Natural antitumor or antiviral substances and their use

 

The invention relates to medicine and provides substances that are effective against tumors and viruses, for which conventional anti-tumor agents and antiviral agents exhibit only insufficient effects, and have cancerostatic action and antiviral effects on different immune tumor. The invention relates to pharmaceutical compositions with anti-tumor or antiviral substance represented by formula 1where R1 represents the basis of nucleic acid, is represented by a specific formula, and R2 represents a hydrogen atom, a hydroxyl group or a methoxy group, or its pharmaceutically acceptable salt. And also relates to a method of obtaining the substance represented by formula 1, or its pharmaceutically acceptable salts, characterized in that cultivate NS-cell, which has the ability to produce the substance represented by formula 1, collect the substance represented by formula 1, from the culture fluid and, if necessary, transform the substance into its pharmaceutically acceptable salt. In addition, the disclosed cell line and treatment. Isopathic-before:always;">

The present invention relates to novel compounds from natural sources, which are useful as pharmaceutical drugs, inhibit the proliferation of tumor cells or viruses and demonstrate antitumor or antiviral effect, method of producing these compounds, their applications and cells for producing these compounds.

In more detail, the present invention relates to products of the cultivation of cell lines derived from the falling of the placenta (placenta deciduae) person, usually cultivation D57-positive, HLA.DR-strongly positive natural cells suppressor (NS) human cell lines D57+HL-DRbrightNS (CPD-1)" (hereinafter referred to as "NS-cells"), which are then clone of cells TTC-1, the method of production of products of cultivation, their application and NS cells.

In the field of cancer chemotherapy have been investigated clinical application of a number of metabolic products of microorganisms, such as bleomycin and adriamycin, and these products are used on almost clinical stages.

However, these products do not always satisfactorily operate on different tumors and clinical applicability complicated, as clinically identified phenomenon ON GAN GAKKAI, pages 12-15 (1988)].

On the other hand, found that maternal immune response to fetal controlled, mostly falling placenta (decidua), and a large number of cell groups of large granular lymphocytes (LGL) with NK cell marker, is accumulated in the layer falling placental mammals, including humans, at the initial stage of pregnancy [Mori T. and others , Immunomolecular mechanism in mammalian implantation. Endocrine J., 41 (send.):S17].

It is recognized that NS-cell is related to the LGL is the cell group, non-immune T cells, b cells or macrophages, as it has a receptor to WGA-lectin in mice and a marker, the sugar chain CD57 in humans.

Described that NS-cell also has a function of suppressing the division of cancer cells, due to its feature strongly suppress fission reaction of lymphocytes, such as the fission reaction of lymphocytes under the influence of MHC-derestricting mitogen or reaction of the mixed culture of lymphocytes (MLR) (Tilden and others, J. Immunol., 130, 1171).

However, with regard to causal factors, managing immunosuppressive influence on the effect of suppressing the proliferation of cancer cells NS-cell is described protein family of TGF-and lepidozona substance with molecular masses is clear up to the present time and requires clarification.

Well-known compounds having a chemical structure similar to the connection according to the invention, and providing antitumor and/or antiviral activity, include fluorouracil (U.S. Patent 2802005 and 2885396), doxifluridine (doxifluridine) (U.S. Patent 4071680), Tegafur (Tegafur) (Patent UK 1168391), AZT (Zidovudine), AZT (German Patent 3608606), DDI (Didanosine; ddl) (European laid open publication 206497) and the like.

However, these antitumor agents and antiviral agents type of nucleic acid is not only effective against the tumor or viral cells limited, but also operate on normal human cells, so these agents are highly toxic and become an object of concern to the public.

The present invention was carried out taking into account the development of similar problems in the prototype, and the purpose of the present invention is a search among metabolites in human cells compounds having efficacy against cancers, viruses and the like, to which the well-known anticancer and antiviral agents exhibit only insufficient effects, and provision of material, which provides cancee reduced side effects, so it does not harm normal cells.

To achieve the goal, the applicants of the present invention have conducted important research and found that the NS cell line induces apoptotic death of cancer cells human K, Molt4, U937, BeWo, GCIY; inhibits cytokinesis cancer cells, and also found the substance type nucleic acids (indicated by the AIF), which are produced by this cell line and induce death of cancer cells due to apoptosis, and separated and purified these substances to determine their structures. Applicants believe that these substances can be developed and used as cancerostatic agents natural type and antiviral agents, as a pharmaceutical preparation based on a completely new idea and have fewer side effects.

The applicants of the present invention were cultured NS-cage, with the ability to produce the compound of formula (1) and obtained from the falling human placenta, gathered in the compound of formula (1) from the culture fluid (supernatant and cells, particularly of the supernatant), turned it, if necessary, pharmaceutically acceptable salt, receiving the connection performance orR2 represents a hydrogen atom, a hydroxyl group or a methoxy group, or its pharmaceutically acceptable salt, and found that the compound of formula (1) or its pharmaceutically acceptable salt induces death of cancer cells due to apoptosis, inhibits proliferation of cancer cells and demonstrates antitumor or antiviral effect. Thus, the purpose of the present invention has been achieved.

That is, the present invention relates to antitumor or antiviral substances represented by the above formula (1), where R1 and R2 have the meanings as defined above, or its pharmaceutically acceptable salt, method thereof, a pharmaceutical preparation containing as an effective ingredient of the antitumor or antiviral substance represented by the above formula (1), or its pharmaceutically acceptable salt, applying the above compounds with the aim of preparing a pharmaceutical product and therapy and D57-positive, HLA.DR-strongly positive NS-cells isolated from the falling human placenta.

Brief description of drawings
The figure 1 shows the NMR spectrum of the compound R the figure 4 shows the NMR spectrum of the compound P4;
the figure 5 shows the NMR spectrum of the compound P5;
the figure 6 shows the NMR spectrum of the compound P6;
the figure 7 presents the UV and mass spectra and the structural formula AIF (P1);
the figure 8 presents the UV and mass spectra and the structural formula AIF (P2);
the figure 9 presents the UV and mass spectra and the structural formula AIF (P3);
the figure 10 presents the UV and mass spectra and the structural formula AIF (P4);
the figure 11 presents the UV and mass spectra and the structural formula AIF (P5);
the figure 12 presents the UV and mass spectra and the structural formula AIF (P6);
the figure 13 shows phase-contrast micrograph NS cell lines of the present invention;
in the figure 14 (A-G) photographs, which show the result of the study, DNA fragmentation;
the figure 15 shows the graphs that show the result of indirect co-culture NS-cells and target cells;
in the figure 16 (a, b) presents graphs that show suppression include3H-thymidine into target cells via AIF from NS (CPD-1) cell supernatant (sup);
in the figure 17 (a, b) presents pictures that show the result of the fragmentation of DNA of the target cells of means is presented graphs that show suppression include3H-thymidine;
in figure 20 (a-C) presents pictures that show the result of the study of fragmented DNA of target cells via AIF;
in figure 21 (A-D) presents pictures that show the result of the study of fragmented DNA of target cells via AIF;
the figure 22 shows a graph that illustrates the effect of suppressing the AIF on cancer cells of the human stomach;
figure 23 presents pictures that show action AIF on the dissimilation cancerous tissue of the human stomach (a, b), and pictures that show the result of the study, DNA fragmentation (C), and
figure 24 presents a graph which shows the suppressive effect of AIF in cell T-cell leukemia human.

The best way of carrying out the invention
Next described in detail the present invention. Primarily used here is described a variety of terms and definitions.

The compounds of formula (1) is obtained by physico-chemical separation and purification of apoptosis-inducing factor (AIF) in the supernatant of the culture of the NS cell line and represents the formula (1)

where R1 represents the battle hydrogen atom, hydroxyl group or a methoxy group, these compounds are designated as P1, P2, P3, P4, P5 and P6, respectively, the active fractions obtained by the method of high performance liquid chromatography with reversed phase.

The terms P1, P2, P3, P4, P5 and P6" here denote specifically 2'-deoxyuridine, ribothymidine, 2'-O-methyluridine, thymidine, 2'-O-methylinosine and 2'-O-methylguanosine respectively.

That is, the compound of formula (1) is R1, if R1 and R2 represent

and hydrogen atom, respectively;
is P2, if R1 and R2 represent

and hydroxyl, respectively;
is R3 if R1 and R2 represent

and a methoxy group, respectively;
is the P4, if R1 and R2 represent

and hydrogen atom, respectively;
is P5, if R1 and R2 represent

and a methoxy group, respectively, and
is P6, if R1 and R2 represent

and a methoxy group, respectively.

The following summarizes the physico-chemical characteristics of typical compounds of the present invention.<H12N2About5;
so pl. 165o;
mass spectrum: FAB-MS (mass spectrometry bombarded with fast electrons) high-resolution m/z 229 [M+1];
UV spectrum:[H2O (pH of 7.2), max 258,5 nm];
an NMR spectrum1H (300 MHz, CDCl3,, M. D.): an NMR spectrum for P1 is shown in figure 1;
solubility: soluble in organic solvents, such as methanol and dimethyl sulfoxide, and water;
diskriminirovaniya of acidic, neutral and basic substances: base material;
thin layer chromatography (Merck, Kieselgel 60F254), the Rf value of 0.64 [manifesting a solvent mixture of chloroform/methanol/water=60:40:8];
high-performance liquid chromatography:
column: TSK gel ODS-80TM, 4,h mm (Tosoh);
mobile phase: water system containing 0.1% triperoxonane acid with a gradient of acetonitrile containing 0.1% triperoxonane acid, 5%/360 min;
a flow rate of 0.5 ml/min;
detection: UV 214 nm;
the retention time of 20 minutes

b) Physico-chemical characteristics P2:
nature is colorless crystal;
molecular formula C10H14N2About6;
so pl. 183-185o;
mass spectrum: FAB-MS (mass spectrometry with bombing bystryk /> an NMR spectrum1H (300 MHz, CDCl3,, M. D.): an NMR spectrum for P2 is shown in figure 2;
solubility: soluble in organic solvents, such as methanol and dimethyl sulfoxide, and water;
diskriminirovaniya of acidic, neutral and basic substances: base material;
thin layer chromatography (Merck, Kieselgel 60F254), the Rf-value of 0.66 [manifesting a solvent mixture of chloroform/methanol/water=60:40:8];
high-performance liquid chromatography:
column: TSK gel ODS-80TM, 4,h mm (Toso);
mobile phase: water system containing 0.1% triperoxonane acid with a gradient of acetonitrile containing 0.1% triperoxonane acid, 5%/360 min;
a flow rate of 0.5 ml/min;
detection: UV 214 nm;
the retention time of 29 minutes

C) Physico-chemical characterization P3:
nature is colorless crystal;
molecular formula C10H14N2About6;
so pl. 159o;
mass spectrum: FAB-MS (mass spectrometry bombarded with fast electrons) high-resolution m/z 259 [M+1];
UV spectrum:[H2O (pH 7,2), max 263 nm];
an NMR spectrum1H (300 MHz, CDCl3,, M. D.): an NMR spectrum for P3 is shown in figure 3;
rastonirovali of sour, neutral and basic substances: base material;
thin layer chromatography (Merck, Kieselgel 60F254), the value of Rf to 0.72 [manifesting a solvent mixture of chloroform/methanol/water=60:40:8];
high-performance liquid chromatography:
column: TSK gel ODS-80TM, 4,h mm (Toso);
mobile phase: water system containing 0.1% triperoxonane acid with a gradient of acetonitrile containing 0.1% triperoxonane acid, 5%/360 min;
a flow rate of 0.5 ml/min;
detection: UV 214 nm;
the retention time of 40 minutes

g) Physico-chemical characterization P4:
nature is colorless crystal;
molecular formula C10H14N2O5;
so pl. 185o;
mass spectrum: FAB-MS (mass spectrometry bombarded with fast electrons) high-resolution m/z 243 [M+1];
UV spectrum:[H2O (pH of 7.2), max 267 nm];
an NMR spectrum1H (300 MHz, Dl3,, M. D.): an NMR spectrum for P4 are shown in figure 4;
solubility: soluble in organic solvents, such as methanol and dimethyl sulfoxide, and water;
diskriminirovaniya of acidic, neutral and basic substances: base material;
thin layer chromatography (Merck, Kieselgel 60F254), the value of Rf 0,69 [about the/> column: TSK gel ODS-80TM, 4,h mm (Toso);
mobile phase: water system containing 0.1% triperoxonane acid with a gradient of acetonitrile containing 0.1% triperoxonane acid, 5%/360 min;
a flow rate of 0.5 ml/min;
detection: UV 214 nm;
the retention time of 50 minutes

d) Physico-chemical characteristics P5:
nature is colorless crystal;
molecular formula C11H14N4O5;
so pl. 210-212o;
mass spectrum: FAB-MS (mass spectrometry bombarded with fast electrons) high-resolution m/z 283 [M+1];
UV spectrum:[H2On (pH 7,2), max 283 nm];
an NMR spectrum1H (300 MHz, CDCl3,, M. D.): an NMR spectrum for the P5 shown in figure 5;
solubility: soluble in organic solvents, such as methanol and dimethyl sulfoxide, and water;
diskriminirovaniya of acidic, neutral and basic substances: base material;
thin layer chromatography (Merck, Kieselgel 60F254), the Rf-value of 0.67 [manifesting a solvent mixture of chloroform/methanol/water=60:40:8];
high-performance liquid chromatography:
column: TSK gel ODS-80TM, 4,6150 mm (Toso);
mobile phase: water system containing 0.1% TST flow 0.5 ml/min;
detection: UV 214 nm;
the retention time of 64 minutes

e) Physical and chemical characteristics P6:
nature is colorless crystal;
molecular formula C11H15N5O5;
so pl. 218-220o;
mass spectrum: FAB-MS (mass spectrometry bombarded with fast electrons) high-resolution m/z 298 [M+1];
UV spectrum:[H2About pH 11, pH 1), max 258 nm at pH 11, 256 nm at pH 1];
an NMR spectrum1H (300 MHz, CDCl3,, M. D.): an NMR spectrum for P6 is shown in figure 6;
solubility: soluble in organic solvents, such as methanol and dimethyl sulfoxide, and water;
diskriminirovaniya of acidic, neutral and basic substances: base material;
thin layer chromatography (Merck, Kieselgel 60F254), the Rf value of 0.59 [manifesting a solvent mixture of chloroform/methanol/water=60:40:8];
high-performance liquid chromatography:
column: TSK gel ODS-80TM, 4,h mm (Toso);
mobile phase: water system containing 0.1% triperoxonane acid with a gradient of acetonitrile containing 0.1% triperoxonane acid, 5%/360 min;
a flow rate of 0.5 ml/min;
detection: UV 214 nm;
the retention time of 83 minutes

The following are cytologica;
2) the origin of the cells: cell tissue falling away of the placenta derived from human placenta at 7 weeks of pregnancy;
3) subculture: capable of continuous proliferation;
4) prerequisite growth factor: capable of proliferation in medium without growth factor normal cells of the endometrium person or heparin;
5) preservation of cells, proliferation conditions, the dependence of the proliferation of this cell line is usually saved and satisfactorily proliferate at a temperature of 36-38oC, preferably at 37oC, and at pH 6.5-7, preferably between 7.0;
6) the ability of cell proliferation: if cultivated 2105/ml of these cells under the above conditions, after 3 days cell density reaches at least 5105/ml;
7) function: it is not interstitial cell falling away of the placenta, because there are no receptors of estrogen and proges-Theron. It produces functional substances such nucleic acids and thereby suppresses the division of lymphocytes due to the reaction of the mixed culture of lymphocytes (MLR) or mitogenic irritation;
8) form colonies: forms a colony on a Petri dish, but not in soft agar;
9) hsome: metacentric;
11) configuration according to chromosomal analysis: cell derived from human tissues;
12) number of chromosomes 99-100, 107-108;
13) cell surface marker is a cell of the immune system, as is D57-positive and HLA.DR-strongly positive;
14) a nutrient medium for the preservation and proliferation: satisfactory persist and proliferate in the environment 10% FCS+RPMI-1640 or medium not containing serum from which removed thymidine.

The cell strain of the present invention can be obtained by using, for example, the following method. That is, the cell falling human placenta can be obtained, for example, by the method described in J. Clin.Invest., 52, 2745-2756 (1973). Very simply this way, the cell can be obtained by taking a sample of the endometrium or the falling human placenta as possible aseptic way (you can use any fabric, making sure that this fabric intrauterine, and samples the falling human placenta preferred due to their easy access), washing it before treatment with trypsin to separate the cells from the connective tissue.

Used in the present invention, the cell can be any cell, it is guaranteed that it is CD57-positive, HLA.DR-strongly positive, produced by the equipment it is a NS-cell or strain her clone, or strain subclone. This cell strain (NS) can be obtained in the usual way, by derivatization in the selected clone strain.

For example, you can obtain the strain clone with a higher capacity of production of the substances of the formula (1), controlling the normal way a number of substances of the formula (1) obtained by cloning NS cells. Specifically, 1105cells TTK-1 pre-cultivated several times, applying the analysis method of the bounding dilution (limiting dilution analysis), at 37oIn the presence of 5% of carbon dioxide gas, so that the number of cells was 0-1 on the cell, and thus selected cell clone with a higher capacity of production of the compounds of formula (1).

In this regard, the cell line of the present invention made in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-Shi, Ibaraki-Ken, Japan) with an incoming number FERM BP6350 (date of initial investment: may 19, 1997) (transferred from FERM P-16233 hip hop Japanese investments (date of transfer: may 13, 1998)).

The figure 13 shows a phase-contrast photomicrograph (400x) NS-cell strain of the present invention. NS-cellular strain adheres to the substrate cultue here is a way to obtain the compounds of the present invention.

Compounds of the present invention can be obtained by making cells derived from the falling human placenta (usually NS-cells) in culture medium containing nutrient, aerobic cultivating these cells in CO2-incubator, collecting the compounds of the present invention represented by the formula (1), from the culture fluid (supernatant and cells, preferably, supernatant) and optionally converting the compounds in pharmaceutically acceptable salt.

Thus obtained NS cells derived from the falling human placenta, usually can be grown in the medium used for culturing animal cells, containing, if necessary, added to the serum, and, specifically, in the normal environment for culturing cells containing 20% fetal bovine serum.

As a medium for culturing cells can mention any environment capable of supporting cells in culture, such as the environment TOGETHER, Wednesday MEM (Earle. Dulbecco, High-GEM), the Ham F-10, F-12), Wednesday ISKOF Wednesday, 119, medium L-15 medium, Msso 5A, Wednesday NCTC135 Wednesday, Williams E, Waymouth medium, among which particularly preferred medium RPMI-1640.

The cultivation can be performed in the same manner as in the production of metal can apply any methods of cultivation, including stationary cultivation, cultivation with stirring, the cultivation with the use of shaking and cultivation with aeration, among which preferred cultivation with application of the shaking and the cultivation of deep aeration-mixing. During culturing of the cells above the environment mainly contains a few percent (specifically about 5%) of carbon dioxide gas.

This environment has a pH of 6-8, appropriate values are near neutral. The cultivation can be carried out at 30-40oWith, preferably about 37oC. Time of cultivation depends on the used medium, pH, temperature and other conditions, but cells can be subcultural, usually after 4-5 days of cultivation.

To obtain from this culture are substances of the formula (1) use appropriate selection methods commonly used to collect produced by the microorganism metabolite from the culture.

Thus obtained compound of formula (1) can be cleaned with known methods of separation-purification method such as solvent extraction, a method using ion exchange resin, the method of absorption chrome or distribution is appropriate to apply the usual methods of separation, such as high performance liquid chromatography with reversed phase thin layer chromatography. A high degree of purification can be achieved, for example, by column chromatography on silica gel, ion exchange chromatography, affinity chromatography and high performance liquid chromatography with reversed phase, especially in combination.

As described here (in the section "confirmation of the applicability of the invention"), is supposed to apply the compounds of the present invention as a pharmaceutical, such as a therapeutic agent for the treatment of cancer and viral diseases in mammals, including humans.

In this regard, pharmacological effect in vivo can provide compounds of the present invention in a phosphorylated form, and needless to say that such phosphorylated compounds are also included in the scope of this invention.

Suitable tumor, which, as expected, the compounds of the present invention have a therapeutic effect include, for example, not only blood cancer in humans, but also epithelial cancer, including cancers of the digestive system such as stomach cancer or thick the new bodies, such as ovarian cancer or horiokartsinoma.

Suitable viruses, which, as expected, the compounds of the present invention have a therapeutic effect include, for example, these types of retroviruses human virus T-cell human leukemia (HTLV), human immunodeficiency virus, (HIV) and the like.

Substances of the formula (1) of the present invention, if they are used as anticancer or antiviral agent, can also be applied in the form of their pharmaceutically acceptable salts.

Non-toxic salts of the compounds of the present invention represented by the formula (1) include, for example, salts with inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid or phosphoric acid, salts with organic acids such as acetic acid, citric acid or tartaric acid, salts with organic sulfonic acids such as methanesulfonate acid or para-toluensulfonate acid, or salts with amino acids such as aspartic acid, glutamic acid or lysine.

Pharmaceutically acceptable salts of the compounds of the present invention can be obtained by combining appropriately alwaysbest alizations titration of a solution of the free compound of the present invention by acid solution.

If the compounds of the present invention is used as an antitumor or antiviral agent, the dosage forms can choose different forms, which include, for example, preparations for oral administration such as tablet, capsule, powder, granule and liquid, and sterile liquid preparations for parenteral use, such as a solution or suspension. The drug of the present invention comprises as an effective ingredient one or more compounds of the present invention and, if necessary, may include various well-known additives such as a carrier, diluent or excipient (pharmaceutical composition).

Solid preparations can be prepared directly in the form of tablets, capsules, granules or powder, and can be prepared using suitable additives.

These additives include, for example, sugars such as lactose and glucose, starches, such as corn, wheat and rice starch, fatty acids such as stearic acid, inorganic salts such as metasilicate sodium aluminate magnesium and anhydrous calcium phosphate, synthetic polymers such as polyvinylpyrrolidone and polyalkyl and benzyl alcohol, synthetic derivatives of cellulose, such as methylcellulose, carboxymethylcellulose, ethylcellulose and hypromellose, as well as the usual additives, such as water, gelatin, talc, vegetable oils, gum Arabic and the like.

Solid preparations such as tablet, capsule, granule or powder, usually can contain the effective ingredient in an amount of 0.1-100% by weight, preferably 5-100% by weight.

Liquid preparations can be prepared in the form of suspensions, syrup, drug injection or the like using water, alcohols or conventional additives, suitable for liquid medications, such as oils, derivatives of vegetable oils such as soybean oil, peanut oil or sesame oil.

Specifically, the solvents suitable for parenteral dosage preparations in the form of a preparation for intramuscular, intravenous, subcutaneous or intratumoral injection include, for example, distilled water for injection, an aqueous solution of lidocaine hydrochloride (for intramuscular injection), saline, aqueous glucose solution, ethanol, liquid for intravenous injection such as an aqueous solution of citric acid or sodium citrate, solutions of the electrolyte is such preparations for injection can also be prepared in the form of a powder or mixture, containing added thereto suitable additives, which forms the solution for the same application, as well as pre-dissolved form. These injections can usually contain the effective ingredient in an amount of 0.1-10% by weight, preferably 1-5% by weight.

Liquid preparations for oral administration, such as suspension or syrup may contain the effective ingredient in the amount of 0.5-10% by weight.

The drug of the present invention may include one or more compounds of the present invention as an effective ingredient, and examples of effective ingredients preferably are typical compounds of the present invention P1-P6, more preferably a combination of two to six of these compounds.

Practically preferred dose of a compound of the present invention can be changed as appropriate, depending on the types of compounds used, types included songs, frequency, dose, exposed to treatment of disorders and severity of condition of patients.

For example, the dose for adults is 10-500 mg/day in the case of oral administration and 10-100 mg/day in the case of parenteral intake, preferably with vnutrivodnaya can be taken once as a single dose or in part, for two to five receptions.

Confirmation of the applicability of the invention
[Summary]
Strain D57-positive, HLA. DR-strongly positive natural cells suppressor (NS) obtained from the fabric falling away of the placenta (placenta deciduae) man, causes apoptosis of cancer cells in humans, such as C, Molt4, U937, GCIY and BeWo, and suppresses proliferation of these cells. Factor causing apoptosis (AIF), is produced and secreted in the culture supernatant NS cell lines. Thus, NS produced by the cells, AIF is separated and cleaned by physical and chemical methods. The activity of AIF is measured by the ability include3H-thymidine into the cells and the method of DNA fragmentation. First of all, the supernatant of the culture of the NS cell line adsorb on C18 column and elute to separate AIF. The crude extract are on the plate for thin-layer chromatography (TLC) and the active fractions are separated and cleaned by high-performance liquid chromatography with reversed phase (HPLC).

Components (P1-P6), derivative peaks obtained by HPLC, induce cell death and inhibit the proliferation of cancer cells C, Molt4, U937, GCIY and BeWo, but does not harm normal cells WI-38 derived lung fetal human. Physical and helices derivatives. Indeed, the structural analysis of these substances by the method of mass spectrometry bombarded with fast electrons (FAB-MS) and NMR shows that P1 is a 2'-deoxyuridine, P2 - ribothymidine, P3 - 2'-O-methyluridine, P4 - thymidine, P5 - 2'-O-methylinosine and P6 - 2'-O-methylguanosine. Also significant degenerative effect against tumors observed in the experiment on animals with mice that have transplantirovannam them human cancer tissue, which impose these six compounds AIF.

[Verification test]
Finally separated and obtained by HPLC connections AIF add to tumor target cells to test in vitro the ability to induce apoptosis (in vitro experiment) and therapeutic effects of the compounds AIF in SCID mice with inoculated them with cancer cells (experiments on animals).

[I] the Experiment in vitro
Next, to illustrate the applicability of the present invention measure the interaction between the NS cell line (as a typical representative of the present invention) and a typical cell from various cancerous human cells as target cells, measured directly or indirectly in the sharing of their culture. Below shows the used cells:
(1) S-cleto the strain by culturing the cells of the tissue falling human placenta at 7 weeks of pregnancy, which is D57-positive, HLA.DR-strongly positive natural immunosuppressive cell derived bone and lymphatic tissues;
(2) Molt4 (cell line leukemia T-cell human);
(3) C (cell line leukemia of erythroblasts person);
(4) U937 (cell line leukemia of histiocytes person);
(5) GCIY (cell line carcinoma of the stomach of a man);
(6) BeWo (cell line choriocarcinoma person);
(7) WI-38 (cell line of normal fibroblasts derived lung tissue of a human fetus).

The above cells are cells obtained by subcultivation in the medium 10% FCS + RPMI-1640 or medium not containing serum from which removed thymidine in the incubator at 37oWith under 5% CO2.

Experimental example 1
The study of direct co-culture NS cell lines and target cells Molt4/K562/U937/GCIY/ BeWo/WI-38 (direct response)
The study of direct co-culture: use NS, Molt4 (cell line leukemia T-cells), C (cell leukemia line of erythroblasts person), U937 (cell leukemia line of histiocytes person), GCIY (cell line carcinoma of the human stomach), BeWo (cell line choriocarcinoma human is, 106and Molt4/K562/U937/GCIY/BeWo/WI-38 (106) cells jointly cultivated in 2 ml of culture medium in 24-hole tablet within 24-48 hours.

As a result of direct interaction (co-cultivation) between the NS cell line target cells after 24 hours, extracted DNA cells and subjected to electrophoresis on 2% agarose gel.

In the end, as shown in figure 14A, lanes 1, 2 and 3 represent the results of co-culture with 104, 105and 106NS cells per cell, respectively, and 106Molt4 on the cell, and the degree of fragmentation of the DNA in the target cell increases, depending on the number of inoculated NS cells.

On the other hand, lane 4 is the result of cultivation only with 106NS cells on the cell, and lane 5 is the result of cultivation only with 106Molt4 on the cell. In these cases we do not observe DNA fragmentation. Lane M is a marker that shows the sizes of the DNA.

As shown in figure 14B, lanes 1, 2 and 3 represent the results of co-culture with 104, 105and 106NS cells per cell , respectively, and 106C on the cell, and the degree of DNA fragmentation in the cell-mission is possessing an effect of the cultivation only with 106C on the cell, and lane 5 is the result of cultivation only with 106NS cells on the cell. In these cases we do not observe DNA fragmentation.

As shown in figure 14C, lanes 1, 2 and 3 represent the results of co-culture with 106, 105and 104C cells on the cell and Molt4 respectively, lane 4 is the result of cultivation only with 106Molt on the cell, and lane 5 is the result of cultivation only with 106C on the cell. In any case, not induced fragmentation of DNA.

As shown in figure 14D, lanes 1, 2 and 3 represent the results of co-culture with 104, 105and 106NS cells per cell, respectively, and 106BeWo on the cell, and the degree of fragmentation of the DNA in the target cell increases, depending on the number of inoculated NS cells. On the other hand, lane 4 is the result of cultivation only with 106NS cells on the cell, and lane 5 is the result of cultivation only with 106BeWo on the cell, lane 6 is the result of co-culture with 106BeWo on the cell and 106GCIY on the cell, and the strip 7 predstaviteli DNA.

As shown in figure 14, lanes 1, 2, 3 and 4 represent
the results of co-culture with 103, 104, 105and 106NS cells per cell, respectively, and 106U937 on the cell, and the degree of fragmentation of the DNA in the target cell increases, depending on the number of inoculated NS cells.

On the other hand, the strip 7 is the result of co-culture with 106Molt4 on the cell and 106U937 on the cell, the strip 8 is the result of cultivation only with 106Molt4 on the cell, the strip 6 is only from 106U937 on the cell, and the strip 5 from 106NS cells on the cell. In none of these cases is not induced fragmentation of DNA.

As shown in figure 14F, lanes 1, 2 and 3 represent the results of co-culture with 104, 105and 106NS cells per cell, respectively, and 106GCIY on the cell, and the degree of fragmentation of the DNA in the target cell increases, depending on the number of inoculated NS cells.

On the other hand, lane 4 is the result of cultivation only with 106GCIY on the cell, and lane 5 is the result of cultivation only with 106NS per cell. In none is the result of co-culture with 104, 105and 106NS cells per cell, respectively, and 106WI-38 cell, and the target cells do not observe fragmentation, regardless of the increase in the number of NS cells. Lane 4 is the result of cultivation only with 106WI-38 cell, and lane 5 is the result of cultivation only with 106NS per cell. In none of these cases is not induced fragmentation of DNA.

That is, the interaction (co-cultivation) between NS-cells and cells Molt4, K, BeWo, U937 and GCIY gives DNA-fragmentation in target cells after 24 hours, and the degree of DNA fragmentation depends on increasing the number of NS cells.

On the other hand, fragmentation of DNA is not induced in any of the cases culturing cells C and Molt4 cells Molt4 and U937 cells BeWo and GCIY. Also not see DNA fragmentation by co-cultivation NS cells and normal cells WI-38. This indicates that the NS cell line does not harm normal cells. Indeed, one can say that the NS cell line induces cancer cell death of a person due to a specific apoptosis (DNA fragmentation).

Experimental example 2
Indirect interaction (indirect smoga culturing. The camera for the cultivation intended for the reaction between the cells and having a bottom in the form of filter (diameter 0.45 μm), is inserted into the cell and add cells Molt4/C (104).

After culturing for 3 days at 37oWith under 5% CO2collecting target cells and expose them radioisotope research on the inclusion of3H-thymidine, to study painting at the inclusion of the dye Trypanosoma blue and research method of DNA fragmentation to determine the interaction between cells.

In this system, the number of cancer cells-targets in the chamber is reduced with the increase in the number of NS cells after 72 hours (figure 15).

This experimental result shows that NS-cell produces soluble low-molecular compound, which penetrates through the membrane millborough filter in the camera and cause the death of cancer cells-targets.

Experimental example 3
The study, confirming the suppression include3H-thymidine in the target cell and DNA fragmentation, through AIF, roughly extracted by thin layer chromatography (TLC)
Sample, liofilizovannye from 50 ml of culture supernatant NS cell lines subjected chromatographia tryout to dryness by evaporation of the solvents by using nitrogen gas. Retained in the column fraction inhibited the proliferation of K562 cells/Molt4 and induces DNA fragmentation.

Next, the fraction shown by TLC. That is, the solution separated on a C18 column connections in a mixture of chloroform: methanol (1:1) is applied in the form of spots on a thin layer and show a mixture of chloroform:methanol:distilled water (60: 40: 8). After developing, the plate is divided into two factions: the below band (Rf 0.5) with the reagent phenol red, contained in the environment (Rf<0.5, the TCX-A), and above this band (Rf>0.5, the TCX-B), and gel with each of the factions soskrebajut and extracted with a mixture of chloroform/methanol, drying the extract using nitrogen gas. In the faction (U) above phenol red (Rf 0.5), and detect the presence of a compound that suppresses the proliferation of K562 cells/Molt4 (figure 16A-C) and induces DNA fragmentation (figure 17A-C).

In the quality control used in fresh medium or in the culture supernatant of target cells K562/Molt4 not detect the production of a compound that inhibits proliferation of target cells or induces DNA fragmentation. Thus confirm that the substance (AIF), which induces apoptosis (cell death) of cancer cells, producyruet, than phenol red (Rf phenol red 0,5).

Experimental example 4
The study of suppression include3H-thymidine in the target cell Molt4 connection AIF, completely isolated and purified by high performance liquid chromatography (HPLC)
The active fraction on TLC separated and cleaned on a column of ODS-80TM (Tosoh), elwira acetonitrile containing 0.1% triperoxonane acid, gradient 0-5%/360 min at a flow rate of 0.5 ml/min, by monitoring the optical density (OD) at 214 nm.

All samples obtained from six major peaks (1-6) (figure 18A, HPLC-chart), inhibit the inclusion of3H-Timi-DIN in the Molt4 cell. In particular, samples of the peaks 1 and 4 demonstrate strong activity and also a strong activity demonstrates a mixed sample dilutions 1/10 of these peaks (0,7 µg/ml) (figure 19).

As shown in column m, the apparent synergistic effect of these peaks (AIF).

Experimental example 5
The critical number of AIF, which is able to induce DNA fragmentation, cells Molt4
As shown in figures 20A and 20B, samples, derivatives of the corresponding active peaks (1-6) separated by HPLC (A: 7 μg/ml, In: 73-2µg/ml), react A 2% agarose gel. At concentrations of 7 mg/ml and 73-2µg/ml register DNA fragmentation, cells Molt4, but, as shown in figure 20C, the effect disappears at low concentrations 73-5µg/ml.

Experimental example 6
The study of the fragmentation of DNA of the target cells BeWo through AIF, finally selected by HPLC
As shown in figure 21A, the samples derived from the respective peaks (1-6) separated by HPLC (21 μg/ml), react with BeWo cells within 48 hours, and DNA BeWo cells extracted and subjected to electrophoresis on 2% agarose gel. All peaks induce DNA fragmentation, BeWo cells (figure 21A).

Experimental example 7
The study of the fragmentation of DNA of the target cells U937 via AIF, finally selected by HPLC
As shown in figure 21B, the samples derived from the respective peaks (1-6) separated by HPLC (21 μg/ml), react with U937 cells within 48 hours, and DNA U937 cells extracted and subjected to electrophoresis on 2% agarose gel. All peaks induce DNA-fragmentation in U937 cells (figure 21B).

Experimental example 8
The study of the fragmentation of DNA of the target cells GCIY through AIF, econchallenge by HPLC (21 μg/ml), react with the GCIY cells within 48 hours, and DNA GCIY cells extracted and subjected to electrophoresis on 2% agarose gel. All peaks induce DNA fragmentation, GCIY cells (figure S).

Experimental example 9
The study of DNA fragmentation of normal human cells WI-38 through AIF, finally selected by HPLC
As shown in figure 21D, does not induce cell proliferation or DNA fragmentation, if the target cell is a normal cell WI-38, even at triple the number (63 μg/ml) of the samples derived from the respective peaks (1-6). As shown in figure 14G, this corresponds to the result that the direct reaction of NS cells and cells WI-38 does not cause any harm to normal cells.

Experimental results in this series are important. That is, these results show that AIF, isolated and purified in accordance with the present invention, specific causes cell death due to apoptosis (DNA fragmentation) of cancer cells, but does not harm normal cells, in other words, a normal cell can be developed as an ideal anticancer agent, does not produce any side effects with the introduction of supressing agent, as immunosuppressive agents prototypes demonstrate their action through inhibition of pharmacological effect on the division of lymphocytes.

If the culture supernatant of cells Molt4 or fresh medium (final inspection) should be treated in a similar manner as the environment, culture NS cell lines, and finally subjected to HPLC, HPLC-the chart does not see such active peaks (1-6), which is obtained from the environment culture NS cell lines (figure 18B, C).

On the other hand, the same sample as the sample supernatant of culture of the NS cell line was observed in the HPLC chart of the sample obtained by the destruction of the NS cell line, followed by extraction. This shows that the NS cell line contains the same active peaks (1-6) that peaks in the supernatant of the culture.

The results from the above in vitro experiments can be summarized as follows.

1. NS cell line induces cancer cell death of target due to apoptosis.

Set that as a result of direct interaction (co-culture) between NS-cells and cells C, Molt4, BeWo, U937 and GCIY 24 hours is fragmented DNA of target cells (figures 14 a, b, C, D, E, F).

The degree of the fragments, fragmentation of DNA is not induced by co-cultivation of cells C and Molt4 (figure 14C), cells Molt4 and U937 or GCIY cells and BeWo. Also not see DNA fragmentation by co-cultivation NS-cells WI-38 (figure 14G). This result shows that NS-cell is not harmful to normal human cells. In other words, we can say that only NS-cell has the ability to induce apoptosis of specific cancerous human cells.

2. NS cell line secretes AIF in the supernatant of the culture.

When indirect interaction (indirect co-cultivation) system, in which NS-cell and the target cell C/Molt4 placed in the camera, the number of leukemic target cells in the chamber is reduced with the increase in the number of NS cells after 72 hours (figure 15). In addition, the camera determine DNA fragmentation, leukemic target cells. The experimental results show that NS-cell produces soluble low-molecular compound, which penetrates through the membrane millborough filter in the camera and cause the death of leukemic target cells.

3. Roughly purified by thin layer chromatography (TCX) AIF suppresses the inclusion of3H-thymidine in target cells and induces FR, will overhaul chromatographicaliy on a C18 column, and retained in the column fraction, which elute with acetonitrile and concentrated, inhibited the proliferation of K562 cells/Molt4 and induces DNA fragmentation.

Further, this fraction is examined by the method of TCX. Determine that a substance that inhibits cell division K562/Molt4 (figure 16A, b) and induces DNA fragmentation (figure 17A, B), is present in this fraction, and the position of his band above bands phenol red (Rf 0.5 in) TCX (Rf>0.5, the TLC-In).

In the quality control used in fresh medium not containing serum (SFM), or in the culture supernatant of target cells K562/Molt4 not detect the production of a compound that induces DNA fragmentation.

Therefore, confirm that the connection (AIF), which induces apoptosis (cell death) of cancer cells, is produced in the culture supernatant NS cell lines, and that AIF find in fraction (Rf>0.5), and moving on TCX faster than phenol red (Rf phenol red 0,5).

4. AIF, completely isolated and purified by HPLC, induces DNA-fragmentation in target cells.

All active fraction (TLC), roughly isolated from 500 ml of culture supernatant NS-cell method TCX, divide and Cista, HPLC-chart).

All samples from the respective peaks 1-6 (7 μg/ml) inhibit the inclusion of3H-thymidine into the cell Molt4, in other words, these samples inhibit cell division Molt4. In particular, samples of the peaks 1 and 4 demonstrate strong activity, and also a strong activity demonstrates a mixed sample dilutions 1/10 of these peaks (0,7 µg/ml, respectively) (figure 19). As shown in column M figure 19, this results in a synergistic effect of these peaks (AIF).

5. The critical number of AIF, which is able to induce DNA fragmentation, cells Molt4".

In accordance with the suppressive effect of active peaks (1-6) on cell division Molt4, samples derived from the respective peaks (1-6) (7 μg/ml), induce DNA fragmentation, cells Molt4 (figure 20A).

During processing of Molt4 cells within 48 hours of each sample (73-2μg/ml) derived from the active peaks separated by HPLC method, identify DNA-fragmentation in target cells (figure 20B), but the effect disappears at low concentrations 73-5µg/ml (figure 20C).

(6) If the target cells used BeWo, U937 and/or GCIY, the samples derived from the peaks (1-6) (21 μg/ml) and diluted 1/10 mixed samples (2.1 and use the normal cell WI-38, no suppression of cell division or DNA fragmentation, even if the target type samples derived from the respective peaks (1-6), in an amount three times greater than add to tumor target cells (figure 21D). This corresponds to the result that the direct interaction of NS cells and cells WI-38 does not cause any harm to normal cells. That is, the result shows that AIF, isolated and purified in accordance with the present invention, specific causes cell death due to apoptosis (DNA fragmentation) of cancer cells, but is unlikely to harm normal cells, in other words, a normal cell can be developed as an ideal anticancer agent, does not produce any side effects with the introduction of cancer patients. In addition, if the culture supernatant of cells Molt4 or a fresh environment handle similar to handle culture NS cell lines, and finally subjected to HPLC, HPLC-charts do not see such an active peaks (1-6), as obtained from environment culture TTK-1 (figure 18B, C).

On the other hand, the same sample as the sample supernatant of culture of the NS cell line was observed in the HPLC diagram of the sample obtained by the destruction of the NS-coboy substance, obtained solely from NS-cell lines and secreted in a nutrient medium.

7. Study of physico-chemical characteristics of the AIF and the structure definition.

The presence of amino acids or hexose in completely isolated and purified by HPLC AIF, are determined by the reaction of arsenosulvanite or reaction of ninhydrin. In addition, we discovered that AIF is a connection type of nucleic acid, since the maximum absorption in the UV spectrum observed around 260 nm. The molecular weight calculated using mass spectrometry (FAB-MASS). Finally define the structure by the method of nuclear magnetic resonance of protons (NMR).

Suppose that the corresponding active peaks (1-6) separated by HPLC, are heat resistant, negative on the reaction of ninhydrin and orcinol and, therefore, contain nonprotein and nhexane connection.

Also found that AIF is a connection type of nucleic acid, since the maximum absorption in the UV spectrum observed around 260 nm (left upper spectra in figures 7-12).

Based on the structural analysis, it was decided that NS cells were cultured in large quantities (300 liters) and samples were prepared from an aliquot of cardamomi mass spectrometry (FAB-MASS) NMR (NMR). Active peaks 1-6 AIF: 2'-deoxyuridine for P1, ribothymidine for P2, 2'-O-methyluridine for P3, thymidine for P4, 2'-O-methylinosine for P5, and 2'-O-methylguanosine for P6 (upper right panel of figures 7-12).

In this regard, under the arrow in the mass spectrum indicated the molecular weight of the (+N) for each of the active peaks (1-6) AIF (bottom spectra in figures 7-12).

[II] Tests on animals
Used typical cancer cells blood Molt4 and typical cells of epithelial cancer GCIY, which experimentally in vitro confirmed the effect of AIF.

1. About 108cancer cells of the human stomach (GCIY) inoculant 20 SCID mice, and when the tumor diameter reaches 0.5 cm (0.25 cm2) (approximately 2 weeks), divide these mice in 4 groups, ranging therapeutic experiments using AIF in mice with tumors. Each group includes 5 mice.

Example research 1
Five mice injected sample solution (fraction TLC), which is fractionated described above by TLC and contains six nucleosides (AIF) in phosphate buffer (PBS). The dose is 1 mg (respectively 90 mcg 2'-dose irradiation on neurogenesis, 110 mcg ribothymidine, 135 μg of 2'-O-methyluridine, 322 µg thymidine, 226 μg of 2'-O-methylinosine and 117 μg of 2'-O-methylguanosine) in 0.5 ml of PBS, the sample solution is injected (caravan described above by TLC and contains a fraction, position in which TLC following provisions phenol red, i.e. does not contain any of the six nukes. This sample injected intravenously (via tail vein), only 18 times. Tumor size was defined as the product of larger to smaller (square). Figure 22 illustrates the average tumor size for five mice after 1, 2 and 3 weeks respectively. As is obvious from the graph, find a significant difference between the group receiving AIF, and the control group: the group receiving AIF, shows a much stronger suppressive effect compared with the control group (t-test, P<0,01).A solution of 1 mg of each fraction TLC and in 0.3 ml of PBS injected with three portions (0.1 ml/dose) directly into the tumors of five mice with a tumor within 3 days. The sample containing 6 nucleosides (AIF) fractions TLC (containing the same components as in example 1), resulting in a complete degeneration of the tumor. Fraction TLC-And not containing AIF as a control, generally does not cause degeneration of the tumor (as a typical example in figures SA and In the control mouse with tumor and treated mice, 3 mice that were injected AIF and in whom the tumor disappears, and 3 control mice). As ocinc, that is, the death of the tumor due to apoptosis.

2. About 108cell leukemia T-cells (Molt4) inoculant 10 SCID mice, and when the tumor diameter reaches 0.3 cm (0,09 cm2) (approximately 2 weeks), divide these mice into 2 groups, ranging therapeutic experiments using AIF in mice with tumors.

Each of the 5 mice injected multiple times (18 introductions, alternately intravenous and direct intratumoral), the sample solution containing three nucleoside AIF (400 mg 2'-dose irradiation on neurogenesis, ribothymidine and thymidine, respectively, only 1.2 mg) in 0.5 ml PBS (total of 21.6 mg). Tumor size was defined as the product of larger to smaller (square) and follow it for 3 weeks. Tumors in three of five mice completely degraded, and the remaining two mice also degrade to an extremely small size. The graph in figure 24 shows the average tumor size for five mice after 1, 2 and 3 weeks respectively. As is obvious from the graph of the control group, the tumor becomes larger in the control group, which was administered PBS, and, thus, registering a significant difference between the group receiving AIF, and the control group (t-test, p<0,001).Maternal immune Rea is x, the applicants of the present invention have found, that a large number of groups of cells, which presumably represent LGL-cell with NK cell marker, is accumulated on the falling placental mammals, including humans, at the initial stage of pregnancy. Also, consider that these groups of NK-cells play an important role in the implantation phase of blastocyte. That is, NK-cell controls the formation of the embryonic placenta, continuously prolifera and, thus, passing to offspring during pregnancy. In other words, the natural immune response against cancer is on the stage of pregnancy.

NS-cell line of the present invention is a CD57-positive, HLA. DR-strongly positive human natural cell suppressor, which was cloned and created a layer of the falling human placenta at 3-month pregnancy. This cell line does not have estrogen receptors or progesterone typical of interstitial cells falling placenta, or not secretes prolactin, therefore, considered that it is a cell line, floating from the bone marrow or lymphatic tissues.

As specific functions of NS cells indicate not only the suppression of the production of antibodies and division limp the aba, H. Ogata, R. Yasumuzu, E. E. Sardina, K. Inaba, S. Kuma, R. A. Good, and S. Ikehara, 1990, inhibition of proliferation of tumor cells by natural cells suppressor cells present in the bone marrow of mice, Cancer Res., 50: 2582). As effector compounds NS-cells, which mediate the control of cell division, indicate protein family of TGF-(D. A. Clark, K. C. Flanders, D. Banwatt, W. Millar-Book, J. Manuel, J. Stedronska-Clark and B. Rowley, 1990, falling placenta of pregnant mice produces a unique immunosuppressive molecule, related to growth factor beta-2, J. Immunol. , 144:3008) and lepidozona compound having a molecular weight of 10,000 or less (Mortari F. and S. K. Singhal, 1988, obtaining suppression factor derived human bone marrow. Influence on the synthesis of antibodies and activated by lectin cell proliferation, J. Immunol., 141:3037), but their essence remained doubtful. Also, as indicated in the previous study authors immunosuppressive mechanism of NS cells (Tatsumi, K., T., Mori, E. Mori, H. Kanzaki, and T. Mori, 1987), immunoregulatory factor, isolated from cell lines derived from tissues falling human placenta, Am. J. Reprod. Immunol. Environ., 13:87), protein compound inhibits the division of T cells through IL-2.

To my surprise, found that the NS cell line induces giekie cancer cells of human stomach GCIY cells and choriocarcinoma human BeWo and inhibits their proliferation.

In addition, to induce apoptosis of cancer cells, these compounds (AIF) was isolated from the supernatant of the culture of this cell line, peeled and determine their structure. This process includes a first elution with acetonitrile compound that liofilizovannye sample of the supernatant of the culture of the NS cell line binds to the hydrophobic C18 column. Erwerbende active factors roughly cleaned by TLC. Active fraction shows a higher Rf value than phenol red, and inhibits cell division C and Molt4, resulting in fragmentation of the DNA. In conclusion, the active molecules are divided and clean as six major peaks by HPLC on a C18 column with reversed phase. All these six peaks samples inhibit the proliferation of cancerous target cells and cause DNA fragmentation. Thus, successfully confirmed that these samples represent new objects, i.e. the AIF. Also discovered that these six AIF are most effective when they are used in a cocktail, combining these factors (a combination of many agents currently used for cancer chemotherapy; in this case, the natural combination of many agents). In addition, these six AIF have psihohimicheskoe svojstvennymi compounds does not contain hexose. It is estimated that these compounds have a molecular weight of 100-500 determined on the basis of it is passed through a dialysis membrane with a threshold of 500 D and the measurements of their mass spectra.

In addition, as shown in the upper left parts of figures 7-12, each of the active peaks, fractionated by HPLC shows UV spectrum with a maximum absorption at 245-265 nm. That is, it is strictly suggests that AIF is a substance-related nucleic acids or their derivatives.

For a sample sufficient for structural analysis, AIF finally divide, clean from the supernatant of a large number of cultural NS cells and finally determine its molecular weight by the method of mass spectrometry bombarded with fast electrons and the structure by NMR on protons (figure 1-12). These six AIF belong to the nucleoside having a unique structure, in which a portion of the base or ribose deoksigenirovanii or methylated.

As far as I know the authors, for the first time in the world found that, as shown in the invention, the NS-human cell secretes substances such nucleic acids and induces apoptosis of cancer cells.

Clinically available protivoraketnoi toxicity and, therefore, when introduced into the human organism creates a social problem due to severe side effects and their effectiveness.

Observations disclosed by the applicants of the present invention AIF present in vitro studies and animal experiments (these experimental systems are conventional systems in vitro and on animals adopted in practice to assess the applicability of man anti-cancer and antiviral agents; see, for example, figures 22, 23, 24). However, in this regard were developed by the Foundation for the development of the ideal cancerostatic agents of natural type, which have no harmful effects on normal human cells, but cause the death of cancer cells by natural pharmacological mechanism, inducyruya specific apoptosis (DNA fragmentation) of cancer cells, whereas it is implied that when introduced into the human body side effects will be reduced.

[IV] Additional description of the drawings
The figure 14 shows the fact that the NS cell line induces death of cancer target cells due to apoptosis, but does not cause apoptosis and, as a consequence thereof, the death of normal cells.

The result p is Airout and subjected to electrophoresis on 2% agarose gel.

As shown in figure 14A, lanes 1, 2 and 3 show the results of co-culture with 104, 105and 106NS cells per cell, respectively, and 106Molt4 on the cell, and the degree of fragmentation of the DNA in the target cell increases, depending on the number of inoculated NS cells. On the other hand, lane 5 is the result of cultivation only with 106Molt4 on the cell, and lane 4 is the result of cultivation only with 106NS cells on the cell. In these cases we do not observe DNA fragmentation. M is a marker.

As shown in figure 14B, lanes 1, 2 and 3 represent the results of co-culture with 104, 105and 106NS cells per cell, respectively, and 106C on the cell, and the degree of fragmentation of the DNA in the target cell increases, depending on the number of inoculated NS cells. On the other hand, lane 4 is the result of cultivation only with 106C on the cell, and lane 5 is the result of cultivation only with 106NS cells on the cell. In these cases we do not observe DNA fragmentation. M represents the marker.

As shown in figure 14C, lanes 1, 2 and 3 represents estwenno, lane 4 is the result of cultivation only with 106Molt4 on the cell, and lane 5 is the result of cultivation only with 106C on the cell. In any case, not induced fragmentation of DNA. M represents the marker.

As shown in figure 14D, lanes 1, 2 and 3 represent the results of co-culture with 104, 105and 106NS cells per cell, respectively, and 106BeWo on the cell, and the degree of fragmentation of the DNA in the target cell increases, depending on the number of inoculated NS cells. On the other hand, lane 4 is the result of cultivation only with 106NS cells on the cell, and lane 5 is the result of cultivation only with 106BeWo on the cell, lane 6 is the result of co-culture with 106BeWo on the cell and 106GCIY on the cell, and lane 7 is the result of cultivation only with 106GCIY on the cell. In none of the cases do not observe DNA fragmentation.

As shown in figure 14, lanes 1, 2, 3 and 4 represent the results of co-culture with 103, 104, 105and 106NS cells per cell, respectively, and 106Molt4 on the cell and 106U937 on the cell, the strip 8 is the result of cultivation only with 106Molt4 on the cell, the strip 6 is only from 106U937 on the cell, and the strip 5 from 106NS per cell. In none of these cases is not induced fragmentation of DNA. M is a marker.

As shown in figure 14F, lanes 1, 2 and 3 represent the results of co-culture with 104, 105and 106NS cells per cell, respectively, and 106GCIY on the cell, and the degree of fragmentation of the DNA in the target cell increases, depending on the number of inoculated NS cells. On the other hand, lane 4 is the result of cultivation only with 106GCIY on the cell, and lane 5 is the result of cultivation only with 106NS per cell. In none of these cases were not observed DNA fragmentation. M is a marker.

As shown in figure 14G, lanes 1, 2 and 3 represent the results of co-culture with 104, 105and 106NS cells per cell, respectively, and 106WI-38 cell, and the target cells do not observe fragmentation, the independent>WI-38 cell, and lane 5 is the result of cultivation only with 106NS per cell. In none of these cases is not induced fragmentation of DNA. M represents the marker.

Figure 15 explains that the NS cell line inhibits proliferation of target cells in the indirect co-cultivation with cells K562/Molt4.

As shown in figure 15, in the system of indirect interactions (indirect co-culture), where NS-cell and cell K562/Molt4 placed in the camera, the number of cancer cells-targets inside the chamber is reduced after 72 hours with the increase in the number of NS cells.

In figures 16-17 explains that AIF, roughly purified by thin layer chromatography (TLC), suppresses the inclusion of1H-thymidine in the target cell and induces DNA fragmentation.

The fraction obtained by means of introducing the sample, liofilizirovannogo approximately 50 ml of the supernatant (TTC-1 Sup) culture NS cells (CPD-1), in column C18, elution of retained fraction of acetonitrile and concentration of the eluate, inhibits cell division K562/Molt4.

Then this fraction is used in TLC, as shown in figure 16A, to obtain a fraction (U) with TLC higher phenol red (Rf 0.5) is contained in the CPE is (SFM), control group (C) and the fraction (L), located on the TLC below phenol red.

On the other hand, this fraction is used in TLC, as shown in figure 16B, to obtain a fraction (U) with TLC higher phenol red (Rf 0.5) is contained in the environment. Found that this fraction (U) significantly inhibits cell division Molt4 compared with the fraction (L), located on the TLC below phenol red.

In the control group (C) and used fresh medium not containing serum (SFM) does not suppress the proliferation of the target cells.

As shown in figures 17A and b, the fraction is obtained by making the specimen, liofilizirovannogo approximately 50 ml of the supernatant NS cell culture (CPD-1), in column C18, elution of retained fraction of acetonitrile and concentration of the eluate. Thus obtained fraction is used in TLC and divide by the fraction (U, lanes 1 and 3) with the position on TLC higher phenol red (Rf 0.5) is contained in the environment, and the fraction (L, lanes 2 and 4) with TLC below phenol red, each of these fractions tested for the presence of a compound that induces DNA fragmentation, cells K562/Molt4. These extracts react with cells K562/Molt4, and cellular DNA extracted h is th figure substance (AIF), which induces apoptosis (cell death) of cancer cells, is produced in the supernatant NS cell culture, and AIF was detected in fractions, which moves on TLC faster than phenol red (Rf>0,5).

The figure 18 shows the HPLC chart AIF, finally separated by HPLC.

As shown in figure 18A, the entire active fraction, roughly purified by TLC from 500 ml of culture supernatant NS cells (CPD-1), is separated and extracted by HPLC with reversed phase on a TSK gel ODS-80TM (Tosoh). Get six main peaks (1-6).

As shown in figures 18B and C, when the supernatant (In) culture of cells Molt4 or fresh medium (C) is subjected to the same treatment as the environment NS-cell culture (CPD-1), and finally used in a method HPLC, HPLC-chart not see active peaks obtained from the environment NS-cell culture (1-6).

Figures 19 and 20 explain the fact that AIF, finally selected by HPLC, suppresses the inclusion of3H-thymidine in cancer the target cell and induces DNA fragmentation, and is a critical number of AIF, which may induce fragmentation.

As shown in figure 19, all samples (7 μg/ml) of the main peaks (1-6) separated by HPLC, pevnost, and diluted 1/10 mixed sample of these peaks (respectively of 0.7 µg/ml) also shows strong activity. As shown in column M figure 19, this indicates a synergistic effect of these peaks.

As shown in figures 20A, samples prepared from the corresponding peaks (1-6), react with the cell Molt4 within 48 hours, and then DNA Molt4 cells extracted and subjected to electrophoresis in 2% agarose gel. According to the suppressive effect of the respective peaks (1-6) on the division of Molt4 cells, both derived from the respective peaks (1-6), and the mixture is diluted 1/10 of the corresponding peaks (1-6) induce DNA fragmentation, cells Molt4 (figure 20A, lanes 1-7).

As shown in figures 20B and C, the samples derived the corresponding peaks (1-6) separated by HPLC (: 73-2ág/ml With: 73-5μg/ml, react with cell Molt4 within 48 hours, and then DNA Molt4 cells extracted and subjected to electrophoresis in 2% agarose gel. As shown in figure 20B, in samples derived the corresponding peaks (1-6) (73-2µg/ml), and mixture of 1/10 dilutions of the respective peaks (1-6) (0,73-2µg/ml) see fragm the each sample separately or a mixture of 1/10 dilutions (0,73-5µg/ml), as shown in figure 20C (lanes 1-7).

Figure 21 explains that AIF, finally selected by HPLC, induces fragmentation of the DNA of the cancerous human cells BeWo/U937/GCIY, but does not induce DNA fragmentation, normal human cells WI-38.

As shown in figures 21A, b and C, samples derived the corresponding peaks (1-6) (21 μg/ml), and the mixture dilutions 1/10 (2,1 mg/ml) samples derived the corresponding peaks (1-6), react with BeWo cells(A)/U937 ()/GCIY (C) within 48 hours, and then the DNA of each cell of the target is extracted and subjected to electrophoresis in 2% agarose gel. In accordance with the effect of suppressing the dividing target cells active peaks (1-6) both samples obtained from the respective peaks (1-6), and a mixture of 1/10 dilutions of the respective peaks (1-6), induce DNA-fragmentation in target cells (lanes 1-7).

As shown in figure 21D, samples derived from the respective peaks (1-6) in a 3-fold amount (63 μg/ml) used for cancer target cells respond within 48 hours with normal human cell WI-38, and then the DNA of a cell WI-38 extracted and subjected to electrophorese in 2% agarose gel. None of the samples derived active peaks, does not cause suppression de the CI and structural definition of the relevant active peaks 1-6 (AIF), separated by HPLC.

Cultivated in a large number of NS cell line (about 300 l) and receive samples, carry out fractionation of the respective peaks (1-6), separation and purification by HPLC.

As shown in the left upper spectra of P1-P6 in figures 7-12, respectively, from the analysis of the absorption maximum in the UV range of about 260 nm found that AIF is a substance of the type of nucleic acid.

As a structural analysis of selected UV-spectral analysis, the molecular weight was determined by mass spectrometrically (mass spectrometry bombarded with fast electrons) (the arrows on the bottom spectra). In addition, the final structure of the AIF was determined by the method of nuclear magnetic resonance (NMR), as shown in figures 7-12 (top right).

The present invention is described hereinafter in more detail with reference to examples, but is not limited to these examples.

Example 1. Description of culture medium and the culture fluid.

The supernatant NS-cellular structures (500 ml) after culturing for 3 days in an atmosphere of 5% CO2at 37oWith lyophilized and divide by the C18 column. Since the active substance is contacted in the column, they are separated and elute with acetonitrile Astor connection allocated on a C18 column, in a mixture of chloroform:methanol (1:1) is applied in the form of spots on a thin layer (Kieselgel) and show a mixture of chloroform:methanol:distilled water (60:40:8). After developing, the plate is divided into factions: the lower strip reagent phenol red (L) and above this band (U), gel soskrebajut to restore the corresponding fractions by extraction with a mixture of chloroform:methanol (1:1) and dried extract using nitrogen gas.

Active TLC fraction (U) is separated and purified column ODS-80TM (Tosoh), elwira acetonitrile containing 0.1% triperoxonane acid, gradient 0-5%/360 min at a flow rate of 0.5 ml/min, monitoring absorbance at 214 nm, and receiving six major peaks (1-6) (figure 18A, HPLC-chart).

Desired products P1, P2, P3, P4, P5 and P6 gain in the amount of 0.014 mg, 0.017 mg, 0,021 mg, 0,050 mg, 0.035 mg and 0,018 mg, respectively.

Below are examples of preparations of the compounds of the present invention, but the preparations of the compounds of the present invention is not limited to these examples.

Example of preparation 1, weight.h.:
This compound (P1) - 10
Bicarbonate magnesium - 15
Lactose - 75
The above ingredients are mixed until homogeneous, getting thin ground or granular> Example of preparation 2, weight.h.:
This compound (P2) - 45
Starch - 15
Lactose - 16
Crystalline cellulose - 21
Polyvinyl alcohol - 3
Distilled water - 30
After mixing the ingredients until a homogeneous mixture is crushed into granules, which are dried and then sieved, receiving granules with a particle size 117-1410 microns.

The example of the drug 3
After receiving the pellets in a similar manner as in example of preparation 2, add 4 parts of calcium stearate to 96 parts of these granules, and from this mixture by direct pressing get tablets with a diameter of 10 mm

Example 4 drug
To 90 parts of the granules obtained by the method described in example of preparation 2, add 10 parts of crystalline cellulose and 3 parts of calcium stearate, and from this mixture by direct pressing get tablets with a diameter of 8 mm, to which add the syrup gelatin/suspension of precipitated calcium carbonate, getting pills.

Sample preparation 5
This compound (P3) to 0.6 (parts)
Non-ionic surface-active agent is 2.4
Saline - 97
After mixing the above ingredients by heating the mixture loaded into ampoules and sterilized by receiving the drug for injection.

Industrial applicability
what are harming normal human cells, and it is expected that when introduced into the body, they treat the disease, such as cancer and viral status, natural mechanism that specifically acts on cancer cells, causing cancer cell death due to apoptosis, this implies reducing its side effects. Thus, the compounds of the present invention are useful as antitumor or antiviral agents in pharmaceutical preparations.


Claims

1. Pharmaceutical preparation containing as an effective ingredient protivoopujolevoe or antiviral substance represented by formula 1

where R1 represents a group



or

R2 represents a hydrogen atom, a hydroxyl group or a methoxy group,
or its pharmaceutically acceptable salt and pharmaceutically acceptable additives.

2. Pharmaceutical drug under item 1, in which R1and R2respectively represent


3. Antitumor or antiviral agent, characterized in that it comprises as an effective ingredient of the antitumor or antiviral substance specified in paragraph 1 or 2, or its pharmaceutically acceptable salt.

4. The method of obtaining antitumor or antiviral substances specified in paragraph 1 or 2, or its pharmaceutically acceptable salt, wherein the cultured cell derived from the falling human placenta and has the ability to produce the substance represented by formula 1 in paragraph 1, is collected from the culture fluid substance represented by formula 1, by elution using a solvent containing acetonitrile, purified chromatographic methods, including either separately or in combination - high-performance liquid chromatography (HPLC), thin layer chromatography, chromatography on a column of silica gel, ion exchange chromatography or affinity chromatography, and active molecules specified in paragraph 1 or 2, and, if necessary, make the connection in pharmaceutically acceptable salt.

5. Line CD57-positive, HLA. DR-strongly positive natural cells, suppressor, p is formula 1, used to obtain antitumor or antiviral agent specified in paragraph 1 or 2.

6. Method of suppressing tumor or virus, wherein the patient in need of suppression of tumor or virus is administered an effective amount of an antitumor or antiviral substances specified in paragraph 1 or 2, or its pharmaceutically acceptable salt.

7. The use of anticancer or antiviral substances specified in paragraph 1 or 2, or its pharmaceutically acceptable salt to obtain antitumor or antiviral agent or drug.

 

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d-arabinofuranosyl)-n-purine, method for their preparation and use and pharmaceutical composition" target="_blank">

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< / BR>
where arabinofuranosyl residue substituted for 2'-, 3'- or 5'-positions, and esters formed by carboxylic acids, in which decarbonising part selected from n-propyl, tert-butyl, n-butyl, methoxymethyl, benzyl, phenoxymethyl, phenyl, methanesulfonyl and succinyl

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< / BR>
where R is C1-C4-alkyl, does not necessarily substituted by one or more groups, or R is:

< / BR>
a benzyl, robotjam, 2-deoxyribosyl or (CH2)n-OR1where n is 1 or 2, and R1is CH2CH2OH or< / BR>
or their salts

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