention relates to uses of viruses capable of reducing undesired cells in ex vivo mixtures of normal marrow or peripheral blood cells and tumor cells, such as leucosis or lymphoma cells due to interaction of abovementioned mixture with vesicular stomatitis virus. Invention also relates to method for malignant tumor treatment in mammalian. Claimed method includes sampling of mammalian marrow or peripheral blood cells; interaction of said cells ex vivo with vesicular stomatitis virus; mieloablative therapy; and transplantation of purified hematopoietic cells into mammalian. In another embodiment invention relates to method for malignant tumor treatment in mammalian having transplant of marrow or peripheral blood stem cells. Said method includes interaction ex vivo of collected transplant cell with vesicular stomatitis virus and administration of these purified cells in mammalian. Method of present invention makes it possible to reduce risk of malignant tumor backset in mammalian with transplanted hematopoietic cells.

EFFECT: new agent for elimination of marrow or peripheral blood tumor cells.

12 cl, 4 ex, 3 tbl

 

The scope of the invention

This invention relates to viruses, which are able to remove (reduce or eliminate undesired cells in the cell mixture. Unwanted cells can include tumor cells, cells mediating graft-versus-host and autoimmune cells. This invention relates also to remove unwanted cells from the collection of bone marrow cells or peripheral blood in the treatment of mammals, including patients with malignant tumors, recipients of transplants and patients with autoimmune disease.

Background of the invention

Ways to remove unwanted cells ex vivo have shown limited success for transplantation of autologous bone marrow or stem cells in patients with leukemia or other malignant diseases. One of the goals of treatment (i.e. removal of unwanted cells) bone marrow or precursor cells of peripheral blood (PBPC) is the destruction of tumor cells with little effect on normal stem cells and hematopoietic precursor cells. Transplantation of purified (subjected to removal of unwanted cells) bone marrow takes place after myeloablative therapy, such as chemotherapy in high doses or radiation (see, for example, Stuart R.K., 1993, Semin Oncol. 20(5 Suppl. 6):40-54); Hammert L.C. and Ball, E.D., 1997, Curr. Opin. Hematol. 4:423-428; Schneidkraut M.J., et al., 1996, J. Hematother. 5:631-646). Transplantation of any tumor cells with bone marrow puts patients at risk of recurrence of malignant tumors (see, for example, Rummel S.A. and Van Zant, G., 1994, J. Hematother. 3:213-218; Kvalheim G. et al., 1996, J. Hematother., 5:427-436). The methods tested in the current research for the selective elimination of tumor cells include the use of chemotherapeutic agents (such as 4-hydroperoxycyclophosphamide; see, for example, Bird, J.M., 1996, Bone Marrow Transplant, 18:309-313), monoclonal antibodies (see, for example, Hammert, L.C. and Ball E.D., 1997, Blood Rev., 11:80-90), photodynamic therapy (see, for example, L. Villeneuve, 1999, Biotechnol. Appl. Biochem. 30:1-17), and viral vectors such as adenovirus (see, for example, M. Hirai et al., 1999, Acta Haematol., 101:97-105; Marini F.C., et al., 1999, Clin. Cancer Res., 5:1557-1568). However, recent experiments have shown that viable tumor cells remained in the bone marrow or RVRS after therapeutic removal of unwanted cells, which led to the recurrence of a malignant tumor. Another major disadvantage of the current methods of removing unwanted cells ex vivo is slow engraftment of the graft due to the damage of normal stem cells and/or early hematopoietic progenitor cells (Rummel S.A. and Van Zant G, 1994, J. Hematother. 3:213-218; Damon et al., 1996, Bone Marrow Transplant, 17:93-99). Cells before Estonica, which actively proliferate, and are therefore very sensitive to the elimination of most chemotherapeutic agents, including 4-hydroperoxycyclophosphamide. The resulting loss of early precursor cells causes prolonged neutropenia and/or thrombocytopenia, which puts the patient in a condition of increased risk for life-threatening infections and/or bleeding. Cytotoxic or cytolytic agent that kills tumor cells, which preserves the normal hematopoietic cells, is an important advance in therapy of malignant tumors.

In addition to tumor cells, fees precursor cells of the bone marrow or peripheral blood may contain other unwanted cells, such as autoimmune cells, for example, people with arthritis or multiple sclerosis. Other unwanted cells include cells, which mediate the response ″graft-versus-host″ (for example, some T-cells in allogeneic grafts. The reduction or elimination of such unwanted cells would be important in the treatment of malignant tumors and autoimmune diseases.

PCT application Roberts et al. (WO 99/18799 and PCT US 99/21230) refer to treatment of tumors by viruses.

The purpose of the invention

The object of the present invention are viruses DL is the reduction or elimination of unwanted cells in mixtures of desirable and undesirable cells.

The next object of this invention is the provision of viruses to reduce or eliminate the tumor cells in mixtures of normal and tumor cells.

The next object is the provision virus removal ex vivo tumor cells from normal hematopoietic cells, such as precursor cells of the bone marrow or peripheral blood.

The next object is the provision virus removal ex vivo autoimmune cells from normal cells, such as precursor cells of the bone marrow or peripheral blood.

Another object is to provide virus removal ex vivo cells, which mediate the response ″graft-versus-host″, from normal hematopoietic cells, such as precursor cells of the bone marrow or peripheral blood.

The next object of this invention is the provision of a method of treating disease in a mammal interaction mixes desirable hematopoietic cells and unwanted cells with the virus and transplantation of such cells into the mammal.

The next object of this invention is the provision of a method of treating a malignant tumor in a mammal interaction of the collected cells with the virus and transplantation of such purified hematopoietic cells of the mammal after myeloablative therapy.

The next object of this invention is the provision of means of preventing reactions ″graft-versus-host″ mammal interaction of the collected cells with the virus and transplantation of such purified hematopoietic stem cells in the mammal after myeloablative therapy.

The next object of this invention is the provision of a method of treating autoimmune disease in a mammal interaction of the collected cells with the virus and transplantation of such purified hematopoietic stem cells in the mammal after myeloablative therapy.

The next object of this invention is the provision of a method of treating a malignant tumor in a mammal receiving a transplant of stem cells from bone marrow or peripheral blood, capable of handling this transplant virus.

The invention

This invention relates to a method for reduction or elimination of unwanted cells in a mixture of desirable and undesirable cells by the interaction of the mixture with the virus.

This invention relates also to a method for reduction or elimination of unwanted cells in a mixture of desirable and undesirable cells by the interaction of the mixture with RNA virus.

This invention relates also to the method is the reduction or elimination of tumor cells in a mixture of ex vivo normal hematopoietic cells and tumor cells by the interaction of the mixture with the virus, such as RNA-virus.

This invention relates also to a method of ex vivo removal of tumor cells from the harvesting of stem cells from bone marrow or peripheral blood collected interaction of cells with the virus, such as an RNA virus.

This invention relates also to a method of ex vivo removal of autoimmune cells from the harvesting of stem cells from bone marrow or peripheral blood collected interaction of cells with the virus, such as an RNA virus.

This invention relates also to a method for removal of ex vivo cells, which mediate the response ″graft-versus-host″, out of a population of stem cells from bone marrow or peripheral blood by the interaction of this cell population with the virus, such as an RNA virus.

This invention relates also to a method of treatment or prevention of diseases such as malignant tumor in a mammal, involving: (a) the taking of bone marrow cells or peripheral blood of the mammal, (b) the interaction of these cells from bone marrow or peripheral blood ex vivo with a virus, such as an RNA virus, (C) the execution myeloablative therapy in the specified mammal and (d) transplantation specified mammal purified hematopoietic cell stage (b).

This invention relates also to a method of treatment of PLN the quality of a tumor in a mammal, receiving the graft precursor cells of the bone marrow or peripheral blood, including interaction of the collected cells of the graft with the virus, such as an RNA virus, and the introduction of these treated cells indicated a mammal.

This invention relates also to a method of treating autoimmune disease in a mammal receiving the graft precursor cells of the bone marrow or peripheral blood, including interaction of the collected cells of the graft with the virus, such as an RNA virus, and the introduction of these treated cells indicated a mammal.

This invention relates also to a method of preventing reaction ″graft-versus-host″ mammal receiving the graft precursor cells of the bone marrow or peripheral blood, including interaction of the collected cells of the graft with the virus, such as an RNA virus, and the introduction of these treated cells indicated a mammal.

Detailed description of the invention

This invention relates to the detection of viruses and the use of viruses for the reduction or elimination of undesirable cells, such as tumor cells, from a mixture of desirable and undesirable cells. This invention provides a virus and ways to remove (reduce the possible number or elimination of undesired cells from normal cells using viruses. Unwanted cells in transplants of hematopoietic cells, which are removed by the viruses in this invention include tumor cells, autoimmune cells (for example, in the case of rheumatoid arthritis or multiple sclerosis)and cells that mediate the graft-versus-host. Treatment of a mammal consists of (a) the taking of bone marrow cells or peripheral blood of the mammal, (b) the interaction of these cells from bone marrow or peripheral blood ex vivo with a virus, (C) conducting myeloablative therapy in the specified mammal and (d) transplantation specified mammal purified hematopoietic cell stage (b).

The methods of the invention

Destruction of tumor cells

Incubation mixtures of normal cells and tumor cells with viruses leads to the selective elimination of tumor cells but not normal cells. Effective way to remove tumor cells from the hematopoietic cells can be used in the treatment of malignant tumors in mammals with transplantation of autologous stem cells from bone marrow or peripheral blood. For example, stem cells from bone marrow or peripheral blood of a mammal with a tumor is subjected to interaction with the virus before transplantation to prevent R is cidia this tumor. Tumor cells, which can be removed by the methods of this invention include, but are not limited to, (1) leukemia, (2) lymphoma (3) carcinomas, such as breast cancer, lung cancer, cancer of the colon, prostate cancer and pancreatic cancer, (4) sarcoma and (5) malignant tumors of neuroepithelial origin, such as melanoma and neuroblastoma.

A variety of viruses, such as RNA viruses [e.g., but not limited to, the vesicular stomatitis virus (VSV), Newcastle disease virus (NDV) and reovirus] can be used to remove tumor cells from normal hematopoietic cells. Normal hematopoietic cells are resistant to infection by many viruses, including RNA viruses. As an example, normal bone marrow cells are resistant to VSV infection, rhabdoviruses, as determined by the production and viability of infectious virus. Normal bone marrow cells from two donors were not produced infectious virus even when infected at high multiplicity of infection (for example, 10 plaque-forming units (PFU) per cell; see example 1). Culture of bone marrow were indistinguishable from monoinfection cultures in their ability to form normal range of types of hematopoietic cells after which Multivitamine in vitro methylcellulose. As another example, CD34+ enriched cells from normal human bone marrow were resistant to infection with NDV virus from another family (paramyxoviruses); see Roberts et al., WO 99/18799.

Many types of tumor cells are highly sensitive to the elimination of cells with numerous viruses, including RNA viruses. For example, using VSV, cell lines of acute myelogenous leukemia OCI/AML3, OCI/AML4 and OCI/AML5 were highly sensitive to VSV infection, and of 0.05 PFU per cell kill 50% of these cells for 24 hours and only 0,003 FIGHT on cell kill 50% of the cells for 48 hours (example 1). The Indiana serotype of VSV used in this experiment were propagated and collected from murine L929 cells. As another example, it was shown that various types of tumor cells were sensitive to VSV, including cell carcinoma of the ovary, fibrosarcoma, lung cancer, melanoma, prostate cancer and leukemia (see example 3). NDV also kills the majority of tumor cells (see Roberts et al., WO 99/18799). Reovirus type 3 killed tumor cells fibrosarcoma person, but not normal fibroblasts (example 4).

Selective elimination of tumor cells from a collaborative culture with normal hematopoietic cells can be achieved using a variety of different viruses. For example, in coculture leukemia cell OCI/AML3 but with the normal bone marrow cells (at a ratio of 1:9 and 1:3 ratio) VSV killing leukemia cells, while he had a minor effect or no significant effect in normal hematopoietic cells (example 2). The Indiana serotype of VSV used in this experiment were propagated and collected from murine L929 cells. These data demonstrate the selective destruction of leukemia cells in a mixed population of normal bone marrow and the applicability of viruses such as VSV, in the purification of bone marrow from unwanted cells. As another example, has been shown selective elimination of tumor cells in a mixed culture of normal cells with the use of NDV. Strain RRMC NDV, three cleared of plaque isolate mesogenic strain MC NDV, selectively killed cancer cells of the oral cavity of a person in a mixed culture with normal fibroblasts (see Roberts et al., WO 99/18799).

Destruction of cells, mediating the graft-versus-host.

Incubation mixtures unwanted cells, such as T-lymphocytes, causing graft-versus-host, and the desired cells with the virus leads to selective elimination of unwanted cells. This effective method of purification of these cells from bone marrow or peripheral blood can be used in the prevention of graft versus host in mammals.

Destruction of cells, mediating autoimmune disease.

Incubation mixtures autoimmune cells that cause autoimmune disease, and the desired cells with the virus leads to selective elimination of autoimmune cells. This effective method of removing these unwanted cells can be used in the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Screening of unwanted cells.

Unwanted cells of this invention can include tumor cells with chromosomal deletions or rearrangeable gene or genes encoding proteins or modulators reactions at the cellular interferon, or to carry otherwise defective response to interferon (Colamonici O.R. et al., 1992, Blood 80:744-749; Heyman M. et al., 1994, Leukemia, 8:425-434; C. Billard et al., 1986, Blood 67:821-826). The suspended nature of the bone marrow or populations RVRS enables easy analysis of sorting cells with excitation fluorescence (FACS analysis) in determining the state of reactivity of the cells to interferon. Probes for responsiveness to interferon include chromosomal hybridization probes for deletions or rurangirwa genes and probes, such as antibodies for analysis of cellular receptors and components of the transduction of signals involved in cellular response to interferon.

Compounds of the invention

The viruses of the present invention is able to distinguish not lateline cells, such as tumor cells, from the desired cells, such as normal hematopoietic cells. RNA viruses of the present invention include, but are not limited to, (1) single-stranded viruses, including negative-sense RNA viruses and positive-sense RNA viruses, and (2) double-stranded RNA viruses. Single-stranded, negative-sense RNA viruses of the present invention include, but are not limited to, a family of non-segmented viruses, such as rhabdovirus [(for example, vesicular stomatitis virus (VSV)] and paramyxoviruses [e.g., Newcastle disease virus (NDV) and parainfluenza virus type 3 person]. Single-stranded positive-sense RNA viruses of the present invention include, but are not limited to, picornaviruses (e.g., rhinovirus) and togavirus (for example, virus fever Sindbis). A family of double-stranded RNA viruses of the present invention include reovirus. This invention enabled replication competent and incompetent for replication of RNA viruses.

In this invention included ″interferon-sensitive viruses″, described by Roberts et al., WO 99/18799, which is incorporated herein by reference in its entirety. These viruses selectively replicate in tumor cells and helps eliminate tumor cells on the basis of a selective deficiency in these cells FN-mediated antiviral response. In addition to RNA viruses, "interferon-sensitive viruses included VA1-mutants of adenovirus, a DNA virus.

Family rhabdoviruses consists of closely related, having a shell, non-segmented RNA viruses, and they include the following genera that infect animals: (1) the genus Vesiculovirus (for example, vesicular stomatitis virus, VSV); (2) the genus Lyssavirus (e.g., rabies virus) and (3) the genus Ephemerovirus [Dietzschold B. et al., 1996. Rhabdoviruses. In: Fields Virology, 3rdEdition (eds. Fields, B.N. et al.), pp 1137-1159].

In a particularly preferred embodiment of this invention rhabdoviruses is the vesicular stomatitis virus (VSV). Identified several serologically distinct strains of VSV together with a number of characterized strains. Natural hosts VSV include insects, rodents, and domestic farm animals. Usually a very small part of the population of North America comes into contact with the virus, and the majority of human infections common among laboratory staff and farmers. Human infections are either asymptomatic or manifest as flu-like symptoms. The VSV strains include, but are not limited to, strains Indiana, New Jersey, Priy, Coccal and Chandipura. Although the described examples relate to the VSV Indiana, it should be clear that specialist in this area, based on the way is, described in this document can easily be screening other strains and derivatives VSV, including VSV mutants that selectively kill tumor cells.

The family of paramyxoviruses, containing non-segmented negative-sense RNA viruses that includes three kinds: (1) paramyxoviruses, including Newcastle disease virus (NDV); (2) viruses such as measles virus (Morbillivirus genus, including the causative agents of measles, the plague dogs and cattle), and (3) respiratory syncytial virus (pneumovirus). NDV is the preferred virus in accordance with this invention. NDV is divided into three distinct groups according to the effect on chickens and chicken embryos. Strains of low virulence" call lentogenic (slow acting) and they require 90-150 hours for the elimination of chick embryos at the minimum lethal dose (MLD); the strains of "moderate virulence" called mesogenic and they require 60-90 hours for the elimination of chick embryos in MLD; strains of high virulence" call villagename (fast action) and they require 40-60 hours for the elimination of chick embryos with MLD. See, for example, Hanson and Bradley, 1955 (Science, 122:156-157) and Diardiri et al., 1961 (Am. J. Vet. Res. 8:918-920). All three classes are applicable, including, preferably, mesogenic strains of NDV, such as MQ.

In a particularly Ave is doctitle embodiment of the present invention, double-stranded RNA virus is reovirus. In the following a preferred embodiment of the present invention reovirus is reovirus type 3.

In another preferred embodiment of the present invention use RNA viruses that can replicate in the tumor, lack of expression of subtilisin-related proteases. Parainfluenza virus type 3 is a virus of this type.

For some purposes it is desirable obtaining clonal virus to guarantee or increase genetic homogeneity of a particular viral strain and removal of defective interfering particles. Removal of defective interfering particles makes possible increased purity in the final product, as measured by the number of all viral particle to infectious particle. Clonal virus can be obtained by purification from plaques or other means, as described in Roberts et al., WO 99/18799.

In another embodiment of the present invention, the virus is genetically modified, for example, to increase its selectivity against tumor cells. Methods of genetic manipulation of rhabdoviruses, such as VSV, are well established (Roberts A. and J.K. Rose, Virology, 1998, 247:1-6), which makes it possible to change the genetic properties of the virus.

In addition, standard methods, well known to the specialist with expertise in this area can be used for genetic option is of VSV and the introduction of desired genes into the genome of VSV to obtain a recombinant VSV (for example, Sambrook et al., 1989, A Laboratory Manual, New York, Cold Spring Harbor Press). In one embodiment of this invention G-protein VSV can be modified to obtain mergers that focus on specific sites on tumor cells. In another embodiment of the present invention VSV genetically modified to Express one or more suicide genes, are able to metabolize the prodrug into a toxic metabolite that allows you to eliminate infected with VSV tumor cells by the introduction of prodrugs. VSV designed for gene expression timedancing or gene sitoindosides herpes virus, can be used to convert ganciclovir or 5-FC, respectively, in the toxic compound. However, it should be clear that can also be used and other suicide genes.

The preparation of compositions and introduction

A preferred variant of the present invention relates to a kit for use in the ex vivo removal of unwanted cells from a mixture of desirable and undesirable cells. This set includes a pre-measured amount of a virus or viruses composition suitable for processing a mixture containing a certain number of desirable and undesirable cells. Preferred compositions include additives that stabilize the virus against loss of infectivity or increase inespo the institutional capacity or the survival of desirable cells. A more preferred set allows the interaction of the viral composition with a mixture of target cells in the avascular stage without the need for special equipment for premises biological components. An example of such device is compartmentalizing collection container intended for a mixture of target cells, which contains in a separate compartment pre-measured amount of virus. The creation of an original path between the compartments makes possible the contact between the virus, the cell population is a target and one or more fillers. Contact with the virus and fillers, if they are separated, can occur simultaneously or sequentially. Additional preferred variant of this invention is the type compartmentalizing container that maintains optimum temperature for the interaction between virus and cells during contact between the virus or viruses and the mixture target of desirable and undesirable cells. Another preferred variant of the present invention refers to the set, which allows to separate kontaktierung mixture of cells from virus, fillers or both after a suitable period of time. Suitable period of time of contact can be known or can be determined specialist is qualified in this field.

Suitable compositions for viruses of the present invention include compositions that are in the list of viruses used in the treatment of tumors (Roberts et al., 1999, PCT WO 99/18799). In addition, preferred compositions include compounds or biological agents that have one or more of the following activities in relation to the desired cells in a mixture of desirable and undesirable cells: differentiating, proliferating, conservation, supportive, protective and inducing a state of calm. For example, compounds and biological agents of these types include cytokines, peptide regulators of the life cycle of cells, interleukins, growth factors, energy sources, vitamins and electrolytes. Additional desirable fillers include createsite connection.

To reduce or eliminate unwanted cells with preservation of desirable cells, you should use an effective amount of the virus of the present invention. Specialists in this field it is clear that the amount used of the virus to reduce or eliminate unwanted cells will vary depending on the virus type and quantity of unwanted cells and the type and quantity of desirable cells that need to be saved. For example, VSV used is 0.0001-10 plaque-forming units (PFU) per cell and more preferably at 0,0003-10 PFU per cell.

In another preferred embodiment of the present invention, the virus uses to interact with a mixture of tumor cells and normal cells that process before, during or after the interaction of interferon. Interferon makes possible enhanced protection of normal cells (see example 3 and Roberts et al., WO 99/18799). Interferon (IFN) is chosen from the group of class I (alpha, beta and omega) and class II (gamma) and their recombinant versions, and analogs, as discussed, for example, in Sreevalsoun, T., 1995 (In: Biologic Therapy of Cancer, second edition, edited by V.T.DeVita, Jr., et al., J.B.Lippincott Company, Philadelphia, pp.347-364).

In another preferred embodiment of the present invention, the virus uses to interact mixture of tumor cells and normal cells that are treated with a chemotherapeutic agent prior to, during or after contact with the indicated virus. Chemotherapeutic agent used to further reduce the viability of tumor cells.

The following examples are illustrative, and not limiting the methods and compositions of the present invention. Other suitable modifications and adaptations of various conditions and parameters normally encountered in clinical therapy, which are obvious for professionals with qualifications in this field, both within ideas and scope of this invention.

Example 1. Election Elim the nation leukemia cells with preservation of normal bone marrow cells using vesicular stomatitis virus defined in separate cell cultures

The Indiana serotype of VSV was isolated from plaques in mouse L929 cells. Individual plaque was used to infect a monolayer of L929 cells after 18 hours collecting the supernatant was subjected to centrifugation at HD within ten minutes. Then the clarified supernatant was filtered through a filter of 0.2 μm (Millipore) and then was titrated on L-cells and kept at 80°in the aliquot. Separate aliquots of virus were used only once.

Culture of normal bone marrow from two separate healthy donors were resistant to infection with this Indiana serotype of vesicular stomatitis virus (VSV). Normal bone marrow cells were not produced infectious VSV particles, even when the infection at multiplicity of infection of 10 PFU per cell. In addition, infected culture bone marrow cells were indistinguishable from monoinfection cultures for their ability to form normal range of types of hematopoietic cells after in vitro cultivation in methylcellulose. In contrast, the cell line AML OCI/AML3, OCI/AML4 and OCI/AML5 were highly sensitive to VSV infection, and of 0.05 PFU per cell kill 50% of these cells for 24 hours and only 0,0003 the FIGHT on the cage killed 50% of the cells for 48 hours.

Example 2. Selective elimination of leukemic cells viruso the vesicular stomatitis in mixed cultures, containing normal cells in the bone marrow

The Indiana serotype of VSV used in this example was prepared as described in example 1. In coculture leukemia cells OCI/AML3, mixed with normal bone marrow cells (ratio 1:9), VSV had oncolytic properties. In this experiment (table 1) coculture VSV infected at a multiplicity of infection of 1 plaque-forming unit (PFU) per cell or 5 PFU per cell for 24 hours and then sown in methylcellulose with growth factors or without growth factors. In the presence of growth factors grew as normal cells of the bone marrow and tumor cells, whereas in the absence of growth factors, cells OCI/AML3 formed colonies. Counting of colonies was performed after 14 days (table 1), and he showed a complete elimination of the independent factors in the growth of leukemia cells and preservation of progenitor cells in normal bone marrow. Identical results were observed when using a mixture of 1:3 cells OCI/AML3 and normal bone marrow. These data demonstrate the selective destruction of leukemia cells in a mixed population of normal bone marrow and the applicability of VSV in the ex vivo purging of unwanted cells.

Table 1.

Selective elimination of cells of acute myeloid leukemia (AML), Sokol tiberuim with normal bone marrow. The number of colonies per Cup (receiving 104cells), observed two weeks after infection with VSV shown in the table below for tumor cells (leukemic and normal hematopoietic cells (neutrophils, mixed and monocytes).
The multiplicity of infection
Type colonies0,01,05,0
Leukemia cells17200*
Neutrophils1275
Mixed cell634
Monocytes1075
* Colony leukemia cells was not found on cups without growth factors even when sowing 105cells on the Cup.

Example 3. VSV selectively grows in tumor cells and kills cancer cells compared with normal cells, as defined in separate cell cultures

Various normal and transformed cell lines either not treated or preoperatively 100 units of IFN-α, infected VSV Indiana at multiplicity of infection (MOI) OF 0.1 PFU/ml and incubated for 18 hours at 37about(Table 2). Culture medium from each sample titre the Ali on the production of VSV. Pretreatment of cultures of normal cells by interferon reduced the production of the virus to <1000 infectious viral particles per ml, while the lines of tumor cells continued to produce copious quantities of viral particles (105-108plaque-forming particles per ml). In tumor cells demonstrated a more rapid and transient increase in VSV than in primary cultures of normal cells fibroblasts or epithelial origin. The differences between the different cell types was reflected not only in the production of viral particles, but also in the cytopathic effect (CPE)observed at the microscopic level.

Table 2.

The output of the virus VSV after a night of infection of different cell lines, either not treated or treated IFN
Cell lineThe titer of virus (PFU/ml)
RawIFN-α
OSF7 (primary normal human fibroblasts)1×106<10
OSF12 (primary normal human fibroblasts)2×105<10
OSF16 (primary normal human fibroblasts)1×105<0
PrEC (primary normal prostate epithelium person)8×106<10
HOSE (primary normal surface epithelium of the ovaries person)1×107<1000
A2780 (ovarian cancer man)2×1081×107
OVCA 420 (ovarian cancer man)1×1083×106
C13 (ovarian cancer man)1×1081×105
LC80 (human lung cancer)2x1096×107
SK-MEL3 (human melanoma)1×1091×109
LNCAP (prostate cancer man)4×1095×109
HCT116 (cancer of the colon of a person)1×1092×109
T (SOME cells, transformed T-antigen and IA Ad-virus)1x1088×107

Example 4. Selective elimination of tumor cells with preservation of normal fibroblastic cells reovirus type 3 in separate cell cultures

Tumor cells (NT, a fiber optic scope is Arkoma) and normal cells (CCD922sk, normal fibroblasts of human skin) were grown to approximately 80%confluently in 24-hole tablets for tissue culture. Culture medium was removed and added PPVR-824, cleaned of plaque clone of reovirus type III human, strain Dearing, at 1E+6 plaque-forming units (PFU) per well of 10 PFU per well in 10-fold dilutions (experiment I) or 7,2E+7 PFU per well and 10-fold dilutions from 107to 100 PFU per well (experiment II). Control wells without the addition of virus were on each tablet. The virus was adsorbing within 90 minutes on a rocking platform at 37°C. At the end of this incubation period the virus dilution was removed and replaced with 1 ml of culture medium. Then the plates were incubated for 5 days at 37°With 5% CO2. Cytotoxicity was quantified using colorimetric analysis using MTT (bromide 2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) (Cell Titer 96, catalog#G4000, Promega Corporation, Madison WI 53711), with monitoring at 570 nm, which detects the activity of the mitochondrial enzyme (Mosman, T., 1983, J. Immunol. Methods 65:55). Viability in the treated virus holes were expressed as percent activity in untreated control wells. The data depicted graphically as a function of viability as a percentage of control from the FIGHT on the hole. IC50rasschitat is whether in the form of the amount of virus in the BATTLE on the hole causing 50%decrease in the number of viable virus. Tumor cells were orders of magnitude more sensitive to elimination PPVR-824 (table 3).

Table 3.

Selective elimination of tumor cells, but not normal fibroblastic cells reovirus type 3 in separate cell cultures
Normal fibroblast (CCD922sk), IC50The tumor cell (T), IC50
Experiment I>1.0 E+06125
Experiment II>7.2 E+07417

The preceding examples are intended to illustrate the present invention and not for limitation. Numerous variations and modifications can be made without deviating from the true scope of this invention.

1. The way to reduce or eliminate the tumor cells in a mixture of ex vivo normal hematopoietic cells and tumor cells by the interaction of the above-mentioned mixture with vesicular stomatitis virus.

2. The method according to claim 1, where these tumor cells are leukemia cells.

3. The method according to claim 1, where the specified hematopoietic cells are bone marrow cells.

4. The method according to claim 1, where the specified gemopoeticheskoi the cells are peripheral blood cells.

5. The method according to claim 1, where the specified RNA vesicular stomatitis virus is replication competent RNA virus.

6. The method according to claim 1, where the specified RNA virus vesicular stomatitis is incompetent for replication of the RNA virus.

7. The method according to claim 1, where these tumor cells are lymphoma cells.

8. The method according to claim 1, additionally introducing a chemotherapeutic agent prior to, during or after the interaction with the indicated virus.

9. The method according to claim 1, additionally providing for the introduction of interferon before, during or after the interaction with the indicated virus.

10. A method of treating a malignant tumor in a mammal, involving (a) the taking of bone marrow cells or peripheral blood of the mammal, (b) the interaction of these cells from bone marrow or peripheral blood ex vivo with vesicular stomatitis virus (c) conducting myeloablative therapy in the specified mammal and (d) transplantation specified mammal purified hematopoietic cell stage (b).

11. The method according to claim 10, where the specified myeloablative therapy is chemotherapy in high doses.

12. A method of treating a malignant tumor in a mammal receiving a transplant of stem cells from bone marrow or peripheral blood, providing the first interaction of the collected cells of the graft ex vivo with vesicular stomatitis virus and the introduction of these treated cells indicated a mammal.



 

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5 cl, 2 ex

FIELD: medicine.

SUBSTANCE: method involves introducing means containing heavy elements into tumor and expose it to X-ray radiation. A heavy chemical element having number of 53, 55-83 is used. The means contains one or several of said elements with additional ligand introduced as iminodiacetic acid or its derivatives or crownethers or porphyrins and a water-soluble polymer. The means is introduced in systemic or direct way into tumor with following X-ray irradiation of power of 10-200 keV being applied.

EFFECT: increased photon therapy dose concentrated in tumor tissue with concurrently reduced radiation exposure upon the unaffected tissues.

4 cl, 4 dwg, 6 tbl

FIELD: medicine, oncology, immunology.

SUBSTANCE: invention relates to humanized antibodies with ErbB2. Invention involves the development of new humanized antibodies raised to tyrosinase receptors of family ErbB2, and to a composition comprising these antibodies. The advantage of invention involves expanding region in using indicated antibodies in cancer treatment wherein receptor of epidermal growth factor, EGFR, is a target of these antibodies.

EFFECT: valuable properties of antibody.

14 cl, 3 tbl, 13 dwg

FIELD: organic chemistry of heterocyclic compounds, biology, medicine, pharmacy.

SUBSTANCE: invention relates to new substituted pyrido[4',3':5,6]pyrano[2,3-d]pyrimidines of the general formula (1): or (2): or their pharmaceutically acceptable salts, N-oxides or hydrate possessing physiologically active properties, in particular, eliciting ability to induce apoptosis in tumor cells causing their death. In the general formula (1) or (2) X represents sulfur or oxygen atom; Y represents sulfur atom, group -SO, group -SO2, group -NH or group -NR6; R1 represents aryl, substituted aryl, heteroaryl; R2 and R5 represent hydrogen atom, alkyl, allyl, substituted benzyl, group -CH2-C(O)R3, group -CH2-C(O)NR3R4 wherein R3, R4 and R6 represent inert substitute. Also, invention relates to new combinatory libraries for search compound-leaders and candidates for medicinal compounds preparing by screening the combinatory libraries.

EFFECT: valuable medicinal properties of compounds.

9 cl, 1 tbl, 9 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to derivative of triazaspiro[5.5]undecane of the formula (I): wherein R1 means compound of the formula (1): or (2): wherein G represents a bond, (C1-C4)-alkylene, (C2-C4)-alkenylene or -CO-; ring A represents: (1) C5-10-membered mono- or bicarbocyclic ring or (2) 5-10-membered mono- or bicyclic heterocycle comprising 1-2 nitrogen atoms and/or 1-2 oxygen atoms; substitute R6 means the following values: (1) (C1-C4)-alkyl, (2) halogen atom, (3) nitrile group, (4) trifluoromethyl group and others; R2 represents: (1) (C1-C4)-alkyl, (2) (C2-C4)alkynyl or (3) (C1-C4)-alkyl substituted with a substitute represented in claim 1 of the invention claim; each R3 and R4 represents independently: (1) hydrogen atom, (2) (C1-C4)-alkyl or (3) (C1-C4)-alkyl substituted with 1-2 substituted taken among: (a) Cyc 2 and (b) hydroxy-group (wherein Cyc 2 represents (1) C5-6-membered monocarbocyclic ring or (2) 5-6-membered monocyclic heterocycle comprising 1-2 nitrogen atoms and/or one oxygen atom), or R3 and R4 form in common group of the formula: wherein R26 represents (C1-C4)-alkyl or Cyc 2; R5 represents hydrogen atom or (C1-C4)-alkyl, its quaternary ammonium salt, its N-oxide or its nontoxic salt. Also, invention relates to pharmaceutical composition inhibiting HIV, regulator of chemokine/chemokine receptor and agent used in treatment and prophylaxis of some diseases, such as inflammatory diseases, asthma, atopic dermatitis, nettle rash, allergic diseases, nephritis, hepatitis, arthritis and other diseases that comprise as an active component above described compound of the formula (I) or its quaternary ammonium salt, its N-oxide or its nontoxic salt. Also, invention relates to (3R)-1-butyl-2,5-dioxo-3-((1R)-1-hydroxy-1-cyclohexylmethyl)-9-(4-(4-carboxyphenyloxy)phenylmethyl)-1,4,9-triazaspiro[5.5]undecane or its pharmaceutically acceptable salt and pharmaceutical composition based on thereof, and to (3R)-1-butyl-2,5-dioxo-3-((1R)-1-hydroxy-1-cyclohexylmethyl)-9-(4-(4-carboxyphenyloxy)phenylmethyl)-1,4,9-triazaspiro[5.5]undecane hydrochloride and pharmaceutical composition based on thereof.

EFFECT: valuable medicinal properties of derivative and composition.

16 cl, 32 ex

FIELD: organic chemistry, medicine.

SUBSTANCE: method relates to piperazinedione compounds of formula I wherein and are independently direct bond or double bond; F represents H or CH(RaRb), when is single bong, or C(RaRb), when is double bond; Z represents R3O-(Ar)-B, wherein B represents CH(Rc), when is single bond or C(Rc), when is double bond; Ar represents piridyl; and R3 represents alkyl, aryl, C(O)Rd, C(O)NRdRe or SO2Rd ; R1 and R2 are independently H, C(O)Rd. Compounds of formula I have antitumor activity. Methods for treatment of tumors and angiogenesis inhibition also are disclosed.

EFFECT: new compounds useful in treatment of tumors and angiogenesis inhibition.

42 cl, 23 ex

FIELD: organic chemistry, medicine, gastroenterology, oncology.

SUBSTANCE: invention relates to a new agent used in treatment of gastroenteric tumors. Invention describes an agent for treatment of gastroenteric tumors representing compounds of the general formula: A-X1-NO2 or their salts wherein A means -RCO(X)t wherein t represents a whole number from 0 to 1; X means oxygen atom (O); R is taken among the following groups: (IA), (IIA), (IIIA), (IVA), (VA) and (VIA) such as given in the invention claim; X1 in the formula A-X1-NO2 means a bivalent binding bridge taken among the following: -YO- wherein Y means a liner or when possible a branched (C1-C20)-alkylene comprising preferably from 2 to 5 carbon atoms, or possibly a substituted cycloalkylene comprising from 5 to 7 carbon atoms of the formula: wherein n3 means a whole number from 0 to 3; wherein nf means a whole number from 1 to 6 but preferably from 2 to 4; wherein R1f means hydrogen atom (H), -CH3; nf means a whole number from 1 to 6 but preferably from 2 to 4. Invention provides a new medicinal agent used in treatment of gastroenteric tumors.

EFFECT: valuable medicinal properties of agent.

2 cl, 11 tbl, 18 ex

FIELD: medicine and veterinary.

SUBSTANCE: invention relates to method for prophylaxis of oncological diseases, or infections mordibidized by bacteria or fungi and protozoa, or arteriosclerosis, or diabetes mellitus, or diseases mediated by delayed hyperresponsiveness reaction, or diseases mediated by somatic cell gene mutations. In the first embodiment of invention blood extracellular DNA destroying agent, such as DNAase, is administered into blood. In the second embodiment agent, binding to blood extracellular DNA, such as anti-DNA antibody is administered into blood. According to the third embodiment enzyme altering of blood extracellular DNA chemical structure is administered into blood. According to the forth embodiment agent, stimulating synthesis and/or activity of endogenic deoxyribonuclease or agent stimulating synthesis of antibody binding to blood extracellular DNA are administered into blood.

EFFECT: effective method for treatment of abovementioned diseases without side effects when prolonged using of preparation affected on blood extracellular DNA.

7 cl, 11 tbl, 18 ex, 5 dwg

FIELD: veterinary medicine.

SUBSTANCE: vaccine has antigenic material produced from La-Sota strain reproduced in 9-10 days old SPF-hen embryos having infectious activity of at least 9.7 lg EID50/cm3 and hemagglutination activity equal to at least 1:512, protective medium and adjuvant of thymogen taken in effective proportions. The protective medium comprises 20% lactalbumin hydrolyzate solution and degreased milk in 1:5 proportion. The vaccine is produced by mixing its ingredients and with following preparation lyophilization. Ready vaccine is dry porous mass of light yellow color. The vaccine is applicable for carrying out specific prophylaxis. The vaccine is introduced as spray at a dose of 1 cm3/head.

EFFECT: high antigenic and immunogenic activity; no adverse side effects.

7 cl, 8 tbl

The invention relates to the field of veterinary Virology

The invention relates to the field of immunology

The invention relates to the field of veterinary biotechnology, Virology and Microbiology

The invention relates to veterinary Virology, in particular strains of paramyxoviruses birds, and can be used to control immunogenic vaccines, as well as in scientific and industrial laboratories in the design of vaccines against Newcastle disease

The invention relates to the field of the biotechnology industry, in particular the production of virus-inducer for the production of human leukocyte interferon

The invention relates to the field of veterinary medicine

FIELD: medical virology and microbiology.

SUBSTANCE: new bacteriophage strain Helicobacter pylori, having lytic activity is disclosed. Method for production of antigastritis and antiulcer drug based on the new bacteriophage strain also is disclosed. Claimed method includes providing of purified suspension of bacteriophage Helicobacter pylori with lytic activity of 109 FFU/ml and addition of 1 % chinozole thereto. Moreover sorbitole and gelatose also may be introduced into said agent. Obtained mixture is frozen and lyophilized.

EFFECT: high effective antigastritis and antiulcer drug useful in medicine.

3 cl, 1 tbl, 2 ex

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