Liposome delivery of compositions based on vitamin e

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to creation and application of aerosol spray compositions for treatment of diseases or disorders requiring lowering of cell proliferation and/or induction of cell apoptosis, such as neoplastic, autoimmune, viral diseases. Agent contains as active substance vitamin E based composition of structural formula , where R1 is carboxylic acid; R2 and R3 are hydrogen or R4; R4 is methyl, and R5 is alkyl; or where R1 is hydrogen or carboxylic acid; R2 and R3 are hydrogen or R4; R4 is methyl, and R5 is alkenyl.

EFFECT: improved delivery of specified composition by inhalation with intensified antiproliferation activity.

59 cl, 17 dwg, 6 tbl, 35 ex

 

Cross-reference to related application

This patent application is a non provisional, based on the useful results obtained previously and described in prior patent applications in the United States: proposal No. 60/416602, registered October 15, 2002, which is now cancelled, application No. 60/406807, registered on August 29, 2002, which is now cancelled, and application No. 60/342156, registered on December 19, 2001, which is now cancelled.

The technical field

This invention relates to the field of pharmacology and cancer treatment. More specifically, the present invention relates to the delivery of liposomal drugs are based on compounds of vitamin E as an effective method for the treatment and prevention of cancer.

The level of technology

Control the regulation of survival (viability) and death (apoptosis) is an extremely complex process, involving many intracellular signal transduction pathways and multiple interacting gene products. Cancer cells may show an increased expression of genes and their products that promote cell proliferation, the increase in the number of cancer cells. In addition to increased expression of genes responsible for survival, cancer cells are regulated by type from Catalinas communications genes and their products, which control signals the death, leading to accumulation and increased life-threatening metastasis of cancer cells. The combination of unregulated cell proliferation and suppression of transmission signals that induce cell death can lead to the growth of cancer cells and their survival.

The increase in the number of cells or depends or does not depend on the balance of expression of positive and negative growth regulators of gene products, and the presence or absence of functional signal transduction pathways to cell death. Negative growth regulators genes contribute to the blocking of the cells in the cell cycle. Positive growth regulators genes stimulate cells to grow throughout the cell cycle. Genes involved in apoptosis, can be either proapoptotic or antiutopicheskim, and the dynamic balance between them determines whether a cell lives or dies.

There is a wide range of pathological conditions associated with cell proliferation, which require new strategies and tools. Such pathological conditions can cover almost all types of cells, demonstrating the ability to abnormal cell proliferation or abnormal response to signals cell death. Among the types of cells that show signs of pathological or abnormal growth, are f broblast, endothelial cells and epithelial cells of blood vessels. Thus, new methods are required for the treatment of localized or spreading of pathological conditions in all or almost all organs and tissues of individuals.

In most cases, malignant tumors, whether they are specific to men, such as prostate cancer or testicular, or specific to women, such as breast cancer, ovarian or cervical cancer, or they equally affect both men and women, such as cancer of the liver, skin or lungs, over time, undergo genetic changes and epigenetic changes that lead, ultimately, to the formation of highly metastatic and difficult treatable tumors. Surgical removal of localized tumors can be effective only when the tumor does not extend beyond the primary lesion. If the cancer has spread to other tissues and organs, surgical procedures should be accompanied by other more specific procedures, contributing to the removal of infected or malignant cells. The most common additional procedures for the treatment of diseased or malignant cells, such as chemotherapy or radiation is not directed only on tumor cells and, although they have soresma is but a higher destructive effect on cancer cells, often affected to some extent on normal cells.

Some natural compounds of vitamin E and some derivatives of vitamin E was used as a proapoptotic funds and inhibitors of DNA synthesis and, thus, effective anti-cancer remedies. Structurally, vitamin E consists of a head group chromanol and alkyl side chain. There are eight main natural forms of vitamin E: alpha (α), beta (β), gamma (γ) and Delta (δ) Tocopherols and α, β, γ and δ tocotrienols. Tocopherols tocotrienols differ from what they have saturated wick side chain, but not unsaturated isoprenyl side chain. Four forms of Tocopherols and tocotrienols differ in the number of methyl groups in chromanones head group (α there β has two and δ has one), as shown in table 1.

Table 1
The General structure of Tocopherols and tocotrienols
R1R2R3
Alpha (α)CH3CH3CH3
Beta (β)CH3NCH3
G is MMA (γ )HCH3CH3
Delta (δ)HNCH3

Some research has focused on the high antitumor activity of RRR-α-tocopherylacetate (succinate vitamin E; VES), hydrolyzable ester derived RRR-α-tocopherol. Researchers Prasad and Edwards-Prasad were the first who described the ability succinate vitamin E, and not other forms of vitamin E, to cause morphological changes and growth inhibition of melanoma cells b-16 mouse, and suggested that succinate vitamin E can be beneficial therapeutic antitumor agent (1). Additional studies have shown that succinate vitamin E is an effective inhibitor of the growth of a wide range of types of epithelial cancer cells, including cells of the breast, prostate, lung and colon, as well as hematopoietic, leukemia and lymphoma cells in vitro (2-7).

Recent studies have shown that succinate vitamin E intraperitoneal injection (I.P. Pavlova.) exhibits antitumor activity in animals, which are models of xenotransplantation and allotransplantation (8-11), which indicates a potential therapeutic activity Dan is on the drug. It was shown that at I.P. Pavlova. or oral (r.o.) introduction succinate vitamin E exhibits inhibitory activity in relation to cancer of the gastric cardia in mice, induced by the carcinogen [benzo(a)pyrene], indicating the potential succinate vitamin E as anticarcinogenic means (12). Studies have shown that succinate vitamin E causes dependent on concentration and time of inhibition of growth of cancer cells by blocking DNA synthesis induction of cell differentiation and the induction of apoptosis (5, 6, 10, 13-15, unpublished data).

Inhibition of cell proliferation includes a lock GO/G1 cell cycle is mediated, in part, MAP kinases MEK and ERK1, and the activation of key regulatory protein in the cell cycle, P21(waf1/cip1) (30). Induction of differentiation is characterized by morphological changes, increased signaling of beta-casein, expression of milk lipids, elevated protein levels of cytokeratin 18 and regulation by the type of feedback the Her2/neu protein (13). Differentiation is mediated, in part, through activation of MEK1, ERK1/2 and phosphorylation of the protein c-Jun (13, 14). Many reactions signal apoptosis modulated RRR-α-tocopherolacetate, especially noteworthy is its ability to transform not sensitive to Fas/Fas ligand on wholesae cells in Fas/Fas landcastle cells, and his ability to transform tumor cells do not respond to transforming growth factor (TGF-α), in cells that are sensitive to TGF-αand both recovered reaction converge on the JNK/c-Jun with subsequent translocation of the protein Bax to the mitochondria, induction passage through the permeable membrane of the mitochondria, release of cytochrome C into the cytoplasm, activation of caspases 9 and 3, cleavage of poly (ADP-ribose)polymerase (PARP) and apoptosis(15, 29, 31).

Succinate vitamin E deserves attention not only because of its induction of inhibition of growth of tumor cells, but also due to lack of toxicity toward normal cells and tissues (2-7, 11). Application neytralinogo derived succinate vitamin E has shown that it is intact connection and none of its cleavage products (namely, RRR-α-tocopherol or succinic acid) are not responsible for the antiproliferative activity (4). Thus, I believe that the antiproliferative effects of this derivative of vitamin E is explained by properties other than antioxidant.

RRR-α-tocopherylacetate (VES) is derived RRR-α-tocopherol, which has been modified by the formation of ester bonds with Succinimidyl fragment instead of the hydroxyl fragment in position 6 chromane. Pointed to by the th succintly fragment, associated ester bond with RRR-α-tocopherol, was the most effective form of vitamin E that affect the biological ability to run apoptosis and to inhibit DNA synthesis. This form of vitamin E causes apoptosis of tumor cells, at the same time does not show apoptotic actions in relation to normal cells. The form of vitamin E with Succinimidyl fragment is an effective antitumor agent and at the same time, the intact agent; however, the cellular and tissue esterase that can split succintly fragment, thus turning succinato the form of RRR-α-tocopherol in free RRR-α-tocopherol, make the connection specified is ineffective as an anti-cancer agent. RRR-α-tocopherol did not show any antiproliferative or proapoptotic biological activity in cells of epithelial or immune origin.

Constructing compounds based on RRR-alpha-tocopherol or RRR-alpha-tocotrienol, modified in the C6 position of the first ring head group chromanol alpha-tocopherol or alpha-tocotrienol through education essential communication, will help to create connections with strong anti-cancer properties. Cellular esterase is not detected in the cells; thus, these connections will remain intact in cell culture, is also in vivo. In U.S. patent 6417223 used commercially available pure RRR-α-tocopherol as the source material from which the synthesized analogues of vitamin E. Modifications were made to the three parts of the molecule RRR-α-tocopherol: 6th carbon phenolic rings in chromane to chromeno consisting of phenolic and heterocyclic rings or wick residue. In RRR-α-tocopherol 6th carbon phenolic ring attached hydroxyl (-OH) fragment, which is important for the manifestation of antioxidant activity.

Check analogues of vitamin E on their ability to induce apoptosis in a wide variety of cancer cells but not normal human cells, as well as the analysis of the structure and function analogues show that based on tocopherol analogues, full length which is 29 Å from donor H-bond to the end of the wick residue, which has a length of 17 Åcontaining the fully methylated closed phenolic ring, a saturated heterocyclic closed ring and neytralizuya fragment acetic acid attached to C6 phenolic rings of essential communication, are the most powerful anticancer activity (figure 1).

The method of delivery of therapeutic agents, whether they are delivered by mouth, with food, through the probe, subcutaneous, intraperitoneal, local, intravenous, NUTRISYSTEM, respiratory way and so on, has a great influence on the levels and tissue distribution of drugs. U.S. patent No. 6090407 describes the anticancer drugs paclitaxel and camptothecin that can be incorporated into liposomes for delivery to the respiratory tract of the individual by means of sputtering. The introduction of these anticancer drugs through inhalation of liposomal is a faster and more efficient delivery system than intramuscular injection or oral administration.

The use of liposomal aerosol for delivery of plant alkaloid 9-nitrocamptothecin is the best method of inhibiting growth of cancer cells in breast cancer (28), the colon and the human lung transplanted immunocompetent naked mice, compared with the delivery of 9-nitrocamptothecin by intramuscular injection. Levels 9-nitrocamptothecin upon delivery to the lungs, liver and brain in the form of liposomal aerosol within thirty minutes amounted to 310 ng/g, 192 ng/g and 61 ng/g, respectively, while levels 9-nitrocamptothecin intramuscular delivery to the lungs, liver and brain within thirty minutes was 2-4 ng/g, 136 ng/g and 0, respectively (16). In addition, the specified delivery method, apparently, is vsokoeffectiven the m way directed against metastasis of melanoma and osteosarcoma in the lungs of mice (18). Aerosol delivery of drugs is of great importance, because it is a highly effective and well tolerated by humans (19). Thus, the method of drug delivery through liposomal spray is an effective way to achieve higher levels and greater distribution in the tissues of drugs.

In addition, the inventors recognize the need for other effective ways of liposomal delivery of anticancer drugs based on vitamin E, which promote longer retention, creating a higher concentration of the drug, to reduce systemic toxicity and reduce dosing requirements. Thus, the prior art is lack of effective means of delivery to the individual anticancer medicines based on vitamin E, by means of liposomes. More precisely, aerosolized liposomal delivery or introduction via a stomach tube liposomal compositions of anti-cancer drugs based on vitamin E, which is desirable. The present invention fulfills this long-standing need and desire in this field.

Brief about isana of the invention

In one aspect of the present invention presents a method of treating diseases associated with cell proliferation, comprising a stage of delivery of a composition containing, based on the vitamin E anti-cancer compound, which is contained in the vesicles delivered to the individual in need of such treatment, where the compound has the structural formula

where R1represents hydrogen or carboxylic acid; R2and R3represent hydrogen or R4; R4is stands and R5is alkyl or alkenyl.

In another aspect of the present invention was developed vesicles for delivery based on vitamin E anti-cancer compounds, as described in the text.

Other and additional aspects, features benefits and advantages of the present invention, will be apparent from the following description presents the preferred aspects of the invention, the data for the purpose of disclosure.

Brief description of drawings

To the essence of the invention in which the above features, advantages and objectives, as well as other features which will become apparent, it was revealed and understood in detail, a more detailed description of the invention briefly summarized with reference to the op is edelenyi aspects of the invention, which is illustrated by the accompanying drawings. Said drawings constitute part of the description. However, it should be noted that the accompanying drawings illustrate preferred aspects of the invention and therefore are not considered as limiting the scope of the invention.

Figure 1 presents the model analogues R,R,R-α-tocopherol, depicting the structural elements that are required for the manifestation of a powerful anti-cancer activity. The group R1the side chain at C6 should have such a length that the total length of the molecule does not exceed 29 Å.

Figure 2 presents the results of a comparison of the anticancer activities of the natural alpha - and gamma-Tocopherols, natural alpha-tocotrienol, fractions, enriched with tocotrienol (TRF), synthetic tocopherol and synthetic derivatives of tocopherol in relation to 66 cl.4 GFP tumor cells of the breast.

On figa-3D presents data on apoptosis, induced α-TEM. Figa: 66 cl.4-GFP cells in the breast mouse was treated with 10 µg/ml α-TEM or succinate vitamin E (positive control) or were not treated and were cultured for 3 days. Cells were collected, nuclei were labeled with a fluorescent dye DAPI binding to the DNA, and cells were examined using a fluorescent microscope Zeiss ICM 405 (×400) with filter 487701. Cell nuclei with condensed matter what Tomatina or fragmented nuclei were counted as apoptotic. Data are presented from numerous experiments. Figv/3C: analysis of cells with DAPI stained nuclei shows that α-tea induces apoptosis depending on concentration and time. Data are presented as averages ± the standard deviation of three experiments. Fig.3D: additional evidence in favor of apoptosis induction by α-TEM with cleavage of poly(ADP-ribose)polymerase (PARP). Cell 66 cl.4-GFP were treated with 5, 10 or 20 μg/ml α-TEM within 48 hours, cell lysates were analyzed for the presence of the product of the cleavage of PARP (R) using immunological analysis Western blotting. Data are from 3 separate experiments.

On figa and 4B it is shown that α-tea induces 66 cl. 4 cells to apoptosis in vivo. The induction of apoptosis by α-TEM was determined using 5 μm tumor sections obtained from mice after treatment liposomal α-tea/aerosol and from the control group of mice (N=4)treated with liposomal spray. Apoptotic cells were determined using a kit ApopTag In Situ Apoptosis Detection kit (Intergen, Purchase, NY). On figa presents data comparing the number of apoptotic nuclei after treatment liposomal spray with α-TEM and after treatment control group spray. Figure 4 presents a positively stained apoptotic cells in the tumor with Utah in mice processed liposomal spray with α-TEM and control-treated aerosol mice.

Figure 5 presents the results of inhibition of clonal growth 66 cl.4-GFP by α-TEM. Processing 66 cl.4-GFP cells were placed 600 cells/cultural tablet) via α-TEM at 1.25, 2.5 and 5 μg/ml for 10 days resulted in inhibition of the formation of colonies. Cells were stained with methylene blue and the number of colonies in the treated and control groups was calculated.

Figure 6 graphically presents the body weight of balb/c mice that were implanted 66 cl.4 GFP tumor cells of the mammary gland of the mouse and which was treated with liposomal composition with α-Thea delivered by aerosol. The weight of the animal was examined, starting with the ninth day after implantation of tumor cells.

Figure 7 graphically presents the levels α-TEM in serum and tissue of balb/c mice after 0, 2, 6 or 24 hours after treatment liposomal/α-Thea 1 spray.

On Fig graphically presents the weight of the tumors in balb/c mice that were implanted 66 cl.4 GFP tumor cells of the mammary gland of the mouse and which was treated with liposomal composition with α-Thea delivered by aerosol. The weight of tumors was examined, starting with the ninth day after implantation of tumor cells.

On figa and 9B graphically presents the results from the ATA inhibition of tumor growth and metastases through liposomal aerosol α -Thea. Figa: 66 cl.4-GFP cells at 2×105/mouse was injected in the groin area to a point located midway between the 4th and 5th nipples. Nine days after inoculation of tumor mice (10/group) were not treated or were treated daily liposomal α-tea/aerosol or spray only for 17 days. Tumor volume/mouse were determined at two-day intervals. Tumor volume (mm3) expressed as averages ± standard error. Figw: during the autopsy, the number of fluorescent metastases in the left lobe of the lung after treatment of mice liposomal α-tea/aerosol (8 mice), aerosol only (10 mice) and untreated mice (10 mice) were determined using a fluorescent microscope Nik-on (TE-200; 200X) and using the software Image Pro-Plus S.E. to determine the size of metastases. Data are presented as averages ± standard error.

Figure 10 graphically presents the results of inhibition of tumor growth 66 cl.4 GFP tumor breast cells by liposomal α-TEM and liposomal succinate vitamin E (VES)to be delivered by aerosol spray.

Figure 11 graphically presents the weight of the tumors in balb/c mice that were implanted 66 cl.4 GFP tumor cells in the breast and mouse which worked liposomal the Noah composition with α -Thea, delivered by means of a gastric probe. The weight of tumors was examined, starting with the ninth day after implantation of tumor cells. 11 differs from Fig only by the fact that mice were treated daily via a stomach tube through 5 mg α-TEM in peanut oil or peanut butter only and were treated for only 13 days.

On figa and 12B shown that α-Thea entered via a stomach tube, did not inhibit tumor growth at the site of inoculation, but inhibits lung metastases. Tumor volume and the size and number of metastases have been described in the caption to figa and 9B.

On Fig graphically presents the results of inhibition of tumor growth 66 cl.4 GFP tumor breast cells by liposomal α-TEM and liposomal succinate vitamin E (VES), delivered via a stomach tube.

On figa and 14B graphically presents the results of the inhibition of lung metastases (figa) and lymph node metastases (pigv) tumor cells 66 cl.4 GFP breast cancer via liposomal α-TEM and liposomal succinate vitamin E (VES), delivered via a stomach tube.

On Fig graphically presents the results of inhibition of tumor growth of MDA-MB-435 cancer cells breast cancer via liposomal α-Thea delivered through ladochy probe.

On figa-16C shows apoptotic action α-TEM or a combination of α-Thea/cisplatin on A cisplatinresistant cell line and cisplatinresistant cf-70 cancer cells human ovarian. Figa: apoptotic effect in vitro α-TEM on A cell line and SR-70 lines of cancer cells human ovarian. Figw: α-Thea restores sensitivity to cisplatin cf-70 in vitro. Figs: inhibition of tumor growth by as α-TE, and combinations α-TEM with cisplatin on A and SR-70 in vivo. Data in vivo show that α-Thea turns cisplatin-insensitive cells cf-70 in cisplatinresistant and that combination α-TEM + cisplatin reduce the growth cf-70 cell xenografts of ovarian cancer in Nude mice.

On Fig shows the action α-TEM, methylselenocysteine and TRANS-resveratrol on the growth of malignant tumor MDA-MB-435 GFP FL breast cancer in vivo.

Detailed description of the invention

In one aspect of the present invention a method of treating diseases associated with cell proliferation, including the stage of delivery of a composition containing, based on the vitamin E anti-cancer compound, which is contained in the vesicles delivered to the individual in need of such treatment, where the compound has the structural formula

where R1represents hydrogen or carboxylic acid; R2and R3represent hydrogen or R4; R4is stands and R5is alkyl or alkenyl.

All aspects of this variant embodiment of the invention the anti-cancer compounds based on vitamin E, can be a tocopherol, such as β-tocopherol, γ-tocopherol, δ-tocopherol or 2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethylacetyl)chroman-6-yloxy)acetic acid. In addition, the anti-cancer compounds based on vitamin E, can be tocotrienol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, or a fraction enriched with tocotrienol, or a synthetic compound based on vitamin E, such as dl-α-tocopherol, acetate dl-α-tocopherol, nicotinate, dl-α-tocopherol or phosphate dl-α-tocopherol.

In this embodiment of the invention, the vesicles, as a delivery vehicle, can be a liposome containing a lipid nanoparticle, a microsphere or neosome. A characteristic example of a suitable lipid in the liposome is 1,2-Dilauroyl-sn-glycero-3-phosphocholine. A preferred example is a liposome with the final concentration of anti-cancer compounds based on vitamin E in liposome, i.e., not higher than 20.0 mg/ml OS is consistent on the vitamin E compound/vesicles for delivery may be delivered by aerosol spray, aerosol inhaler, gastric probe, oral ingestion, oral with a soft gel capsule, transdermal patch, subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection. The preferred delivery vehicle is a liposomal aerosol delivered via jet nebulizer.

In the aspect of this variant embodiment of the invention the method may also comprise a stage of introduction of the second composition anticancer funds contained in deliver the vesicles. The second composition may be introduced in combination with based on the vitamin E compound/deliver vesicular composition or consistently for the past. When the composition is administered jointly, based on the vitamin E compound, and an anticancer drug can be in the same delivery of the vesicles. Typical examples of anti-cancer drugs are 9-nitrocamptothecin, cisplatin, paclitaxel, doxorubicin or celecoxib.

Anticancer compounds based on vitamin E, according to the present invention exert an antiproliferative effect expressed in apoptosis, the inhibition of DNA synthesis, inhibition of cell cycle or cell differentiation. In this version done by the means of the invention for quantitative and/or qualitative analysis of antiproliferative action can be performed by detecting the biomarker. A preferred example of the biomarker is a marker KI-67 cell proliferation. Alternatively can be applied immunohistochemical analysis.

Delivery of compounds based on vitamin E, or other anti-cancer compounds according to the present invention can be applied for the treatment of neoplastic diseases and non-neoplastic diseases. Typical examples of neoplastic diseases are ovarian cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, breast cancer, prostate cancer, testicular cancer, glioma, fibrosarcoma, retinoblastoma, melanoma, sarcoma of soft tissue, osteosarcoma, cancer of the colon, carcinoma, kidney cancer, pancreatic cancer, basal cell carcinoma and squamous cell carcinoma. Typical examples of diseases, non-neoplastic diseases are diseases selected from the group consisting of psoriasis, benign proliferative skin diseases, ichthyosis, papilloma, restenosis, scleroderma and hemangioma and leukoplakia.

The methods according to the present invention can be applied for the treatment of diseases, non-neoplastic diseases that develop due to the inability of the selected cells to undergo normal programmed to emochnoy death or apoptosis. Typical examples of diseases and disorders that occur due to inability of the cells to death, are autoimmune diseases. Autoimmune diseases are destruction of the immune system cells, tissues and organs. A characteristic group of autoimmune diseases include autoimmune thyroiditis, multiple sclerosis, severe pseudoparalysis the gravis, systemic lupus erythematosus, dermatitis herpetiformis, coeliac disease and rheumatoid arthritis. The present invention is not limited to autoimmune diseases, but includes all disorders with immune component, such as an inflammatory process involved in the formation of plaques in the cardiovascular system, or a skin lesion caused by ultraviolet radiation.

The methods according to the present invention can be applied for the treatment of disorders and diseases that develop due to viral infections. Typical examples of diseases and disorders that occur due to viral infections are diseases caused by the human immunodeficiency virus (HIV). Because of the connection based on the vitamin E effect on vnutrikletochnykh apoptotic network signal transmission, delivery of the vesicles, such as liposomal aerosol containing contradictory, the new connection based on vitamin E, according to the present invention, has the ability to affect the signal transmission of any type of external cellular signals, such as cytokines, viruses, bacteria, toxins, heavy metals, etc.

In another embodiment, the present invention was developed vesicles for delivery of anticancer compounds based on the vitamin E contained in the vesicles. In the aspect of this variant embodiment of the invention, the vesicles may also contain anti-cancer drug. In a preferred aspect of the delivery of the vesicles is a liposome, in which the ratio of anti-cancer compounds based on vitamin E, lipid is about 1:3 (wt.:wt). Anticancer compounds based on vitamin E, anti-cancer drugs, types of vesicles and methods of delivery can be as described above.

The following definitions are given to facilitate understanding of the invention described in this text. Any terms that are not clearly defined, shall be construed accordingly the meaning of the term in this field.

As used herein, the terms "aerosol", "gastric tube", "liposome", "vesicles for delivery" and "vesicles" will include a variety of chemical compositions for the preparation the Oia preparations vesicles/liposome and a variety of methodologies in relation to the dispersion of the aerosol or oral delivery of these drugs.

As used herein, the term "individual" shall refer to animals and man.

As used herein, the term "biologically inhibiting" or "inhibition of growth of syngeneic tumor grafts will include partial or complete inhibition of growth and also include reducing the rate of proliferation or growth of tumor cells. The dose of the composition according to the present invention, providing biological inhibitory effect can be determined by evaluating the effect of the test item on the growth target malignant cells or cells with impaired proliferation in tissue culture, tumor growth in animals and cell culture, or any other method known qualified specialists in this field.

Used in the text, the term "inhibition of metastasis" will include partial or complete inhibition of the migration of tumor cells from the primary site to other organs, especially to the lungs, as described above. The dose of the composition according to the present invention, providing biological inhibiting metastasis effect can be determined by evaluating the effect of the test item on the growth target malignant cells or cells with impaired proliferation in tissue culture, tumor growth in animals kletochnoi culture or in any other way, known for well-qualified specialists in this field.

Used in the text, the term "inhibiting angiogenesis" will include partial or complete inhibition of the formation of blood vessels in the tumor or reducing the bandwidth of the blood vessels supplying blood to the tumor.

Used in the text the term "induction of programmed cell death or apoptosis will include partial or complete cell death in those cells, which are characteristic of apoptosis morphological and biochemical properties. The dose of the composition according to the present invention, which triggers apoptosis, can be determined by evaluating the effect of the test item on the growth target malignant cells or cells with impaired proliferation in tissue culture, tumor growth in animals and cell culture, or any other method known qualified specialists in this field.

Used in the text the term "induction suppression of DNA synthesis" will include growth suppression due to processing of cells blocked in the cell cycle phases GO/G1, S or G2/M. the Dose of the composition according to the present invention, which causes inhibition of DNA synthesis, can be determined by evaluating the effect of the test item on the growth of the target issue for lighting the x malignant cells or cells with impaired proliferation in tissue culture, tumor growth in animals and cell culture, or any other method known qualified specialists in this field.

Used in the text the term "induction of cellular differentiation" will include inhibition of cell growth due to treatment of cells undergoing cell differentiation according to morphological and biochemical characteristics of differentiation, the stage at which cell proliferation does not occur. The dose of the composition according to the present invention, which induces cell differentiation, can be determined by evaluating the effect of the test item on the growth target malignant cells or cells with impaired proliferation in tissue culture, tumor growth in animals and cell culture, or any other method known qualified specialists in this field.

Used in the text, the termα-Thea" will include similar RRR-α-tocopherol associated with essential communication acetic acid, which is neytralizuya essential similar RRR-α-tocopherol, i.e. 2,5,7,8-tetramethyl-2R-(4R,8R-12-trimethylacetyl)chroman-6-alexianna acid, which can be abbreviated written as RRR-α-tocopherylacetate acid.

The invention is a method of treating Zab the diseases, associated with cell proliferation, via aerosol delivery of liposomal composition, or by delivery via gastric tube liposomal compositions containing natural or synthetic anticancer drug based on vitamin E, and lipid. Compounds according to the present invention, based on vitamin E, exhibit antiproliferative action; typical examples of these antiproliferative effects are apoptosis, inhibition of DNA synthesis, inhibition of cell cycle or cell differentiation. These compounds exhibit anti-metastatic effect and do not show toxicity towards normal cells and tissues in vivo with the introduction of clinically appropriate manner, such as aerosol delivery or via gastric tube.

Perhaps anticancer drug based on vitamin E, and the second anti-cancer drug, such as 9-nitrocamptothecin (9NC), doxorubicin, paclitaxel, celecoxib or cisplatin, but not limited to them, in a liposomal composition or introduced by other means, such as intraperitoneal injection, can be introduced in combination with each other or sequentially one after another. For example, 9-nitrocamptothecin, as topoisomerase inhibitor-1, which breaks one of the TLD is the DNA chains, that leads to breaks in two chains of the DNA replication process, operates by the mechanism of cell death that is distinct from the mechanism α-TEM. In addition, 9NC can activate traditional dependent D95/D95-ligand (FADD/caspase 8) apoptotic pathway (29), which can act together with non-traditional way, i.e. D95/Daxx/JNK/mitochondria. This combined action enhances cell death.

These connections are based on vitamin E, according to the present invention include natural or synthetic Tocopherols or tocotrienols and their derivatives exhibiting chemical functionality at position R1chromane and R5matchlock or isoprenaline side chain. Preferably R1is a carboxylic acid, for example acetic acid. Usually the synthesis of these compounds is accompanied by the interaction of R,R,R-alpha-tocopherol with the appropriate romancenovel acid by methods customary in this field.

Derivatives of natural Tocopherols and tocotrienols are not hydrolyzed by cellular esterases and, although not limited to such derivative with side chain at C6 position, preferably contain a fragment of acetic acid in this position. The preferred compound, which satisfies the structural elements, demand is for the manifestation of a strong anti-cancer actions, is α-TEA, which is different from RRR-α-tocopherol fragment of acetic acid, the associated simple essential connection with the phenolic oxygen at carbon 6 of the head group Romana. VES is different from α-TEA that fragment, succinic acid, binds to a complex of essential communication with phenol at carbon 6 of the head group Romana. Since the antioxidant properties of the parent compound, RRR-α-tocopherol, belong-IT piece of carbon is 6, antitumor properties α-TEA are not mediated antioxidant.

The present invention can be applicable as a therapeutic agent. The methods according to the present invention may be applicable to treatment of any animal. Most preferably, the methods according to the present invention is applicable to humans. Normally, in order to achieve a pharmacologically effective cell death and antiproliferative effect of liposomal compositions with anti-cancer compound can be entered in any therapeutically effective dose. Enter the dosage depends on age, clinical stage and extent of disease or genetic predisposition of the individual, location, weight, type of competitive treatment, if it takes place, and nature of pathological or malignant condition.

Preferably d is serowka is approximately from 0.1 to 100 mg/kg Specialist having ordinary skill in this field will be able to easily determine, without undue experimentation, the appropriate dosages.

In vivo studies of tumor growth and metastasis of human tumor cells, transplanted ectopiceski or orthotropic immunocompetent animals such as Nude mice or in vivo studies, using well-studied animal model, allow you to get pre-clinical results for clinical trials. Not limited to these animal models, such in vivo studies can be focused on other neoplastic and non-neoplastic models of diseases associated with cell proliferation. For example, studies can be used metastatic, not estrogen-dependent MDA-MB-435 cancer cells in the breast, 66 cl.4 GFP cells or resistant to this drug called cisplatin cf-70 cancer cells human ovarian.

In particular, researchers are considering the use of new derivatives of tocopherol, tocotrienol and other derivatives chromane, whether or not containing saturated wick or unsaturated isoprenaline side chains, or their analogues in liposomal compositions used for delivery to the individual, such as an aerosol delivery to the respiratory tract sludge is delivered via a stomach tube. Such molecules contain chemical functional group, R1-R5patterns chromane and chemically functional wick and isoprenaline side chains, in particular, compounds based on Tocopherols and tocotrienols. It also covers the connection with the substitution of oxygen Romanovka rings and oxygen 6-hydroxy-group on heteroatom (N or S).

Using the methods of alkylation, a large number of compounds containing different R1group, can be synthesized, especially those in which X represents oxygen. After alkylation further chemical modification of R1groups allow you to synthesize a large variety of new compounds. Substituents R1can be alkyl, alkenyl, quinil, aryl, heteroaryl, carboxylic acid, carboxylate, carboxamide, ester, thioamide, timeslot, thioether, saccharide, saccharide associated with alkoxygroup, amine, sulfonate, sulfate, phosphate, alcohol, ethers, and NITRILES.

Bromination of benzyl methyl groups chromane leads to the production of intermediate compounds with which it is possible to obtain compounds with different R2, R3and R4groups. Substituents for R2and R3can be hydrogen or the optional substituents for R4for example, methyl, benzylcarbamoyl acid, gasoline is carboxylate, benzylcarbamoyl, benzyl ester, saccharide and amine. Options group, R5such as alkyl, alkenyl, quinil, aryl, heteroaryl, carboxyl, amide and ester, it is also possible, especially in the case when the initial connection using commercially available 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

When heteroatomic substitution with nitrogen oxygen Romanovka ring, then the nitrogen may be substituted by a group R6that represents hydrogen or methyl. Change X groups other than oxygen, which is identical to the X group in Tocopherols and tocotrienols, can be produced through reaction using palladium (X=CH2) and nucleophilic aromatic substitution (X=N or S). Other possible modifications of the structure chromane include desaturation at 3-4 provisions and the reduction of the five-membered ring to furnishing rings.

Obviously, other lipids can be used in liposomal compositions. Any lipid that may contain these anti-cancer compounds based on vitamin E, or other anti-cancer compounds and to deliver a therapeutic dose would be suitable. Various methods of delivery of liposomes used, such as aerosol delivery or delivery via gastric tube. For example the EP, methods of delivery of liposomes by aerosol and spray or ways of liposomal injection by means of a gastric probe can be applied.

Obviously that α-Thea or other derivatives of Tocopherols and tocotrienols can be used in the preparations of the NREN, microspheres or nanoparticles and delivered in a therapeutic dose by aerosol inhalation or aerosol spray to the respiratory tract or entered via a stomach tube through local application, subcutaneously, intraperitoneally, intravenously, intramuscularly, or other established techniques. For example, liposomal composition α-TEM or the composition in the form of nanoparticles in a soft gel capsule for oral delivery may be ideal for chemoprophylaxis person, however, not be considered as limiting the invention. Compositions in the form of nanoparticles, administered orally in soft gel capsules can to stay longer in the digestive tract and may be better absorbed. In addition, the composition in the form of nanoparticles may be suitable for delivery by inhalation. Liposomal composition or compositions in the form of nanoparticles can be used in the transdermal delivery system, such as a patch.

The following examples are intended to illustrate different variants from the Britania and are not intended to limit the present invention in any form.

EXAMPLE 1

Synthesis and characterization of 2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethylacetyl)chroman-6-yloxy)acetic acid (α-TEM)

In large scale α-TEM was prepared as follows. Added NaH (5.0 g, was 124.9 mmol) in dry THF (THF) (300 ml) and stirred in an argon atmosphere at 0°C for 10 min before adding through assistive device α-tocopherol (41,3 g, for 96.1 mmol), which was dissolved in 100 ml of dry THF. The resulting mixture was stirred at 0°C for 15 min in an argon atmosphere, and then with a syringe was added ethylbromoacetate (g (Jn 19 : 26, 115,3 mmol). The reaction course was monitored by TLC (hexane:ethyl acetate = 10:1, Rf=0,65), and the reaction was completed after 3.5 hours. The reaction mixture was diluted with 150 ml of CH2Cl2off , washed with a saturated NaCl solution (150 ml×3) up until the organic phase became transparent, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The crude product still contained a small amount of free α-tocopherol, which you can delete column chromatography on silica gel using a solvent mixture of hexane:ethyl acetate (30:1 to 20:1), to obtain the pure product ethyl ester α-TEM (41,6 g, 84%).

Ethyl ester α-TEM (21,0 g of 40.7 mmol) was dissolved in 250 ml of THF, then add the Lyali 75 ml of 10% KOH solution (122,1 mmol) and the mixture was stirred at room temperature for 6 hours. The reaction course was monitored by TLC (CHCl3:Meon:CH3COOH=97:2,5:0,5, Rf=0,18), and the reaction was suppressed by addition of 100 ml of water. the pH of the solution was brought to pH 3 using 1 N. HCl and the product was extracted by CH2Cl2(100 ml×4), washed with saturated solution of NaCl, dried over Na2SO4and the solvent was removed under reduced pressure, obtaining the final product α-TEM in the form of a waxy solid (18.5 g, 93%). The melting point 54-55°S; molecular weight 488,8.

EXAMPLE 2

Line of tumor cells of the mammary gland of the mouse

Cell 66 cl.4-GFP represent the line of tumor cells of the mammary gland of the mouse produced from spontaneous tumors in the mammary gland of Balb/cfC3H mice and later isolated as a clone resistant to 6-Tg (20,21). Subsequently, these cells were subjected to stable transfection of green fluorescent protein (GFP). Cell 66 cl.4-GFP are highly metastatic 100% of metastases in the lungs. Before use in this study, the cells were sent to the University of Missouri Research Animal Diagnostic and Investigative Laboratory (RADIL; Columbia, MO), where they were checked for the absence of pathogens.

Cell 66 cl.4-GFP were maintained as monolayer cultures in the medium for cell growth: Wednesday McCoy (Invitrogen Life Taechnologies, Carlsbad, CA.), supplemented with 10% fetal serum of bovine (FBS, Hyclne Lab, Logan, UT), 100 μg/ml streptomycin, 100 ME/ml penicillin, 1X (about./about.) nonessential amino acids, 1X (about./about.) MEM vitamins, 1.5 mm sodium pyruvate and 50 μg/ml gentamicin (Sigma Chemical Co., St. Louis, MO). Processing was performed using the same medium supplemented McCoy except that the content of FBS in the medium was reduced to 5%. The culture was checked by the usual method, to verify the absence of contamination with Mycoplasma.

EXAMPLE 3

Determination of apoptosis by morphological evaluation of DAPI-stained nuclei

Apoptosis was determined using previously published procedures (22). Briefly, 1×105cells/well in 12-hole tablet cultivated during the night to carry out the attachment of cells. Next, cells were processed through α-TEM, succinate vitamin E (Sigma) or ethanol as a control (0.1% ethanol F.C. about./about.) in the experimental medium at various concentrations α-TEM and succinate vitamin E during different time intervals. After processing surfaced cells and adherent cells, released by scraping, besieged by centrifugation for 5 min at 350 × g, washed once with phosphate buffer solution (PBS; 137 mm NaCl, 2.7 mm KCl, 10,4 mm Na2HPO4, 10.5 mm KH2PO4; pH of 7.2) and stained with 2 μg/ml dihydrochloride 4,6-diamidino-2-phenylindole (DAPI, Boehringer Mannheim, Indianapols, IN 100% methanol for 15 min at 37°C.

The cells were viewed under 400X magnification using a fluorescent microscope Zeiss ICM 405 filter 487701. Cells in which the nucleus contained condensed chromatin, or cells with fragmented nuclei were counted as apoptotic. Data are as a percentage of apoptotic cells in the cell population, i.e. the number of apoptotic cells/total number of counted cells. Three different fields were examined with a microscope and 200 cells were counted at each position relative to at least 600 cells counted on one glass slide. These aporosa represented as mean ± the standard deviation for the three independently conducted experiments.

EXAMPLE 4

The definition of fragments cleavage of poly(ADP-ribose)polymerase (PARP) by Western blotting

Cleavage of poly(ADP-ribose)polymerase were analyzed as an alternative method for determination of apoptosis. Cell 66 cl.4-GFP were treated as described above for analysis using DAPI. After washing by PBS, the cells suspended in buffer for lysis (IX PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 μg/ml leupeptin, 1 μg/ml Aprotinin, 1 mm dithiothreitol (DTT), 2 mm orthovanadate sodium, 10 μg/ml of phenylmethylsulfonyl (PMSF)for 30 min at 4°who, mixed and supernatant collected by centrifugation at 15000 × g for 20 minutes. Protein concentration was determined by Bio-Rad (Bradford) protein assay (Bio-Rad Laboratories, Hercules, CA) and samples (100 µg/lane) were subjected to electrophoresis in 7.5% SDS-polyacrylamide gel under reducing conditions.

Proteins were subjected to transfer on nitrocellulose membrane Optitran nitrocellulose VA-S-substrate, the pores of 0.2 μm, Schleicher and Schuell, Keene, NH) using electrophoresis. After transfer the membrane was treated with a buffer to block [25 mm Tris-HCl (pH 8.0), 125 mm NaCl, 0.5% tween-20 and 5% non-fat dry milk] for 45 min at room temperature. The Western blot turns was performed using 1 µg of the first rabbit antibodies against poly(ADP-ribose)polymerase [PARP (H-250), Santa Cruz Biotechnology, Santa Cruz, CA], and conjugated with horseradish peroxidase goat antibodies against rabbit immunoglobulin was used as second antibody (Jackson Immunoresearch Laboratory, West Grove, PA) at a dilution of 1:3000. Horseradish peroxidase strips after carrying out Western blot turns and washing of immunoblotted" was investigated using enhanced chemiluminescence (Pierce, Rockford, IL) and autoradiography (Kodak BioMax film; Rochester, NY).

EXAMPLE 5

Analysis of the formation of colonies

Cell 66 cl.4-GFP were seeded at a concentration of 600 cells in h mm tablet for tissue culture (Nunclon, Rochester, NY) and left p is triplets over night at 37° C. On the following day environment for the growth of cells was removed and replaced by medium for processing cells, containing α-TE at a concentration of 1.25, 2.5, 5, and 10 μg/ml, ethanol control (0.1% ethanol F.C. about./vol.), or only medium (untreated cells). Processing was performed on the cells for 10 days without changing the medium. After 10 days, the medium was removed and the tablets were washed by PBS three times. Cells were stained for 30 min with 1% methylene blue in PBS and colonies >0.5 mm manually counted.

EXAMPLE 6

Mouse balb/C

Mice females of Balb/cJ at the age of 6 weeks (25 g body weight) were obtained from Jackson Labs (Bar Harbor, ME) and were left to acclimatize for at least one week. Animals were kept in the Animal Resource Center at the University of Texas at Austin with 74±2°F with a humidity of 30-70% and a 12-hour peremejayutsya cycle light-darkness. Animals were kept at 5 per cage and given water and standard laboratory food ad libitum. Applicants followed the instructions regarding the humane treatment of animals by the University of Texas Institutional Animal Care and Use Committee.

EXAMPLE 7

Inoculation of tumor cells

Cell 66 cl.4-GFP were collected by trypsinization, centrifugation, re-suspension in the environment Msso that does not contain additives at a density of 2×105/100 μl. The mice were injected through the injection of cells in the groin area at the point, finding the action scene at an equal distance between the 4th and 5th nipples on the right side, using needle gauge 23.

50 mice (10 mice per group) were subjected to processing by aerosol, aerosol control, were subjected to oral treatment, control sample was also given oral or was not subjected to any processing so that the average tumor volumes for all groups closely correspond to each other. In each group, the tumor size was 2×2-4×4 mm at the beginning of the treatments that were started nine days after inoculation of tumor cells. Ten additional mice that were not injected with tumor cells were treated with aerosol or oral by α-TEM (5 each) for 17 days, the treatment was finished and watched for an additional 11 months to assess the safety of the drug for a long time. Tumors were measured with calipers every other day and the volume was calculated using the formula: volume (mm3) = [width (mm)2× length (mm)]/2. Body weight was determined weekly.

EXAMPLE 8

Preparation and introduction α-TEM, solubilizing in peanut oil for delivery via gastric tube

α-TEM was dissolved in 100% ethanol (400 mg/ml) and then mixed with peanut butter (100% peanut oil; nSpired Natural Foods, San Leandro, CA) at a ratio of 1:8 (vol./vol.). The control sample contained an equivalent amount of ethanol and ar is heaveho oil, contained in the sample with α-TEM. The mixture was vigorously stirred, and then kept at 4°s to use. The temperature of the mixture α-Thea/peanut butter brought to room temperature and injected 100 μl/mouse per day by gastric probe. This number corresponds to a final concentration of 5 mg α-Thea/mouse/day.

EXAMPLE 9

Preparation of liposomal compositions containing 2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethylacetyl)chroman-6-yloxy)acetic acid (α-TEM)

Ratio α-Thea/liposome of 1:3 (wt./wt.), it was determined empirically as the optimal methods described previously (17). In order to prepare a combination of α-Thea/lipid, the temperature of the components is first brought up to room temperature. Lipid [1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC); Avanti Polar-Lipids, Inc., Alabaster, AL] at a concentration of 120 mg/ml was dissolved in tertiary butanol (Fisher Scientific, Houston, TX), then dispersively using ultrasound to obtain a transparent solution. Also α-TEM was dissolved at a concentration of 40 mg/ml tertiary butanol and vigorously stirred until then, until it all dissolved solids. Then the two solutions were combined in equal amounts (about./about.) with vigorous stirring to give the desired ratio α-Thea/liposome 1:3 frozen at -80°C for 1-2 hour and liabili Aravali during the night to obtain a dry powder before storage at -20° With up until not needed. Each vial contains 75 mg α-TEM.

EXAMPLE 10

Cooking spray and introduction of liposomes containing 2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethylacetyl)chroman-6-yloxy)acetic acid (α-TEM)

The aerosol was injected into mice as described previously (17). Briefly, an air compressor (Easy Air 15 Air Compressor (Precision Medical, Northampton, PA)), which produces an air flow of 10 l/min was used with a spray type AeroTech II (CIS-US, Inc. Bedford, MA)to obtain an aerosol. The size of the particles containing α-Thea liposomal aerosol released from the sprayer AeroTech II, were determined using a cascade impactor Anderson, and he was 2.01 microns average mass aerodynamic diameter (NMAD) with geometric standard deviation 2,04. Approximately 30% of such particles when inhaled will be deposited in the respiratory tract of the mouse and the remaining 70% will fizzle out (17).

Before spraying powder α-Thea/lipid brought to room temperature, then was restored by the addition of 3.75 ml of distilled water to obtain the final desired concentration α-Thea 20 mg/ml and the Mixture was left to swell at room temperature for 30 min with occasional stirring and then added to the spray. The resulting composition with α-TEM can be administered orally using a stomach probe at levels of 4 mg αThea/0,1 ml Mice were placed in plastic cages (7×11×5 inch) sealed in protective plastic. The aerosol was injected into the cell through a 1-inch corrugated tube at one end and released at the opposite end, using a one-way valve for pressure relief. Animals were subjected to processing by aerosol to until the entire composition α-Thea/liposome was not sprayed, approximately 15 minutes

EXAMPLE 11

Aerosol properties α-Thea included in liposomes

Analyses containing α-Thea liposomes obtained from aerosol in All Glass Impinger (ACE Glass Co., Vineland, NJ), was performed by HPLC. Delivered dose = concentration (µg/l) × minute volume mouse (1 min/kg) × duration of delivery (min) x estimated otorisasi fraction (30%; 17). On the basis of this formula, the applicants have found that approximately 36 ug g α-Thea deposited in the respiratory tract of each mouse every day (316,2 µg g/l × 1 min/kg × 15 min × 0,30=1422,9 µg g/kg/day).

EXAMPLE 12

Tests α-TEM in tumors by HPLC

Tumors were removed at autopsy and half of each tumor was rapidly frozen in liquid nitrogen, then stored at -70°until then, until he started HPLC analyses. Tumors for HPLC analysis were processed by homogenization and extraction of lipids hexane is m Briefly, weighted tumors were placed in a 5 ml disposable conical tubes (Sarstedt, Newton, NC) along with 5-7 Kimble glass ball (4 mm), 1 ml of 1% SDS in water, 1-2 ml of 100% ethanol and 1 ml of hexane. Then the samples were mixed using a mixer type Crescent Wig-L-Bug (model V Densply International, Elgin, IL), twice for 1 min each time. Then the samples were centrifuged for 5 min at 1000 rpm, and the organic layer was collected, the aqueous layer was again mixed with hexane, vigorously stirred, centrifuged and processed more than two times before the organic layer was dried in nitrogen atmosphere. Samples "chased away" immediately. Analyses HPLC with reversed phase with fluorometrically registration ether analogue of tocopherol was performed as described Tirmenstein, M.A., et al. (23).

EXAMPLE 13

Metastases in the lung and lymph node

Lesions metastases in five lobes of the lung were counted visually at the time of autopsy. Fluorescent green micrometastatic colonies of cancer cells in the lobe of the left lung and lymph nodes were counted using a fluorescent microscope (Nikon TE-200) with a 20X objective lens (200X magnification) and the program Image-Pro Plus (version 4.1; Media Cybernetics, Silver Spring, MD), combined with a microscope. Fluorescent lesions were divided into three groups according to their sizes: <5 μm, 5-10 μm and >10 μm.

EXAMPLE 14

Analysis of TUNEL determine apoptosis in vivo

DEPA is Afanasievna sections (5 μm) tumor tissue was used for assessment of apoptosis using reagents in the kit ApopTag in situ Apoptosis Detection kit (Intergen, Purchase, NY)according to the manufacturer's instructions. Kernel, painted brown, considered as positive in relation to apoptosis and nuclei, which were painted blue, was considered as negative. At least sixteen 400X field microscope was used to count on the tumor. Data are presented as averages ± standard error in the number of apoptotic cells counted in three separate tumors from each group. Picture of the tumor tissue were examined at 1000X magnification.

EXAMPLE 15

H&E staining of tumor tissue

Tumors were fixed with 10% neutral buffered formalin solution and included in paraffin according to standard histological procedures. H&E stained sections with a thickness of 5 μm was used for morphological verification of the tumor.

EXAMPLE 16

Histological evaluation 66 cl.4-GFP cells

The tumor cells of the mammary gland of the mouse, has been described as spindle cell carcinoma, poorly differentiated, with high mitotic index.

EXAMPLE 17

Statistical analysis

Statistical analysis was performed using Prism version 3.0 (Graphpad, San Diego, CA). The number of animals for experiments was determined by calculating the degrees. The animal's weight and volume of tumours analysed is ovali by the t student test.

EXAMPLE 18

Evaluation of the anticancer properties of natural and synthetic Tocopherols, tocotrienols and their derivatives

Mice balb/c mice by injection was injected cells 66 cl.4-GFP in the amount of 200,000 subcutaneously in the accumulation of adipose tissue in the thoracic region between the 4th and 5th nipples on the right side of the body. 9 days after injection, animals were divided into groups (10 animals per group) with comparable tumor size, ranging between 0.5×0.5 mm - 1×2 mm of Animals was treated with liposomal compositions with natural alpha - and gamma-tocopherol, a natural alpha-tocotrienol, a fraction enriched with tocotrienol (TRF; this fraction contains approximately 32% of alpha-tocopherol, 20% alpha-tocotrienol, 31% gamma-tocotrienol and 12% Delta-tocotrienol; Gould, M.N. (30)), synthetic dl alpha-tocopherol and synthetic derived acetate dl-alpha-tocopherol by aerosol delivery for 19 days or 21 days. Animals were given 75 mg of each compound by spraying in a day, and each animal was delivered to 36 μg of each compound per day. Animals were checked every other day and the volume was calculated as (w2×1) /2.

Data are presented only for RRR-gamma-tocopherol and dl-alpha-tocopherol (figure 2). Liposomal composition containing dl-alpha-tocopherol, delivered via aerosol, inhibited p the article cells 66 cl.4-GFP 81 and 73% of the 15-th and 17-th day of treatment, respectively. Although not so effective as dl-alpha-tocopherol, other compounds, such as acetate dl-alpha-tocopherol, RRR-Delta-tocopherol, RRR-alpha-tocotrienol, tocotrienol rich fraction (TRF), inhibited tumor growth on day 15 of the handle 29, 25, 34, and 25%, respectively, and on the 17th day of processing - 57, 40, 27 and 40%, respectively.

Liposomal preparations with RRR-α-tocopherol and RRR-γ-tocopherol, delivered via aerosol, increased tumor growth. The increased tumor growth, defined as the volume of the tumor when treatment with drugs with RRR-α-tocopherol was slightly higher than the growth of tumors in the controls; however, the tumor volume in mice treated with RRR-γ-tocopherol was significantly higher than the tumor volume in control mice (figure 2).

Table 2 presents the percentages of inhibition of visible lung metastases in the processing of liposomal preparations of vitamin E or its derivatives. Data show that liposomal preparation with dl-α-tocopherol, delivered via aerosol, significantly reduced the number of visible metastatic lesions of the lung compared to control.

Table 2
Visible lung metastases/
Processingalive is Noah Inhibition (%)
Control1,70
dl-α-tocopherol0,194**
Acetate dl-α-tocopherol0,382
RRR-Delta-tocopherol0,382
RRR-α-tocopherol1,70
RRR-γ-tocopherol1,1532
RRR-α-tocotrienol0,477
TRF1,041
**significantly higher than controls

Immunohistochemical analysis of tumor sections from mice treated with liposomal drug with dl-alpha-tocopherol, delivered via aerosol, showed that this form of vitamin E inhibits tumor growth by reducing the number of blood vessels in tumors by 51%, induction of apoptosis by 44% and reduce cell proliferation by 33%. Table 3 lists the mechanisms through which liposomal preparation with dl-alpha-tocopherol delivered by aerosol, inhibited the growth of 66 cl.4-GFP tumors.

Table 3
Mechanisms of inhibition of growth of tumors through the m liposomes, contains dl-alpha-tocopherol
ProcessingThe number of CD31-blood vessels/provinceTUNEL positive apoptotic cells/fieldKI-67 Inhibition of proliferation (%)
Control2271,460
dl-α-tocopherol, %1122,540
% reduction51-33
% gain-44-

In addition, the study of the mechanisms of action α-TEA on the model of syngeneic mice with transplanted cancer breast cancer showed that α-TEA reduces cell proliferation and induces apoptosis. More precisely, the median level of the biomarker KI-67 proliferation, specific immunochemical methods, were significantly reduced by 56%, and the average level of apoptosis, certain TUNEL immunohistochemical methods were greatly improved by 30%compared to controls (unpublished data). Thus, I believe that these pseudomarket can be important biomarkers for the qualitative and/or quantitative determination of the effectiveness of chemoprophylaxis by α-TEA.

EXAMPLE 19

VES α-TEA indusiry the apoptosis in cells 66 cl.4-GFP in vitro

Previous studies suggest that succinate vitamin E is a strong apoptotic inducer in many lines of cancer cells, including breast cancer. For comparison, the applicants have included succinate vitamin E in in vitro studies of apoptosis induced α-TEA. Cancer cells 66 cl.4-GFP mammary gland of balb/C mice were treated with succinate vitamin E or α-TEA and the level of apoptosis was assessed by morphological analysis of cells stained DAPI, relatively condensed nuclei and fragmented DNA.

The kernel of 66 cl.4-GFP cells treated by 10 μg/ml α-TEM or succinate vitamin E for three days, was condensed with DNA fragments that are signs of apoptosis, while the nucleus of untreated cells did not show these features (figa). Cell 66 cl.4-GFP treated by means of 2.5, 5, 10 and 20 μg/ml α-TEM or succinate vitamin E for three days, were apoptotic, depending on the dose of 5, 6, 34, and 50% when machining α-TEM and for 3, 5, 16, and 34% when processed by succinate vitamin E (pigv). Raw, VEH and EtOH controls found the background level of apoptosis at 2, 2 and 3%, respectively (pigv).

It was shown that α-tea induces apoptosis in a time-dependent manner. Cell 66 cl.4-GFP treated by 10 μg/ml α-Team 2-5 days were apoptotic at 20, 35, 47 and 58%, respectively (figs). Induction of apoptosis was confirmed by the presence of PARP cleavage after treatment 66 cl.4-GFP cells by 5, 10 and 20 μg/ml α-TEM within 48 hours (fig.3D). Fragment of PARP cleavage with a molecular mass of 84 kDa was detected while processing through 10 μg/ml, and at processing through 20 μg/ml α-TEM, while only intact PARP protein was determined in cells treated by 5 mcg/ml α-TEM or in untreated control cells (fig.3D).

EXAMPLE 20

Induction of apoptosis by α-TEA in vivo

Based on in vitro data showing that α-tea inhibits the growth of 66 cl.4-GFP tumor cells through induction of apoptosis, three tumors from each treatment liposomal aerosol containing α-TEM, and a control group subjected to processing only the aerosol was tested on apoptosis using TUNEL staining of tumor sections of 5 micron. Tumors from mice treated by α-TEM, contained on average ± standard error 2,04±0,23 apoptotic cells/field, while tumors from control mice subjected to processing only the aerosol contained in the middle ± standard error of 0.67±0,15 apoptotic cells/field (p<0,03; figa). Positively stained adoption the Kie cells in tumor sections from mice processed liposomal aerosol containing α-TEM, and a control aerosol, can be seen on figv.

EXAMPLE 21

α-tea inhibits the growth of 66 cl.4-GFP cell clone

α-TE at a concentration of 1.25, 2.5 and 5 μg/ml reduced the formation of colonies 30, 85 and 100% relative to control with EtOH (figure 5). Untreated cells (data not shown) and EtOH controls was formed in the middle ± standard deviation 146±11 and 140±22 colonies, respectively. Cells treated through α-TEM at concentrations of 1.25 and 2.5 µg/ml, was formed in the middle ± standard deviation 98±20 and 21±6 colonies, respectively. No colonies were not formed when cells were treated α-TE at a concentration of 5 (figure 5) or 10 µg/ml

EXAMPLE 22

The process action of liposomal spray with α-TEM on the weight of the body

Mice balb/c mice, 10 mice /group (4 groups, a total of 40 mice), were made of cells 66 cl.4-GFP in the amount of 200,000, as described on day 0. Nine days after inoculation began treatment groups: treatment spray (TX) = aerosol delivery of liposomes with α-TEM (5 mg/mouse)/daily on day 23. Aerosol control = aerosol delivery of liposomal compositions only daily on day 23. Processing by means of a gastric probe = probe through a α-TEM at 5 mg/mouse in a mixture of ethanol/peanut butter, ejednevnogo 23 day. Control by means of a gastric probe = probe through a mixture of ethanol/peanut butter only on day 23. Mice were weighed at the beginning of treatment (day 9) and after treatment on days 13, 16, 20 and 23 (6). Data are presented as averages ± the standard deviation. Significant differences in weight among the four groups was not found.

EXAMPLE 23

The process action of liposomal spray with α-Thea levels α-TEM in serum and tissues

Eight mice were treated with aerosol through delivery to the lungs 40 mg α-TEM (5 mg/mouse) in 6 ml of liposomes. Mice inhaled aerosol during the period 30 minutes before the end delivery. Mice were killed after 0, 2, 6 or 24 hours after treatment. Levels α-TEM in serum and tissues was determined by HPLC (Fig.7). During the first experimental period (time 0) α-TEM was found only in the tissue of the stomach. α-TEM was found in the serum and stomach of mice killed 2 hours after the end of treatment. α-TEM was found in the liver and stomach of mice killed 6 hours after the end of treatment. α-TEM was found in the liver and stomach of mice killed 24 hours after processing.

EXAMPLE 24

The process action of liposomal spray with α-TEM on the weight of the tumor

The mice were injected via the injection 66 cl.4-GFP tumor cells of the mammary gland of the mouse is the number 200000, as described on day 0. Treatment was started on the 9th day, when the tumors had reached a size of 1-3 mm Aerosol processing liposomes with α-TEM (5 mg α-Thea/mouse) produced daily for 16 days (Fig). Data are presented as averages ± standard error (SE), N=10 mice for the control group and groups exposed to treatment. When mice were killed at 25, 16 days after the start of treatment, the tumor size in the group subjected to the processing of liposomal spray with α-TEM, was 61% less than the tumor size in the control group subjected to processing only the aerosol.

EXAMPLE 25

Processing through liposomal α-TEA/aerosol inhibited the growth 66 cl.4-GFP tumors in balb/c mice and reduced lung metastases

Balb/c mice by the injection was administered s.c. 2×105cells 66 cl.4-GFP in the groin area between the 4th and 5th the nipple on the right side of the body. When the tumors had reached a size of 2×2-4×4 mm (9 days after injection of the tumor), the mice were divided into 5 groups(group 1: untreated control, group 2: control liposome/aerosol, group 3: treatment of liposomal spray with α-TE, group 4: control of peanut butter/gastric tube, group 5: processing by α-TEM in peanut oil/gastric tube) from 10 mice per group)so that the average tumor volume in each group corresponded to a friend on the uh-huh. Daily treatment was started on the 9-th day after injection of the tumor.

The average tumor volume in the group subjected to the processing of liposomal spray with α-TEM, in comparison with the aerosol control, was reduced by 23, 41, 50, 67, and 61% for 9, 11, 13, 15 and 17 days of treatment, respectively (figa). After mice were killed, they had removed the light, checked visually for the presence of metastatic lesions and froze to analyze the metastases using fluorescence. In the group subjected to processing by α-TEM, no visible tumors were not detected, while the untreated group and the control group treated with aerosol only had visible tumors/animals volume of 3.25±1,7 and 4.25±0,5 respectively, with metastases in the lungs, as shown in table 4.

The use of fluorescent microscope Nikon and program Image-Pro Plus allowed us to group the metastases in three groups by size <5 μm, 5-10 μm and >10 μm based on the definition of green fluorescent metastases. This analysis showed a highly significant decrease in the number of metastases in all three dimensions in the group subjected to processing by α-TEM, compared with untreated control groups and groups subjected to processing by aerosol only (pigv). Average cyclometalation in the group, subjected to processing by α-TEM (11,4±3.5 standard error; N=8), compared with the control group subjected to the processing by aerosol only (60,0±15 standard error; N=10), was reduced by 81% (p<0,2). Although the average number of metastases in the control group treated with aerosol versus untreated control group (N=10) was reduced (60±15,2 standard error against 101,7±17,0 standard error; p<0,9)suggest that the difference was insignificant due to the large range in the number of metastases among mice within these two groups (pigv).

Table 4
Pulmonary metastases in balb/c mice, cultured cancer cells 66 cl.4-GFP breast cancer
ProcessingNumber of animals/group with visible metastases in the lungaThe number of visible tumor foci in the lung/animalb
No processing4/103,25±1,7
Control spray/liposome4/104,25±0,5
Aerosol /liposome/α-TE0/100
andMetastatic lesions in all five lung lobes for each of the CSOs animal in all groups, subjected to processing, was estimated visually during slaughter mice.
bData are expressed as averages ± the standard deviation of the visible tumor foci in the lung observed in four animals with pulmonary metastases in the two control groups.

EXAMPLE 26

Comparison of the action of liposomes containing α-TEM and succinate vitamin E (VES), delivered by sputtering, on the growth of tumor cells 66 cl.4-GFP

Mice balb/c mice by the injection was administered cells 66 cl.4-GFP in the amount of 200,000 subcutaneously in the accumulation of adipose tissue in the thoracic region between the 4th and 5th the nipple on the right side of the body. 9 days after injection, animals were divided into groups (10 animals per group) with comparable tumor size, ranging between 0.5×0.5 mm - 1×2 mm of Animals processed through α-TEM in the liposome, succinate vitamin E in liposome or control liposomes only in the form of an aerosol, 7 days a week for 21 days. Animals were given 75 mg of the compounds per day with a spray so that each animal received 36 μg of the compound per day. Animals were defined through the day and the volume was calculated as (w2×1)/2. Liposomal aerosol containing α-TEM, reduced tumor growth by 64, 76, 69, and 67% on days 15, 17, 19 and 21, respectively (values statistically significantly of various the of between n α -Thea and the control on days 17, 19 and 21, p=0.03, 0,048 and 0.03, respectively). Liposomal aerosol containing VES, reduced tumor growth by 76, 76, 69, and 68% on days 15, 17, 19 and 21, respectively (figure 10). Values statistically significantly differed between succinate vitamin E and control on days 17 and 21, p=0,029 0,029 and respectively.

EXAMPLE 27

The delivery action α-TEM via a stomach tube on the weight of the tumor

Shipping α-TEM through the gastric tube is ineffective in preventing the growth of 66 cl.4-GFP tumor cells of the mammary gland of the mouse at the site of inoculation (11). Applicants determined the size of tumors through 9-21 day after the injection 66 cl.4-GFP cells in the amount of 200,000 between the 4-m and 5-m nipple on the right side of the body of each mouse. Each group consisted of n=10. Processing through probe = processing 5 mg α-TEM in ethanol and peanut butter (volume 0.1 ml) was carried out daily, starting from the 9th day after inoculation of the tumor and continuing for 21 days. Control through probe = mouse received peanut oil and ethanol only (volume 0.1 ml)/day, starting from the 9th day after inoculation of the tumor and continuing for 21 days. Data (tumor size) was expressed as the average ± the standard error relative to days 9, 11, 13, 15, 17, 19 and 21. Significant differences in the weight of the tumors between the control group and the group subjected to processing α-TEM through zones is, all time points were not observed.

EXAMPLE 28

Shipping α-TEM through the probe did not reduce tumor growth at the site of inoculation, but reduced the level of lung metastases

In contrast to the treatment of liposomal spray with α-TEM, the average volume of tumors in mice treated with 5 mg/day/mouse song α-TEM in EtOH/peanut oil, introduced through the probe, did not differ from the average tumor volume of mice treated with control through the probe (figa). However, the introduction of α-TEM through the probe reduced the number of lung metastases by 68%. The number of metastases, based on three groups by size (<5 μm, 5-10 μm, >10 μm), was 6,8±1,5, 11,3±and 1,8 3,1±1,2 standard error in mice that α-TEM was introduced through the probe, while the number of metastases in the control group of mice was 27,9±9,0, 29,2±and 6.3 and 8.4±1,5 standard error, respectively (pigv).

No differences in average body weight among any of the control groups or groups subjected to treatment were observed (data not shown).

In mice without tumor, which was treated with either spray/α-tea, either through the probe for 17 days and then contained within eleven months, to assess the duration of action of the drug, not discovered the harmful effects of the treatments by α-TEM.

Although the introduction of α-TEM through aerosol was significantly better than the introduction of the probe that is introduced through the probe α-Thea did not reduce the size of the tumor at the site of inoculation in comparison with the size of the tumors of control mice, interest is the fact that the number of lung metastases was decreased compared to control, when α-TEM was introduced through the probe. I think that α-TEM was bioavailable. Because α-TEM is not hydrolyzed, and, in addition, tests have shown that by putting her through a tube it should be an effective antitumor agent, I believe that is introduced through the probe α-TEM at 5 mg/ml/day/mouse did not reduce tumor growth at the site of tumor inoculation due to the low absorption through the digestive tract. Thus, suggested that the low levels of α-TEM can be effective in preventing the formation of tumor foci in the lungs.

EXAMPLE 29

Comparison of the degree of inhibition of tumor growth and metastasis to lung and lymph node 66 cl.4 breast tumors when processing through α-Thea/probe, liposomal α-Thea/probe and liposomal α-tea/aerosol

Although the song α-TEA/EtOH/peanut butter, introduced through the probe, was effective in inhibiting metastases of the lung, it is not inhibit the shaft tumor growth in syngeneic animal models transplanted with breast cancer. However, liposomal composition containing α-tea, administered orally through a feeding tube twice a day at 6 mg α-tea/day, is an effective delivery vehicle for preventing tumor growth and metastasis 66 cl.4 balb/c tumor cells of the mammary gland in female balb/c mice. Liposomal composition α-TEM, delivered through the probe, inhibited tumor growth by 70% and inhibited lung metastases and lymph node 59% and 56%. It has been estimated that 36 mcg α-TEM were deposited in the lungs of mice/a day when α-Thea/liposomal preparations were administered by aerosol. Number α-TEM, pending in the tissues when α-Thea/liposomal composition, or song α-TEA/EtOH/peanut butter was introduced through the probe, is not known. Comparison of antitumor effectiveness of different processing modes shown in table 5.

Table 5
Comparison of drugs with α-TEM and techniques in preventing tumor growth and metastasis of tumor cells 66 cl.4 breast cancer mouse balb/c
Inhibition (%)
TrackShipping method Concentration α-TEM (mouse)Tumor growthMetastases
LightLymph node
LiposomalProbe3 mg/2× day70%59%56%
LiposomalAerosol5 mg/day70%81%94%
EtOH/peanut butterProbe5 mg/day0%62%Not defined

EXAMPLE 30

Comparison of the action of liposomes containing α-TEM and succinate vitamin E delivered through the probe, and the growth of 66 cl.4-GFP tumor

Liposomal preparation containing α-TEA, taken orally through a feeding tube, inhibited the growth of cancer cells of the mammary gland of the mouse, transplanted in balb/c mice. Liposomal preparation containing succinate vitamin E taken orally through a feeding tube, not inhibit the growth of cancer cells of the mammary gland of the mouse, transplanted in balb/c mice. Balb/c mice by the injection was administered 66 cl.4-GFP cells in the amount of 200,000 subcutaneously in the accumulation of adipose tissue in the thoracic region between the 4th and 5th the nipple on the right side of the body. 9 days after injection, animals were divided into groups (10 animals per group) with the comparable tumor size, oscillating between 0.5×0.5 mm - 1×2 mm of Animals processed through α-TEM in the liposome, succinate vitamin E in liposome or control liposomes only through oral probe 2 times a day, 7 days a week for 21 days. Animals were given a total of 6 mg of the compounds per day. Animals were defined through the day, and the volume was calculated as (w2×1)/2. Processing through α-TEM in the liposome through the probe was reduced tumor growth 85, 85, 80, and 70% at days 15, 17, 19 and 21, respectively (values are statistically significantly differed between α-TEM and control on days 15, 17 and 19, p=0.03, 0,039 and 0,029 respectively). Processing by VES in the liposome did not reduce tumor growth (Fig).

EXAMPLE 31

Processing liposomal drug α-TEM through the probe was reduced metastases 66 cl.4-GFP lymph node and lung

Balb/c mice by the injection was administered 66 cl.4-GFP cells in the amount of 200,000 subcutaneously in the accumulation of adipose tissue in the thoracic region between the 4th and 5th the nipple on the right side of the body. 9 days after injection, animals were divided into groups (10 animals per group) with comparable tumor size, ranging between 0.5×0.5 mm - 1×2 mm of Animals processed through α-TEM in the liposome, succinate vitamin E in liposome or control liposomes only through oral probe 2 times a day, 7 days a week in those is giving 21 days. Animals were given a total of 6 mg of the compounds per day. After slaughter lobe of the left lung of each mouse was instantly frozen and used to determine micrometastatic lesions through the expression of GFP using a fluorescent microscope.

In animals that were given liposomal drug α-TEM through the probe, found a significant decrease in micrometastatic lesions of the lung compared with control (21,5/easy against 52,7/lung, respectively). Liposomal drug succinate vitamin E delivered through the probe, not affecting the lung metastases (figa). After slaughter mice lymph nodes were collected and examined on the subject micrometastatic lesions of the lung. In animals that were given α-TEM through the probe, found in an average of 3.1±0,8 micrometastatic lesions in the lymph node compared to 7.1±1,7 micrometastatic lesions in the lymph node in the control group animals (p=being 0.036). The drug succinate vitamin E delivered through the probe, was not effective in reducing micrometastatic lesions of the lung (pigv).

EXAMPLE 32

Processing liposomal drug α-TEA over the probe was reduced tumor growth of MDA-MB-435 breast cancer cells in vivo

Nude naked mice Nu/Nu through the injection was administered 1,0×106cells MDA-MB-435 GFP FL podkoren is in the accumulation of adipose tissue in the thoracic region between the 4th and 5th the nipple on the right side of the body. 8 days after injection, animals were divided into groups (10 animals per group) with comparable tumor size, ranging between 0.5×0.5 mm - 2×2 mm. Animals were treated by alpha-TEM in the liposome or the control liposome only through oral probe 2 times a day, 7 days a week for 21 days. Animals were given a total of 8 mg of the compounds per day. Animals were defined through the day and the volume was calculated as (w2×1)/2. Processing through α-TEM in the liposome through the probe was reduced tumor growth by 68, 75, 78, and 79% on days 27, 29, 31 and 35, respectively (Fig). Values statistically significantly differed between α-TEM and control on days 33 and 35 (p=0.045 and 0,033 respectively).

EXAMPLE 33

Inhibition of tumor growth and metastasis 66 cl.4-GFP tumors in balb/c mice in the processing of aerosol by a combination of liposomal/α-TEM + 9NC

Female balb/c mice aged 6 weeks through the injection was administered subcutaneously 66 4-GFP clone of tumor cells of the breast between the 4th and 5th nipples on the right side. When the tumors had reached a size of 1×1 mm, started processing the aerosol. α-Thea entered separately and in combination with the low levels 0,403 µg/day 9-nitrocamptothecin, is an effective inhibitor of tumor growth. Aerosol delivery α-tea + 9-nitrocamptothecin inhibited growth op the Holi breast cancer mouse 90%, while α-TEA and 9-nitrocamptothecin only inhibited tumor growth by 65% and 58%, respectively. Aerosol delivery α-tea + 9-nitrocamptothecin inhibited micrometastatic lesions of the axillary lymph node and lung of 87% and 71%, while α-TEM and 9-nitrocamptothecin entered only through aerosol, inhibited micrometastatic lymph node 94% and 60% and micrometastatic lung lesions by 71% and 55%, respectively. Thus, α-tea + 9-nitrocamptothecin entered sequentially, inhibited tumor growth better than the introduction of the two drugs separately. α-TEM is an effective drug for the inhibition of metastasis of lung and lymph node as introduced separately, and in combination with 9-nitrocamptothecin. Comparative data with respect to the action α-Thea entered separately and in combination with 9-nitrocamptothecin aimed at preventing tumor growth and metastasis, are presented in table 6 below.

Table 6
Comparison of action α-tea alone and in combination with 9-nitrocamptothecin (9NC) in preventing tumor growth and metastasis
Inhibition (%)
TrackShipping methodThe concentration of the drug in the nebulizer (mouse)Metastases
Tumor growthEasyLymph node
Liposome/α- TEAAerosol5 mg/day65%71%94%
Liposome/9NAerosol2 mg/day58%55%60%
Liposome/α-TEA + 9NCAerosol5 mg + 2 mg/day90%71%87%

EXAMPLE 34

α-Thea turns are resistant to this drug called cisplatin Cf-70 cancer cells human ovarian cells that are sensitive to this drug called cisplatin

Combination α-TEM + cisplatin increased destruction of tumor cells in vitro and in vivo. Cell line cf-70, ovarian cancer man, shows resistance to this drug called cisplatin, drug-drug platinum, which is usually used primarily for the treatment of tumors of the ovary. Subclan cf-70 was obtained in vitro from A cisplatinresistant cell line by periodic exposure to the impact of increasing the I levels of cisplatin, up to 70 mm. When both cell lines, A and SR-70, was subjected to processing in the culture through α-TEM, they underwent apoptosis (figa). Processing cf-70 cancer cells human ovarian suboptimal levels of cisplatin (of 0.625 and 1.25 mg/ml) and α-TEM (10 µg/ml) restored the sensitivity to cisplatin cf-70 cells have increased levels of apoptosis in A cell lines and restore cisplatin sensitivity in cf-70 cell lines (pigv). α-TEM in combination with cisplatin inhibited growth of cf-70 cells in vivo. Gene green fluorescent protein (GFP) expressively in cf-70 cells by viral infection so that the cells were light and could be used to study the effects of combined treatment in a murine model of xenotransplantation. Processing the combination of cisplatin and α-Thea produced in vivo model naked mouse xenotransplantation. In this model, female Nude mice were inoculable 1×106cells subcutaneously between the 4th and 5th nipples. When the average tumor size reached 1.0 mm3mice were selected for processing or only α-TEM, delivered by aerosol in liposomes, only cisplatin (5 mg/kg), introduced by injection I.P. once a week for 3 weeks, cisplatin + α-TEM, or control aerosol (liposome). The data is showed that treatment by α-TEM + cisplatin is an effective way of inhibiting the growth resistant to this drug called cisplatin cf-70 cancer cells human ovarian (figs).

EXAMPLE 35

Effect of alpha-TEM, methylselenocysteine and TRANS-resveratrol on cancer cells MDA-MB-435-GFP-FL breast cancer

Female NU/NU homozygous mice were injected through the injection of one million MDA-MB-435 GFP FL cells subcutaneously in the accumulation of adipose tissue in the thoracic region between the 4th and 5th nipples on the right side of the body. Nine days after injection, animals were divided into five groups of ten animals with comparable tumor size ranging from 0.5 mm × 0.5 mm to 2.0 mm × 2,0 mm Animals through the probe gave 3 parts per million methylselenocysteine (MSC) in water, α-TEM in the liposome by aerosol or through the probe were given 10 mg/kg body weight of TRANS-resveratrol (t-RES) first in a mixture of 1:1 EtOH:saline, and since the 24th day 6.5% EtOH and 93.5% oil Neobee. A group of animals was treated with each of the above three compounds and the control group were treated with liposomes in the form of an aerosol, 100 μl of water and 50 μl of a solution of t-Res. Animals were handled seven days a week for 35 days. Tumors were defined through the day and the volume was calculated by the formula (w2×1) /2.

On the 35th day of the inspection it was found that in the group, Podwale the Oh processing α -Thea, tumor growth was reduced by 47.8%in the group treated MSC, tumor growth was reduced by 47.2%, in the group treated with t-RES, tumor growth was increased to 61.2% in the group exposed to the combined treatment, tumor growth was increased by 19.8% from control (Fig). Group statistically analyzed relative to control by t-test, and p values were p=0,0487, 0,0347, 0,2221 and 0,6255 for α-TEM, MSC, t-RES, and the combined treatment, respectively. Thus, only in the groups treated alpha-TEM and MSC, tested separately, the observed reduction in tumor growth, which significantly differed from the controls. Treatment of TRANS-resveratrol and only a combination of three compounds increased tumor growth.

The following references are cited in the text:

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2. Prasad, et al., Vitamin E and cancer prevention: Recent advances and future potentials. J. Am. Coll. Nutr., 11: 487-500, 1992.

3. Schwartz, J., and Shklar, G. The selective cytotoxic effect of carotenoids and α-tocopherol on human cancer cell lines in vitro. J. Oral Maxillofac. Surg., 50: 367-373, 1992, conserved homolog, Bax, that accelerates programmed cell death. Cell 74: 609-619, 1993.

4. Fariss, et al., The selective antiproliferative effects of α-tocopheryl hemisuccinate and cholesteryl hemisuccinate on murine leukemia cells result from the action of the intact compounds. Cancer Res., 54: 3346-3351, 1994.

5. Kline, K., Yu, W., and Sanders, B.G.Vitamin E Mechanisms of action as tumor cell growth inhibitors. In: K.N.Prasad and W.C.Cole (eds.). Proceeding of te International Conference on Nutrition and Cancer, pp 37-53. Amsterdam: IOS Press, 1998.

6. Kline, K., Yu, W, and Sanders, B.G.Vitamin E Mechanisms of action as tumor cell growth inhibitors. J.Nutr., 131: 161S-163S, 2001.

7. Neuzil, J., Weber, T., Gellert, N., and Weber, C.Selective cancer cell killing by α-tocopheryl succinate. Br.J.Cancer, 84: 87-89, 2000.

8. Malafa, M.P., and Neitzel, L..Vitamin E succinate promotes breast cancer tumor dormancy. J.Surg. Res., 93: 163-170, 2000.

9. Malafa, et al., Vitamin E inhibits melanoma growth in mice. Surgery, 131: 85-91, 2002.

10. Neuzil, et al., Induction of cancer cell apoptosis by α-tocopheryl succinate: molecular pathways and structural requirements. FASEB J., 15: 403-415, 2001.

11. Weber, et al., Vitamin E succinate is a potent novel antineoplastic agent with high selectivity values and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) in vivo. Clin. Cancer Res., 8: 863-869, 2002.

12. Wu, et al., Inhibitory effects of RRR-alpha-tocopheryl succinate on benzo(a)pyrene (B(a)P)-induced forestomach carcinogenesis in female mice. World J.Gastroenterol, 7: 60-65. 2001.

13. You, H., Yu, W., Sanders, B.G., and Kline. K. RRR-α-tocopheryl succinate dosage MDA-MB-435 and MCF-7 human breast cancer cells to undergo differentiation. Cell Growth Differ., 12: 471-480, 2001.

14. You, H., Yu, W., Munoz-Medellin, D., Brown, P.H., Sanders, B.G., and Kline, K. Role of increasing interest among signal-regulated kinase pathway in RRR-α-tocopheryl succinate-induced differentiation of human MDA-MB-435 breast cancer cells. Mol. Carcinogenesis, 33: 228-236, 2002.

15. Yu, et al., Activation of increasing interest among signal-regulated kinase and c-Jun-NH2-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-α-tocopheryl succinate-induced apoptosis of human breast cancer cells. Cancer Res., 61: 6569-6576, 2001.

16. Koshkina, et al., Distribution of camptothecin after delivery as a liposome aerosol or following intramuscular injection in mice. Cancer Chemother. Pharmacol., 44: 187-192, 1999.

17. Knight, et al., Anticancer effect of 9-nitrocamptothecin liposome aerosol on human cancer xenografts in nude mice. Cancer Chemother. Pharmacol., 44: 177-186, 1999.

18. Koshkina, et al., 9-Nitrocamptothecin liposome aeosol treatment of melanoma and osteosarcoma lung metastasis in mice. Clin. Cancer Res,. 6: 2876-2880, 2000.

19. Walrep, et al., Pulmonary delivery of beclomethasone liposome aerosol in volunteers. Chest., 111: 316-23, 1997.

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21. Miller, V.E., Roi, L.D., Howard, L.M., and Miller, F.R.Quantitative selectivity values of contact-mediated intercellular communication in a metastatic mouse mammary tumor line. Cancer Res., 43: 4102-4107, 1983.

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23. Tirmenstein, et al. Sensitive method for measuring tissue alpha-tocopherol and alpha-tocopheryloxybutyric acid by high-performance liquid chromatography with fluorometric detection. J. Chromatogr. In Biomed. Sci. Appl, 707: 308-311, 1998.

24. Schwenke, D. .Does lack of tocopherols and tocotrienols put women at increased risk of breast cancer? J.Nutr. Biochem. 13:2-20, 2002.

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All patents and publications referred to in this description reflect the level of knowledge and professionalism of those experts in the field to which the invention relates. In addition, the above-mentioned patents and publications incorporated in this text by reference to the same extent as if each individual publication was a special and individually incorporated by reference.

Specialists in this field will readily appreciate that the present invention is sufficiently adapted to the implementation of the objectives and outcomes and benefits mentioned in the description. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described in this text, are now preferred aspects, are illustrative and do not seek to limit the scope of the invention. Changes and other applications can be made by those skilled in the field that cover the scope of the invention defined by the scope of the claims.

1. The application is based on the vitamin E compounds having the structural formula

where R1is a carboxylic acid;

R2and R3represent hydrogen or R ;

R4is stands; and

R5is alkyl; or

where R1represents hydrogen or carboxylic acid;

R2and R3represent hydrogen or R4;

R4is stands; and

R3is alkenyl,

as the active substance of the medicinal product sprayed in aerosol form for the treatment of diseases or disorders, the treatment of which requires a reduction of cell proliferation and/or induction of cell apoptosis in a patient.

2. The use according to claim 1, where specified based on the vitamin E compound is a 2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethylacetyl)chroman-6-yloxy)acetic acid.

3. The use according to claim 1, where specified based on the vitamin E compound is a tocotrienol.

4. The use according to claim 3, where the specified tocotrienol is a α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol.

5. The use according to claim 1, where the specified medicinal product also contains anti-cancer drug.

6. The use according to claim 5, where the specified anti-cancer drug is a 9-nitrocamptothecin, cisplatin, paclitaxel, doxorubicin or celecoxib.

7. The use according to claim 1, where the specified Leka is the only tool contains lipid.

8. The use according to claim 7, where the ratio is based on the vitamin E compounds and lipid is about 1:3 by weight.

9. The use according to claim 7, where the specified lipid represents a 1,2-Dilauroyl-sn-glycero-3-phosphocholine.

10. The use according to claim 7, where specified based on the vitamin E compound is presented in liposomal form.

11. The use of claim 10, where the final concentration specified based on the vitamin E compound in the liposome is not more than 20.0 mg/ml

12. The use according to claim 7, where specified based on the vitamin E compound is presented in the form of nanoparticles, microspheres or Nozomi.

13. The use according to any one of claims 1 to 12, where the specified medicinal product is administered to the patient by aerosol inhalation.

14. Use item 13, where the specified aerosol inhalation is carried out by inkjet aerosol spray.

15. The use according to claim 1, where the disease is a neoplastic disease selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, breast cancer, testicular cancer, prostate cancer, gliomas, fibrosarcoma, retinoblastoma, melanoma, sarcoma of soft tissue, osteosarcoma, leukemia, colon cancer, carcinoma of kidney, pancreatic cancer, basal cell kartz the bromeliad and squamous cell carcinoma.

16. The use according to claim 1, where the disease is selected from the group consisting of psoriasis, benign proliferative skin diseases, ichthyosis, papilloma, restenosis, scleroderma, hemangioma, leukoplakia, viral infections and autoimmune disorders.

17. The use according to claim 1, where the disease is an autoimmune disease selected from the group consisting of autoimmune thyroiditis, multiple sclerosis, severe pseudoparallelism gravis, systemic lupus erythematosus, dermatitis herpetiformis, gluten disease and rheumatoid arthritis.

18. The application of article 16, where the specified autoimmune disorder selected from the group consisting of inflammatory process involved in the formation of plaques in the cardiovascular system, skin lesions caused by ultraviolet radiation, and disease, including immune component.

19. The use according to claim 1, where the disease is a viral disease.

20. The application of claim 19, where the specified viral disease caused by human immunodeficiency virus.

21. The application is based on the vitamin E compounds having the structural formula

where R1is a carboxylic acid;

R2and R 3represent hydrogen or R4;

R4is stands; and

R5is alkyl; or

where R1represents hydrogen or carboxylic acid;

R2and R3represent hydrogen or R4;

R4is stands; and

R5is alkenyl,

as active ingredient to obtain medicines sprayed in aerosol form for the treatment of diseases or disorders, the treatment of which requires a reduction of cell proliferation and/or induction of cell apoptosis in a patient.

22. Use item 21, where specified based on the vitamin E compound is a 2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethylacetyl)chroman-6-yloxy)acetic acid.

23. Use item 21, where specified based on the vitamin E compound is a tocotrienol.

24. The application of item 23, where the specified tocotrienol is a α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol.

25. Use item 21, where the specified medicinal product also contains anti-cancer drug.

26. Use A.25, where the specified anti-cancer drug is a 9-nitrocamptothecin, cisplatin, paclitaxel, doxer bicin or celecoxib.

27. Use item 21, where the specified medication contains lipid.

28. The application of item 27, where the ratio is based on the vitamin E compounds and lipid is about 1:3 by weight.

29. The application of item 27, where the specified lipid represents a 1,2-Dilauroyl-sn-glycero-3-phosphocholine.

30. The application of item 27, where specified based on the vitamin E compound is presented in liposomal form.

31. The application of article 30, where the final concentration specified based on the vitamin E compound in the liposome is not more than 20.0 mg/ml

32. The application of item 27, where specified based on the vitamin E compound is presented in the form of nanoparticles, microspheres or Nozomi.

33. The use according to any one of p-32, where the specified medicinal product is administered to the patient by aerosol inhalation.

34. Use p where the specified aerosol inhalation is carried out by inkjet aerosol spray.

35. Use item 21, where the disease is a neoplastic disease selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, breast cancer, testicular cancer, prostate cancer, gliomas, fibrosarcoma, retinoblastoma, melanoma, sarcoma of soft tissue, osteosarcoma, leukemia, cancer about the colonic ulcers, carcinoma of kidney, pancreatic cancer, basal cell carcinoma and squamous cell carcinoma.

36. Use item 21, where the disease is selected from the group consisting of psoriasis, benign proliferative skin diseases, ichthyosis, papilloma, restenosis, scleroderma, hemangioma, leukoplakia, viral infections and autoimmune disorders.

37. Use item 21, where the disease is an autoimmune disease selected from the group consisting of autoimmune thyroiditis, multiple sclerosis, severe pseudoparallelism gravis, systemic lupus erythematosus, dermatitis herpetiformis, gluten disease and rheumatoid arthritis.

38. Use item 21, where the specified autoimmune disorder selected from the group consisting of inflammatory process involved in the formation of plaques in the cardiovascular system, skin lesions caused by ultraviolet radiation, and disease, including immune component.

39. Use item 21, where the disease is a viral disease.

40. The application of § 39, where the specified viral disease caused by human immunodeficiency virus.

41. Aerosol spray composition for treating diseases or disorders whose treatment required is the duty to regulate the reduction of cell proliferation and/or induction of cell apoptosis in a patient, where the composition comprises based on the vitamin E compound having the structural formula

where R1is a carboxylic acid;

R2and R3represent hydrogen or R;

R4is stands; and

R5is alkyl; or

where R1represents hydrogen or carboxylic acid;

R2and R3represent hydrogen or R4;

R4is stands; and

R5is alkenyl,

as the active substance.

42. The composition according to paragraph 41, where specified based on the vitamin E compound is a 2,5,7,8-tetramethyl-(2R-(4R,8R,12-trimethylacetyl)chroman-6-yloxy)acetic acid.

43. The composition according to paragraph 41, where specified based on the vitamin E compound is a tocotrienol.

44. The composition according to item 43, where the specified tocotrienol is a α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol.

45. The composition according to paragraph 41, optionally containing anticancer drug.

46. The composition according to item 45, where the specified anti-cancer drug is a 9-nitrocamptothecin, cisplatin, paclitaxel, doxorubicin or celecoxib.

47. The composition according to the .41, optionally containing lipid.

48. The composition according to p, where the specified lipid represents a 1,2-Dilauroyl-sn-glycero-3-phosphocholine.

49. The composition according to p where specified based on the vitamin E compound is presented in liposomal form, this lipid is a 1,2-Dilauroyl-sn-glycero-3-phosphocholine.

50. The composition according to p, where the ratio is based on the vitamin E compounds and lipid is about 1:3 by weight.

51. The composition according to p where specified based on the vitamin E compound is presented in the form of nanoparticles, microspheres or Nozomi.

52. The composition according to paragraph 41, where the disease is a neoplastic disease selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, breast cancer, testicular cancer, prostate cancer, gliomas, fibrosarcoma, retinoblastoma, melanoma, sarcoma of soft tissue, osteosarcoma, leukemia, colon cancer, carcinoma of kidney, pancreatic cancer, basal cell carcinoma and squamous cell carcinoma.

53. The composition according to claim 1, where the disease is selected from the group consisting of psoriasis, benign proliferative skin diseases, ichthyosis, papilloma, restenosis, scleroderma, hemangioma, leukoplakia, viral diseases and autoimmune disorders.

54. The composition according to paragraph 41, where the disease is an autoimmune disease selected from the group consisting of autoimmune thyroiditis, multiple sclerosis, severe pseudoparallelism gravis, systemic lupus erythematosus, dermatitis herpetiformis, gluten disease and rheumatoid arthritis.

55. The composition according to item 53, where the specified autoimmune disorder selected from the group consisting of inflammatory process involved in the formation of plaques in the cardiovascular system, skin lesions caused by ultraviolet radiation, and disease, including immune component.

56. The composition according to paragraph 41, where the disease is a viral disease.

57. The composition according to p, where the specified viral disease caused by human immunodeficiency virus.

58. Composition according to any one of p-57, which is administered to the patient by aerosol inhalation.

59. The composition according to § 58 where the specified aerosol inhalation is carried out by inkjet aerosol spray.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention pertains to new derivatives of indole with general formula 1: where R is unsubstituted or substituted quinolyl, pyridopyrazinyl, indazolyl or pyridyl and which is directly bonded to nitrogen of the amide group; R1 is unsubstituted or substituted alkly-aryl; R2 represents hydrogen; R3-R6 represent hydrogen, R7 represents (C1-C6)-alkylcarbonyl or (C1-C6)- alkoxycarbonyl, and X, Y represent oxygen or sulphur, under the condition that, when R is an unsubstituted or substituted 2-, 3-, 4-, 5- and 6-pyridyl group and R1-R6 have the above mentioned values, R7 is not an acetyl radical or tert-butyloxycarbonyl group. The invention also relates to physiologically tolerant salts of the indole derivatives, as well as to pharmaceutical compositions based on them and their use in obtaining medicinal preparations.

EFFECT: obtaining of medicinal preparations, used as medicines for curing tumorous diseases, especially in case of resistance to other drugs and metastasising carcinomas.

14 cl, 7 tbl, 6 dwg, 25 ex

FIELD: chemistry.

SUBSTANCE: invention pertains to derivatives of quinoline with general formula Ia or Ib their stereoisomers and pharmaceutical salts, where X represents oxygen or sulphur, Z-CH2, Y-NO2, -C(O)OR5, -NR5SO2R5, -SO2R5 (for Ia) and -NO2 or -C(O)OR5 (for Ib). Description is also given of the method of obtaining Ia and Ib compounds, pharmaceutical compositions based on them, and their use when making medicinal preparations.

EFFECT: compounds can be used for treating lesions, related to inhibition of migration of magrophage, for example, during treatment of septic shock or arthritis.

175 cl, 16 tbl, 22 ex, 16 dwg

FIELD: technological processes; medicine.

SUBSTANCE: invention is related to preparation of recombinant analogues of human gamma-interferon and may be used in medicine for prophylactics and treatment of oncological diseases, neoplasms and inflammatory processes of humans. Highly pure genetically engineered analogue of human gamma-interferon - deltaferon with molecular mass of 16.2 kilo Daltons is produced by microbiological synthesis with further chromatographic purification on "KM"-sepharose - cationic-exchange sorbent with high linear speed of flow and suitable for preparative loads, at different pH values. On the basis of deltaferon, which contains by data of polyacrylamide gel-sodium dodecyl sulfate electrophoresis at least 98% of main substance in the form of monomer that possesses antiproliferative and anti-inflammatory activities inherent in gamma-interferon, medical product is prepared, which also includes low molecular polymer filler-stabiliser (reopolyglukine or polyvinylpyrrolidone) and salt buffer system with pH 7.0-7.1.

EFFECT: highly active and stable preparation of deltaferon is produced.

2 cl, 6 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention refers to the improved method of preparation of paclitaxel, which contains: (a) acetylization of 10-deacetyl baccatin III in position C-10 with tertiary amine base for baccatin III extraction; (b) protection of baccatin III in the position C-7 by means of baccatin III reacting with the protective group such as 2,2,2-trichloroethylchloroformates with tertiary amine as a catalyst; (c) reforming of product which was prepared on the stage (b) into paclitaxel. Paclytaxel is a well-known antitumor drug.

EFFECT: process simplification and increase of yield.

11 cl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention refers to cyanoguanidine derivatives of the general formula I in which A, A, X1, X2, X3, Y1, Y2, Y3, R1, R2, R5, R6 and n have the values indicated in the formula of the invention. The invention also refers to the pharmaceutical composition which has antiproliferative activity on the basis of the compound of the formula I, and application of these compounds for medicinal preparation aimed at treatment and alleviation of proliferative disease and conditions.

EFFECT: new compounds can be useful at proliferative disease treatment.

25 cl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention pertains to tricyclic derivatives, represented by formula (I) or to their pharmaceutical salts, which have proliferative activity and ability to inhibit angiogenesis, to the method of obtaining them (alternatives), the proliferative agent and an angiogenesis inhibitor on their base. , where R1 represents T1B1; Ti represents -N(R5)C(O)-, where R5 represents H or C1-C5 alkyl group; and B1 is chosen from a group, consisting of the following values (a),(b),(f),(g): , where represents , or ; R6 represents H, halogen or hydroxy group; R7 represents a hydroxy or -ONO2 group, under the condition that R6 represents H, R7 is different from hydroxyl; T2 represents -O-C(O)-; B2 the stated (a) group or -Z1-R7, where Z1 represents a linear C2-C5alkyl group, n2 represents an integer 0-3; and n3 represents an integer 0-5; R2 represents CH3; R3 represents C1-C4 unbranched or branched alkyl, or C3-C7 cycloalkyl, under the condition that, if B1 represents (a), R6 represents H, R7 represents an -ONO2 group and R3 is not CH3; R4 represents SCH3 or OCH3; X represents O.

EFFECT: perfection of the method of obtaining compounds.

7 cl, 6 tbl, 14 dwg, 48 ex

FIELD: medicine; oncology.

SUBSTANCE: respiratory conditions are changed between hypoxic and hyperoxic by means of device furnished with corrugated respiratory tube (CRT) with smooth length variation within 50 cm to 200 cm and auxiliary dead zone (ADZ) within 500 cm3 to 2000 cm3. During 2-3 weeks latent and compensated hypoxia and hyperoxia are interchanged. For this purpose within 15-30 minutes 3-5 times a day patient daily breaths in and out gas mixture of initial content O2 21% accompanying with gradual reduction to 16-12% by ADZ changing. Then in hyperoxic respiratory conditions patient breaths in and out atmospheric air within 5-10 minutes. Then within at least 10 days subcompensated and decompensated hypoxia and hyperoxia are interchanged. For this purpose patient once a day within 10-30 minutes breaths in and out gas mixture of falling content O2 within 21% to 5%, in closed circuit under hypercapnia 3-5% CO2- Then patient is transferred to hyperoxic breathing with 100% content of O2 in gas mixture within 10-30 minutes. Simultaneously oxidant inhalations are taken. Device contains mask, specified CRT and inhalational chamber (IC). IC is made of corrugated flexible tube. Inner and outer diameters of CRT are 3 and 5 cm respectively. IC is fixed U-shaped bend of CRT liquid filled. Distal end is equipped with oxygen container or respiratory sac.

EFFECT: reduced toxicity and number of traumas.

7 cl, 2 dwg, 2 ex

FIELD: medicine; pharmaceutical.

SUBSTANCE: invention refers to pharmaceutical formulation and therapeutic method including introduction to related patient of composition 2-alkyliden derivative of 19-nor-vitamin D and bisphosphonate. In particular, this invention refers to pharmaceutical formulation and therapeutic methods including introduction to related patient of 2-methylene-19-nor-20(S)-1α,25- dihydroxyvitamin D3 and bisphosphonate selected from tyludronate, alendronate, zoledronate, ibanedronate, risedronate, ethydronate, clodronate or pamydronate. Stated invention allows increasing of therapeutic efficiency of such diseases as senile osteoporosis, postclimacteric osteoporosis, bone fracture, bone graft, osteopeny and male osteoporosis.

EFFECT: increased therapeutic efficiency.

18 cl, 2 ex, 2 tbl

FIELD: medicine; pharmacology.

SUBSTANCE: invention can be applied for tumours treatment and diagnostics. This composition contains urease enzyme and target fragment directly conjugated with urease, and optionally weak-base antitumor compound.

EFFECT: improved enzyme delivery to tumour cells is provided as composition is injected.

6 cl, 6 ex, 3 tbl, 5 dwg

FIELD: pharmacology.

SUBSTANCE: refers to application of compounds of common formula 1 for production of medicinal agent for inhibition of cdc25-phosphatases and/or CD45- phosphatase, medicinal agent for inhibition of cdc25-phosphatases or CD45- phosphatase, pharmaceutical formulations for inhibition of cdc25-phosphatases or CD45-phosphatase; invention also refers to compounds of formula 1 and provides high-degree inhibition of cdc25-phosphatases and/or CD45- phosphatase.

EFFECT: high degree of inhibition.

22 cl, 1 dwg, 3 tbl, 131 ex

FIELD: medicine.

SUBSTANCE: method of production of dry polyvalent virus-vaccine includes separate infection of cell culture with strain PC-126 of turkey herpesvirus (virus of Marek's disease 3rd serotype) and one-day chicken infected with chicken herpesvirus (virus of Marek's disease 2nd serotype), incubation, turkey herpesvirus harvest, and sampling of double flag follicles of chicken herpesvirus infected chicken, protective medium addition, separate ultrasonic processing of virus cell mass and flag follicle mass, freezing and drying of end product followed with their mixing. At that chicken herpesvirus strain are sampled for (VMD 2nd serotype) strains "42", "50", "SB-1", inoculated in dosage 10000-50000 functional residual capacity (FRC) for chicken and grown in body within 12-25 days. Follicles processed with ultrasonic is removes, and protective medium processed with ultrasonic and containing released chicken herpesvirus is added equal proportion of processed with ultrasonic clean cell cultures of bird embryos grown within 24-72 hours. Dry polyvalent virus-vaccine contains cell-free lyophilized strain FC-126 of turkey herpesvirus - 3rd serotype of Marek's disease virus in protective medium. In addition virus-vaccine includes cell-free lyophilised strains of chicken herpesvirus - 2nd serotype of Marek's disease virus, produced by any cl.1-5, in protective medium at ratio 2000 FRC /units: 100-5 00 FRC /units, respectively.

EFFECT: vaccine has high immunogenic activity and storage stability.

10 cl, 3 tbl, 4 ex

Poplar-aspen oil // 2326685

FIELD: medicine; pharmacology.

SUBSTANCE: agent contains oil extract of poplar buds and sprouts and oil extract of aspen buds and sprouts with component ratio as follows, mass.%: oil extract of poplar buds and sprouts 20-90, oil extract of aspen buds and sprouts 10-80, mixed oil extract of poplar buds and sprouts and oil extract of aspen buds and sprouts, with component ratio as follows, mass.%: oil extract of poplar buds and sprouts 20-90, oil extract of aspen buds and sprouts 10-80, as well as fat extract of plant raw with plant component ratio as follows, mass.: marsh tea 3, comfrey 3, blueash 2, silver fir 1, common burdock (root) 2, horseheal (root) 1, deer's-tongue 1, horseradish 1, musquash-poison 2, hog bean (herb) 2, hop (cones) 2, cowberry (root) 2, mountain arnica (blossom) 2, ratio of oil extracts and fat extract of plant raw is 1:3 respectively.

EFFECT: agent allows widening range of preventive and therapeutic herbal medicinal agents of antiviral and anti-inflammatory action.

2 cl, 9 ex

FIELD: veterinary; veterinarian virology.

SUBSTANCE: production of dry cultural rinderpest virus-vaccine for minor ruminants includes growing of virus containing raw materials from "45G37/35-K" rinderpest virus strain in cell culture, introduction of protective medium and production of the end product. Passaged cell culture of saiga kidney is used as a cell culture. Infected culture is incubated under roller conditions during 5-7 days with the change of supporting medium each 2-3 days. Virus containing culture is mixed before freeze drying in proportion 1:1, and then freeze dried until moisture mass fraction is no more than 4%. Finally, it is packed into ampoules. The end product contains virus raw materials, peptone, sucrose, gelatine and demineralised water.

EFFECT: high-performance production of standard and innocuous rinderpest virus-vaccine for minor ruminants stable for storage conditions.

2 cl, 3 ex

FIELD: medicine, pharmacology.

SUBSTANCE: mixture of chitosonium glutamate obtained from high molecular chitosan with molecular mass 100-500 kD, and restrictedly depolymerised low polymeric chitosan with molecular mass 0.5-20 kD, is added to the inactivated flue vaccine as an adjuvant; the molecules of chitosan and chitosonium glutamate have free aldehydic groups at one of their free end, and have the deacetylation degree within 60-90%; the concentration of high molecular chitosan is brought to 0.5%, and low polymeric chitosan - to 0.05%, and the parenteral vaccination is performed.

EFFECT: method increases the immunogenicity of inactivated flue accine.

4 dwg, 4 ex

FIELD: medicine, veterinary.

SUBSTANCE: vaccine contains active substance and food additive. Active substance consists of efficient amount of avirulent antigenic material from "Novosibirsky" bird flue strain Influenzae virus avicum, Orthomyxoviridae family, serotype A, subtype H5N1, collection of FGU "VGNKI" VNIISZH N. 125 - deposition "Novosibirsky".

EFFECT: vaccine provides the efficient bird protection against the epizootic virus and prevents the environmental propagation of infectious agent from immunized bird.

6 cl, 12 tbl, 7 ex

FIELD: organic chemistry, medicine, pharmacy, chemical technology.

SUBSTANCE: invention relates to novel substituted esters of 1,2,3,7-tetrahydropyrrolo[3,2-f][1,3]benzoxazin-5-carboxylic acids of the general formula (1): or their racemates, or their optical isomers, or their pharmaceutically acceptable salts and/or hydrates possessing the antiviral effect. In compounds of the general formula (1) each R1 and R4 represents independently of one another a substitutes of amino group chosen from hydrogen atom, optionally substituted linear or branched alkyl comprising 3-12 carbon atoms, optionally substituted cycloalkyl comprising 3-10 carbon atoms, optionally substituted aryl or optionally substituted and possibly an annelated heterocyclyl that can be aromatic or nonaromatic and comprising from 3 to 10 atoms in ring with one or some heteroatoms chosen from nitrogen, oxygen or sulfur atoms or their oxides; R2 represents alkyl substitute chosen from hydrogen atom, optionally substituted mercapto group, optionally substituted amino group, optionally substituted hydroxyl; R3 represents lower alkyl or cycloalkyl; R5 represents a substitute of cyclic system chosen from hydrogen atom, optionally substituted linear or branched alkyl comprising 3-12 carbon atoms, optionally substituted cycloalkyl comprising 3-10 carbon atoms, optionally substituted aryl or optionally substituted and optionally an annelated heterocyclyl that can be aromatic or nonaromatic and comprising from 3 to 10 atoms in ring with one or some heteroatoms chosen from nitrogen, oxygen or sulfur atoms or their oxides; R6 represents a substitute of cyclic system chosen from hydrogen atom, halogen atom, cyano group, optionally substituted aryl or optionally substituted and optionally annelated heterocyclyl that can be aromatic or nonaromatic and comprising from 3 to 10 atoms in ring with one or some heteroatoms chosen from nitrogen, oxygen or sulfur atoms or their oxides. Also, invention relates to methods for treatment, drugs and pharmaceutical compositions using compounds of this invention. Proposed compounds can be used as active components of drugs used in treatment of such diseases as infectious hepatitis, human immunodeficiency, atypical pneumonia and avian influenza.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition, improved methods of synthesis.

22 cl, 3 tbl, 6 dwg, 7 ex

FIELD: organic chemistry, medicine, pharmacy, chemical technology.

SUBSTANCE: invention relates to novel substituted esters of 1H-indol-3-carboxylic acids of the general formula (1): or their racemates, or their optical isomers, or their pharmaceutical acceptable salts and/or hydrates. Compounds can be used in treatment of such diseases as infectious hepatitis, human immunodeficiency, atypical pneumonia and avian influenza. In compound of the general formula (1) R1, R41 and R42 each represents independently of one another a substitute of amino group chosen from hydrogen atom, optionally linear or branched alkyl comprising 3-12 carbon atoms, optionally substituted cycloalkyl comprising 3-10 carbon atoms, optionally substituted aryl or optionally substituted and possibly an annelated heterocyclyl that can be aromatic or nonaromatic and comprising from 3 to 10 carbon atom in ring with one or some heteroatoms chosen from nitrogen oxygen or sulfur atoms; or R41 and R42 in common with nitrogen atom to which they are bound form 5-10-membered azaheterocycle or guanidyl through R41 and R42; R2 represents an alkyl substitute chosen from hydrogen atom, optionally substituted mercapto group, optionally substituted amino group, optionally substituted hydroxyl; R3 represents lower alkyl; R5 represents a substitute of cyclic system chosen from hydrogen atom, halogen atom, cyano group, optionally substituted aryl or optionally substituted and possibly an annelated heterocycle that can be aromatic or nonaromatic and comprising from 3 to 10 atoms in ring with one or some heteroatoms chosen from nitrogen, oxygen or sulfur atoms. Also, invention relates to methods for treatment, drugs and pharmaceutical compositions using compounds of this invention.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition, improved method of synthesis.

22 cl, 3 tbl, 8 dwg, 6 ex

FIELD: biotechnology, preparative biochemistry.

SUBSTANCE: method involves hyperimmunization of animal-producers with inactivated, purified and concentrated rabies virus with aluminum phosphate and/or sodium nucleinate as an adjuvant. Virus obtained in culture transplantable cells VERO and in primary culture of Syrian hamster kidney cells is concentrated by adsorption of aluminum phosphate gel or centrifugation, or ultrafiltration methods and purified passing through porous silica, or by ion-exchange chromatography. For immunization antigen with the immunogenicity index 9.5 IU/ml, not less, is used and with the content of protein less 200 mcg/ml, bovine serum albumin less 0.5 mcg/ml Syrian hamster kidney cells and VERO cells less 0.5 mcg/ml, cellular DNA 5.0-10.0 ng/ml, aluminum ions concentration in antigen 0.2-1.25 mcg/ml, and sodium nucleinate 0.002-0.5 mg/ml. Scheme of grund-immunization - 1-3 injections of antigen in the dose 3-15 ml with interval for 14-30 days, the main immunization cycle - on 45-60 day involving 10-15 injections with interval for 5-15 days and in the antigen dose 5-70 ml. Then producers are immunized once per a month with the dose 20-70 ml, and once per 6 month the shortened cycle of immunization is carried out involving 2-4 injections of antigen with interval for 5-15 days in the dose 20-70 ml. Method provides preparing heterologous antirabies serum of high specific activity.

EFFECT: improved preparing method, improved and valuable properties of serum.

12 cl, 1 tbl, 4 ex

Drug // 2320333

FIELD: medicine, pharmacy.

SUBSTANCE: invention relates to drugs and concerns using pharmaceutically acceptable 2,4-dichlorophenoxyacetic acid alkaline metal salts and mixture of these salts as a drug possessing anti-tumor, immunomodulating and anti-inflammatory properties, and antiviral activity also. Invention provides expanding assortment of agents possessing the broad spectrum of curative effect being without adverse symptoms.

EFFECT: improved and valuable medicinal properties of drug.

8 cl, 18 ex

FIELD: medicine, pharmacy.

SUBSTANCE: invention proposes two variants of composition. By the first variant the composition comprises silver nitrate, hexamethylenetetramine, sodium thiosulfate, alpha-aspartic acid or asparagine, nicotinic acid and water in the amounts given in the invention claim. By the second variant the composition comprises silver nitrate, hexamethylenetetramine or imidazole, imidazole-containing compound of platinum cis-[Pt(NH3)2Im2]Cl2 or cis-[Pt(NH2OH)2Im2]Cl2, alpha-aspartic acid or asparagines, nicotinic acid and water taken in the amount given in the invention claim. Proposed compositions possess the enhanced antitumor activity and simultaneous antibacterial and antiviral effect on body.

EFFECT: improved, enhanced and valuable medicinal properties of pharmaceutical composition.

3 cl, 6 tbl, 3 dwg, 11 ex

FIELD: medicine; pharmacology.

SUBSTANCE: given invention refers to pharmaceutical field, specifically to pharmaceutical formulation of gel dosage form applied for prevention and treatment of osteoporosis including as an active component bisphosphonate incorporated in phospholipid vesicles generated from lipid and hydrophilic phases, including components in the following proportions, mass %: bisphosphonate 0.01-2.0; egg lecithin 1.0-6.0; pine essence 0.05-0.2; camphor oil 0.01-1.0; olive oil 0.01-5.0; vitamin E 0.01-0.15; vitamin D 0.01-0.2; vitamin F 0.2-0.4; carbopole 0.4-0.6; NaOH 0.42; glycerol 2.0-4.0; nipagine 0.3; nypazole 0.1; water and others. In addition invention refers to treatment of bone tissue resorption of any etiology and osteoporosis for patients suffering from gastrointestinal disturbance with this composition.

EFFECT: high concentration of bisphosphonate inside of tissues and bones directly in and around medicine application onto the skin without necessity to apply additional medical equipment or techniques.

10 cl, 2 dwg, 3 ex

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