Anticancer drug based on nanoparticles bearing recombinant human tumour necrosis factor alpha
SUBSTANCE: invention refers to medicine and biotechnology and concerns an anticancer drug based on nanoparticles bearing recombinant human tumour necrosis factor alpha. Substance of the invention includes the anticancer drug representing nanoparticles each of which contains a nucleus consisting of polynucleotide complex representing double-helical RNA (dhRNA) - an interferonogenesis inducer, and coated with a layer of spermidine conjugate with polyglucin held by ionic interaction between negative polynucleotide complex and positive spermidine, while recombinant human tumour necrosis factor alpha is covalently bound with activated polyglucin. As double-helical RNA, the anticancer drug contains double-helical RNA of Saccharomyces cerevisiae yeast. Nanoparticles are ball shaped and sized about 50-70 nm; 60-80 molecules of recombinant human TNF-α of cytolytic activity 106 ME/mg of protein and higher, 60-80 molecules of polyglucin and 1000-1300 molecules of spermidine are necessary for one molecule of double-helical RNA of Saccharomyces cerevisiae yeast.
EFFECT: reduced dose of TNF-α and lower toxicity.
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The invention relates to anticancer agents to be used in the field of medicine and pharmaceuticals.
As is known, the incidence of malignant tumors in many countries continues to grow steadily. The increase in the number of cancers caused by several factors, most important of which are the increasing number of elderly and adverse environmental conditions. Over the last 25-30 years the rate of increase of frequency of diseases of this type exceeded the annual rate of population growth in developed countries. By 2010 the number of cases in the world is projected to 12.5 million people. In Russia the incidence of primary malignant neoplasms in 2004 amounted to 473 thousand (326,3 per 100 thousand population) and continues to grow steadily. Serious is the problem of deterioration of the epidemiological situation in many countries of the world, which is associated with a progressive weakening of the immunoreactivity of the population in these countries, increased frequency and severity of acquired immunodeficiencies, due to environmental and social causes, chronic infectious diseases.
In this regard, the relevance of the search for new means and methods of treatment of malignant tumors in our country is extremely high. This is due to both teenaging disease (histological heterogeneity of tumors, their different sensitivity to chemotherapy, as well as individual characteristics of the functioning of the immune system of patients, the degree of development of the disease), and the current limited Arsenal of effective chemotherapeutic agents.
In recent years considerable attention from the viewpoint of therapeutic applications on cytokines are biologically active factors, peptide, which is produced by cells of the immune system and are both products of vital activity of this system, and its main regulators. Currently, a number of cytokines, such as interferons and interleukins, are used in Oncology as immunomodulators and anticancer agents .
Among the promising anticancer drugs include representatives of the family factors tumor necrosis, in particular tumor necrosis factor alpha (TNF-α). As is known, the factors tumor necrosis represent poly potent proteins-cytokines that play a key role in many physiological and pathological processes in the body. The tumor necrosis factor alpha is able to exert a direct cytotoxic effect on tumor cells of different types to activate antitumor immune response and cause damage to the endothelial cells of tumor vessels, achiev what tatom what is hemorrhagic necrosis [2-4].
Currently, the drugs TNF-α production by pharmaceutical companies Asahi, Knoll, Genentech, Wadley Inst. Cetus, Dainippon and other clinical trials conducted in different countries of the world. Clinical studies of recombinant human TNF-α human abroad showed a positive effect of the drug on more than 10 kinds of tumors, such as melanoma, liposarcoma, neuroblastoma, lung cancer and several other . In 2000, Europe was a registered product on the basis of TNF-α and melphalan for the treatment of unresectable soft tissue sarcoma of the extremities by the method of regional perfusion .
The technology of recombinant TNF-α human was developed in Russia, in particular, in the fsri SRC VB "Vector" of Rospotrebnadzor [7, 8]. Currently completed phase II clinical trials of medicinal forms of TNF-α, drug Alorin. Studies performed at WANTS Sciences (Moscow), suggests that the introduction of Algerina followed by chemotherapy improves therapeutic effect in patients with disseminated melanoma of the skin (up to 59%), resulting in complete or partial remission of metastases or the stabilization process . These data suggest a promising use of the drug TNF-α as a modern tool for immunotherapy of cancer.
However, it should be noted that still presen the th remains the problem of rapid degradation of TNF-α in the bloodstream, and also the limited selectivity of accumulation of the cytokine in the tumor tissue, which necessitates repeated injections to maintain the required effective dose. It was found that long-term administration of TNF-α in high doses is accompanied by various side effects and serves as a barrier to the introduction of this drug in medical practice [10-12].
In this regard, for several years there have been numerous attempts to reduce the systemic toxicity of TNF-α as by directly weakening its proinflammatory properties, and indirectly, through increased proteolytic stability or antitumor activity. One of the most common methods used for this purpose is the creation of chimeric forms of TNF-α or composite preparations from substances-biological response modifiers.
Thus, it was shown that obtaining chimeric variants of TNF-α with α1-Timoshina , leukocidin  allows to increase the antitumor activity of the drug by increasing its immunomodulatory activity. Conjugate protein monometoksipolietilenglikolya differed longer, compared to TNF-α, the period of removal from the blood of experimental animals, with a resulting increase in protivoopujoleve the activity of the drug against transplanted tumors of Meth fibrosarcoma and reducing the intensity of the toxic manifestations . The conjugation of TNF-α with polymers, such as poliglyukin, polyvinylpyrrolidone, divinely ether, allowed to increase the stability of proteins to proteolysis and antitumor activity [16-20]. Received a number of positive results related changes in the pharmacokinetics of the drug and enhance its antitumor properties in the case of conjugation TNF-α with fragments of antibodies against tumor antigens , as well as ligands of tumor receptors, such as ligand integranova receptor V-type  or transferrin .
Among the compounds that were used as dietary supplements in the creation of composite preparations, we can distinguish two groups of compounds that differ in their mechanism of action on the organism.
The first group to form a compound-synergists TNF-α. These include, for example, interferons different types (α, β and γ) [24, 25], transforming growth factor beta . Proposed drug for inhibiting the growth of tumors, active principle which, in addition to TNF-α, is the connection that is blocking the protein C system (antibodies against protein C and S, C4δ-binding protein) . O Conner with co-authors  was developed antitumor preparation containing an effective amount of tumor necrosis factor-man and C-reactive protein, reinforcing antiplasmodial activity of TNF-α.
Drugs : the first group are characterized by the presence in its composition of biologically active compounds, reduce side effects of TNF-α. To prevent cell damage induced by cytotoxic action of lymphokines, are used accesory free radicals or metabolic inhibitors (uric acid, butanesulfonic, vitamin C and others) . To reduce or suppress the toxic effects of high doses of TNF used in the treatment of malignant tumors, the proposed introduction of the integrated drug NSAID such as indomethacin, ibuprofen .
Known antitumor composition comprising interleukin-2, alpha-2-interferon and Dunaeva RNA (DN-RNA) . It was shown that the addition of the composition DN-RNA as a catalyst that increases the activity of cytokines, allowed lower doses of IL-2 and TNF necessary to achieve therapeutic effect. However, it should be noted that to obtain a positive effect of the introduction of composite drug when using this DN-RNA/citicentre was conducted using doses of the order of 103-104ME per kg of body weight, i.e. doses, which, as is well known , already can cause toxic reactions.
For all the undoubted advantages of this approach it should be noted that the introduction of the complex or composition of the drug is a biological modifier is directed, typically, the only solution to any one problem: increased proteolytic stability, enhance the antitumor activity of TNF or reduce the severity of its side effects.
Attempt holistic solution proteolytic stability of TNF-α and enhanced accumulation in tumor tissue is the creation of preparations containing TNF-α in the composition of the nanoparticles. As you know, abnormal vascular wall of a tumor, in contrast to normal, permeable to large molecules with molecular weight of 40 kDa or higher and small particles that accumulate in the extracellular space of the tumor . In oncopathology accumulation also contributes to the failure of the lymphatic system which is responsible for the drainage of macromolecules in normal tissues. The pore size of the endothelium of blood vessels of most tumors ranges from 200 to 600 nm in diameter, which allows the particles of the appropriate size to penetrate directly into the tumor tissue.
Currently studied and developed two types of nanoparticles carrying TNF-α: nanoparticles containing as a carrier of inorganic components, and liposomes.
Visaria R.K. al. demonstrated the possibility of using nanoparticles consisting of coated polyethylene glycol particles, colloidal gold, as systems is the supplying of TNF-α for thermal therapy of tumors . It was shown that this design slows down as the speed of blood flow in the vascular system of the tumor and its growth. Increased accumulation of TNF-α in the composition of the gold particles, coated with modified polyethylene glycol, tumors and a decrease in the bodies enriched with cells of the RES (liver, spleen, and other), noted by the authors of . The nanoparticles, the core of which was formed by particles of silicon, and an organic shell contained maleimide group to bind TNF-α, initiated immune response, which intensity was characteristic of membrane-bound TNF-α .
However, it should be noted that the General lack of structures [32-34], which contains inorganic particles, is the lack of systems for their biodegradation, resulting in accumulation of these particles in the body leads to the development of serious toxic effects, including cytotoxicity .
Another option of nanoconstructs that were designed to protect TNF-α from degradation, are liposomes [36-40]. Unfortunately, it was found that a significant drawback of liposomal forms is their rapid opsonization with intravenous and subsequent capture by cells of the reticuloendothelial system . Liposomes containing TNF-α, quickly recognizable by mononuclear cells faguoqitirute system, had little time circulation and selek is actively destroyed in the reticulo-endothelial system tumors.
To overcome the capture of liposomes by the mononuclear cells of the blood and tumors were developed so-called "liposomes"invisible containing polyethylene glycol (PEG), which led to increased osmotic pressure around the liposomes and inhibited its rapprochement with the cell [42-45]. Pegylated liposomes invisible to the cells of the reticuloendothelial system and for a long time circulate in the blood, but have a major drawback - the bad accumulate in the target cells. In addition, it was found that "liposomes"invisible containing TNF-α, unstable when stored. Finally, it is known that the vast majority of types of liposomes accumulate in the liver (hepatocytes, in particular, to 97% phosphatidylcholine liposomes) and spleen . Therefore, the use of liposomes as carriers of TNF-α has the serious disadvantage associated with the fact that TNF-α, due to its anti-inflammatory properties, may contribute to the development of hepatotoxicity.
The literature data indicate the possibility of changes in pharmaco-toxic properties of TNF-α in conjugation, the inclusion in the composition of the drug or the introduction of nanoconstruction (nanoparticles). As he and other approaches have their advantages and disadvantages. The creation of chimeric forms and compositions TNF-α is aimed at raising about politicheskoi sustainability, strengthening antiplasmodial activity or reducing the toxicity of the protein, but not the goal to increase the accumulation of TNF-α in the tumor tissue. The introduction of TNF-α in the composition of nanoconstructs allows to approach the problem of selectivity of accumulation of the substance in the tumor tissue, but the components of nanoconstructs are mainly biologically inert substances, and there is often the problem of their biodegradation.
For complex problems, you may have to get biodegrading nanoconstruction increasing proteolytic stability of TNF-α, its accumulation in the tumor and containing in its composition, in addition to TNF-α, substance-synergist with antitumor and immunomodulatory properties.
Earlier in fsri SRC VB "Vector" were created two-layer molecular structures containing in the Central part of the nucleotide material (double-stranded yeast RNA), coated from a spermidine-poliglyukina, and on the surface antigens of the infectious agent. On the basis of these structures were obtained from experimental samples of candidate vaccines against HIV, tuberculosis, encephalitis, shown their high efficiency for the immunization of laboratory animals [48-50]. Created design containing polynucleotide and a polysaccharide material, physiological services is under have a spherical virus-like shape, in connection with what is called "virus-like particles (HPV), and size from 25 to 40 nm, which allows you to take it to the category of nanomaterials.
The closest analogue (prototype), according to the applicant, is to design nanoparticles containing TNF-α proposed by Ya-Ping LI and co-authors . Implemented by encapsulating recombinant TNF-α of human rights in polycyanoacrylate nanocapsules (poly(methoxypolyethyleneglycol cyanoacry-late-co-n-hexadecyl cyanoacrylate, PEG-PHDCA) resulted in prolonged half-life of TNF-α in 7 times. Containing TNF-α nanoparticles accumulated predominantly in tumor cells and inhibited tumor growth by 78.3%, while free TNF-α in the same dose caused inhibition of tumor only 15.4%.
However, in this publication, no information about possible degradation of this type of polymeric materials in the biological environment of the body, the possibility of cumulation and, as a consequence, there are no data on the longer-specific toxicity of this formulation.
Information about known nanoconstruction, containing in its composition polynucleotide complex (double-stranded RNA, dsRNA) and TNF-α in the literature currently available.
The technical result of the proposed technical solution is the creation of such antitumor agents based on nanocast is, bearing recombinant tumor necrosis factor alpha man, which is biodegradable nanoparticles with anticancer activity at lower effective doses of TNF-α and reduced toxicity compared to known the above analogs.
This technical result is achieved by the fact that in antitumor agent based on nanoparticles that carry recombinant tumor necrosis factor alpha, according to the invention, each nanoparticle includes a core consisting of a polynucleotide complex, representing double-stranded RNA (dsRNA) - inductor interferonogenesis, and covered with a layer of spermidine conjugate with polyglycine held due to ionic interactions between the negatively charged polynucleotide complex and the positively charged spermidine, and recombinant tumor necrosis factor alpha man is covalently bonded carbon polyglycine. Poliglyukin activated by periodate sodium.
As double-stranded RNA antitumor agent contains double-stranded RNA of the yeast Saccharomyces cerevisiae. The nanoparticles have a spherical shape with a size of about 50-70 nm, which allows to avoid their capture by cells of the reticulo-endothelial system. And one molecule of double-stranded RNA of the yeast Saccharomyces cerevisiae coming up with what is 60-80 molecules of recombinant human TNF-α with cytolytic activity is not lower than 106 IU/mg protein, 60-80 molecules poliglyukina and 1000-1300 molecules spermidine.
Therapeutic effect means is that dsRNA and TNF-α in the composition of the nanoparticles mutually potentiate the effects on malignant cells, thereby reducing the effective dose of TNF-α and reduce the toxic effects of the cytokine.
A sample of the drug nanoparticles with TNF-α by scanning probe microscopy showed that the nanoparticles have a spherical shape with a particle size of about 50-70 nm.
The study of antitumor activity of the drug nanoparticles with TNF-α in experiments on mice transplanted with Ehrlich carcinoma showed that the drug has a high potential to inhibit tumor growth, compared with monotherapy TNF-α. Effective dose of TNF-α in the composition of the nanoparticles in the 10-100 times lower than the effective dose is included in its composition TNF-α.
Evaluation of the toxic properties of the drug nanoparticles with TNF-α in comparison with the preparation of TNF-α in mice-the carriers of tumour transplanted with Ehrlich carcinoma showed that the maximum tolerated dose for nanoparticles with TNF-α 1.5 times higher than the values obtained for TNF-α.
Therefore, nanoparticles, representing the composition of two biologically active substances dsRNA and TNF-α, has a higher antitumor activity and reduced toxicity, with whom avanyu with TNF-α.
Justification of the criterion of "inventive step"
The creation of the claimed design nanoparticles as a means of depositing and delivery of TNF-α with high antitumor and low toxic effect is not obvious to a person skilled in the field of medicine and biotechnology, as it took more research and obtain the following data:
1) can the introduction of TNF-α in the composition of the proposed nanoconstructs to improve its accumulation in tumor tissue, which, in turn, is due both to the size and characteristics of the penetration of the inventive nanoparticles through the vessels of the tumor;
2) it is known that the drug double-stranded RNA of the yeast S. cerevisiae, which forms the core of the nanoparticles, has anti-tumor and immunomodulatory properties , however it was required to install and experimentally substantiate whether dsRNA lead to modulation and enhancement of the antitumor properties of TNF-α and consequently, reduction of toxic properties of the cytokine;
3) as a shell surrounding the core of the nanoparticles and providing exposure of TNF-α on the surface, it is proposed to use poliglyukin. On the one hand, it is known to Deposit and protection from degradation of therapeutic agents , and stimulation of the immune response , and with drugoystorony it was necessary to establish the possibility of creating conjugate poliglyukin-spermidine - TNF-α and the ability to create entire nanoconstruction with the desired pharmacological properties. Obvious is the quantitative content of the proposed components in the composition of the nanoparticles.
The invention is illustrated by the following drawings.
Figure 1. Appearance nanoparticles carrying TNF-α where:
And diagram design nanoparticles;
B - image of the particles obtained by the method of scanning probe microscopy. A sample of nanoparticles with TNF-α, 10 µg/ml Conditions scan: cantilever NSG10, the scanned area of 2.5×2.5 μm.
Figure 2. Electrophoregram drug nanoparticles with TNF-α and its components in a 1%agarose gel, where tracks:
And - the original double-stranded RNA (dsRNA);
1 - dsRNA in the sheath of the conjugate "spermidine-poliglyukin";
2 - dsRNA in the sheath of the conjugate "spermidine-poliglyukin-TNF-α.
Figure 3. The impact of drug nanoparticles with TNF-α and TNF-α on the growth of transplantable tumors of Ehrlich carcinoma.
The abscissa axis is the dose of drugs, E/mouse; on the y - axis the inhibition of tumor growth, %, with the introduction of the name-α (dark bars) or nanoparticles with TNF-α (bars with hatching), * - differences from control are statistically significant, p≤0,05.
Figure 4. The death of animals with transplantable Ehrlich carcinoma after administration of drugs is ancaster with TNF-α and TNF-α.
On the x - axis dose, mg/kg; on the y - axis the death of animals %, with the introduction of TNF-α (dark bars) or nanoparticles with TNF-α (bars with hatching).
The design of nanoparticles with TNF-α
Each of the nanoparticles (figure 1) contains the core 1 consisting of the polynucleotide complex, representing double-stranded RNA (dsRNA) - inductor interferonogenesis, covered by a layer 2 conjugate spermidine/poliglyukina and attached molecules 3 recombinant tumor necrosis factor alpha man (TNF-α), held due to ionic interactions between the negatively charged polynucleotide complex and the positively charged spermidine. As double-stranded RNA antitumor agent contains double-stranded RNA of the yeast Saccharomyces cerevisiae. The nanoparticles have a spherical shape with a size of about 50-70 nm, which allows to avoid their capture by cells of the reticulo-endothelial system of the body. And one molecule of double-stranded RNA of the yeast Saccharomyces cerevisiae have 60-80 molecules of recombinant human TNF-α with cytolytic activity is not lower than 106 IU/mg protein, 60-80 molecules poliglyukina and 1000-1300 molecules spermidine.
For a better understanding of the invention the following are examples of nanoparticles with TNF-α.
Example 1. Synthesis of conjugate poliglyukin-which-is-BUT-α
For the synthesis of conjugate 60 mg (1.2 µm) poliglyukina with molecular weight of 50 000 Da dissolved in 0.5 ml of 50 mm solution of periodate sodium and incubated for 50 min at room temperature. The activated polysaccharide is separated from periodate sodium gel filtration on a column of Sephadex G-50 (V=2 ml) in 50 mm bicarbonate buffer, pH 8.6 and add a solution of 50 mm bicarbonate buffer, pH 8,6 (4-6 ml), containing 17 mg of recombinant TNF-α. After incubation for 3 h at 6°C in the solution contribute to 0.05 ml of a solution containing 2.25 mg (15 μm) spermidine, mix thoroughly. After another 5 h add 0.1 ml of freshly prepared 10 mm solution of periodate sodium. After incubation for one hour at 6°C unreacted components are removed by gel-filtration on a column of Sephadex G-50 (V=20 ml) in phosphate-buffered saline containing 0.15 M NaCl, pH to 7.2. Then additional sample cialiswhat against two changes of the same buffer, 200 ml each. The preparation is sterilized by filtration (0.2 μm).
In the analysis of the conjugate on electrophoregram a 10% polyacrylamide gel was observed cloud with an apparent molecular mass of 120 kDa. When gel filtration on Sephadex G-50 conjugate was elyuirovaniya in the area of molecular masses 40-45 kDa.
Example 2. Assembling nanoparticles with TNF-α
For nanoparticles containing dsRNA and TNF-α, to a solution of yeast dsRNA to relax is whether the conjugate solution poliglyukin/spermidine/TNF-α ratio: 1 mg dsRNA - 2 mg (protein component) conjugate. For the formation of particles, the solution was incubated at 4°C for 2 hours. Analysis of the obtained nanoparticles was performed by gel-filtration on a column of separate CL-6B and electrophoresis in 1%agarose gel.
The quantitative composition of the nanoparticles containing tumor necrosis factor alpha and double-stranded RNA of the yeast Saccharomyces cerevisiae (Central part): one molecule of double-stranded RNA of the yeast Saccharomyces cerevisiae have 60-80 molecules of recombinant human TNF-α with cytolytic activity is not lower than 106 IU/mg protein, 60-80 molecules polyglycine 1000-1300 molecules spermidine.
Example 3. Characterization of nanoparticles with TNF-α
theoretical model of molecular structure, carrier TNF-α, presented at Figo. Sample analysis carried out by the method of probe microscopy, showed that the structure has a spherical shape with a diameter of about 50-70 nm (Figb.).
In the analysis of the drug by gel-filtration on a column of separate CL-6B shows that the collected nanobiotech eluted earlier original RNA in the free volume (free volume for sepharose CL-6B, according passport characteristics of the producer, from 4 MDA and above). Analysis of nanoparticles and its component, dsRNA, coated from poliglyukina, electrophoresis in 1%agarose gel showed that each sample has two in the Yes of the particles, the corresponding L - and M-forms dsRNA (Figure 2). Judging by the size of the zones, the particles retain a compact and uniform shape. Their mobility in electrophoresis is reduced, compared with the components of the original dsRNA, indicating that the increase in their mass and volume, i.e. about the formation of nanostructures.
After treatment with RNase (0.05 mg/ml) by the method of gel electrophoresis, it was found that if the original double-stranded RNA complete degradation is observed after 30 min of incubation, the collected construction nucleotide material remains intact for at least one day. This proves the completeness of the packing in the membrane and reduce the availability of nucleotide material for degrading factors. Particles in sterile drug retains its compact structure when stored in a domestic refrigerator for at least 6 months.
Example 4. The study of antitumor activity of nanoparticles with TNF-α
The influence of the drug nanoparticles with TNF-α on tumor development study on outbred ICR mice, male, from the transplanted tumor of Ehrlich carcinoma. Tumor cells perejivaut intramuscularly at a dose of 105 cells per animal. The drug nanoparticles with TNF-α injected intraperitoneally in the dose range from 102up to 104E/mouse weight of 20 g as the comparison drug use TNF-α in equivalent doses, control of inim animals injected with saline. Injection of the tested preparations carried out three times, with an interval of one day, a course of injections starting on day 7 after transplantation of tumor cells.
Effect of drugs on tumor growth assessed by change in mass of the tumor site by the end of the experiment (day 15 after transplantation of the tumor). The percent inhibition of tumor growth on the background of the introduction of drugs calculated by the following formula:
where VK is the average tumor growth in the control group;
The average of the experimental group.
Experimental data are treated by methods of variation statistics using the software package "Statgraphics, Vers. 5.0" (Statistical Graphics Corp., USA). The reliability of the detected differences assessed by t-criterion of student.
The results of the study of antitumor activity of the drug nanoparticles with TNF-α in comparison with the drug, TNF-α, shown in Fig 3. The addition of TNF-α in any of the used doses did not significantly pronounced inhibition of tumor growth. The trend towards reduction in the average weight of the tumor was observed only in the group of mice to which the drug was administered at the highest dose of 104E/20 g, and the percent inhibition of tumor growth did not exceed 20%. The drug nanoparticles with TNF-α weakened the growth of tumors already in the dose of 102E/20 g (TNF-α, 19%). Trackr is the things the introduction of the drug nanoparticles with TNF-α doses of 10 3-104E/20 g caused a statistically significant inhibition of tumor growth. The average weight of tumor in the experimental group animals at the end of the experiment were, respectively, 28, and 29% lower than the benchmark (the difference is significant, p<0,05) (figure 3).
Thus, in experimental tumor models have shown that the inhibition of tumor growth with the introduction of nanoparticles with TNF-α was observed at doses of 10-100 times lower than after injection of the drug TNF-α.
Example 5. The study of the toxic properties of the drug nanoparticles with TNF-α
The level of toxicity is determined in experiments on outbred ICR mice, male, with transplantable Ehrlich tumor, in comparison with TNF-α. Tumor cells perejivaut intramuscularly at a dose of 5·105cells in the animal. Drugs nanoparticles with TNF-α and TNF-α was dissolved in 0.9% sodium chloride solution and injected intraperitoneally once daily, in doses ranging from 50 to 750 mg/kg in a volume of 0.2 ml per 20 g of body weight of the animals. The count of dead animals and the appearance of clinical signs of poisoning should be performed within days after drug injection.
Data obtained during the study is presented in Figure 4. It is evident that the dose of 750 mg/kg for drug nanoparticles with TNF-α and TNF-α was absolutely lethal and resulted in 100% mortality within 4 hours after injection. M is ximala portable dose for nanoparticles with TNF-α was 150 mg/kg, for TNF-α (100 mg/kg body weight. Thus, the results of Toxicological experiments indicate that the drug nanoparticles with TNF-α has a more low toxicity, compared with drug TNF-α.
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50. RF patent №2190018, IPC C12N 15/87, 27.09.2002.
51. Masycheva VI, Danilenko DU, Pustoshilova NM, November VA // Vestnik St.Petersburg University. The RAMS. - 1998. No. 4. - S-17.
52. Sakenfeld G.K. Immunological mechanism of action of polysaccharides yeast cells Saccharomyces cerevisiae. Riga: 1990; 152
53. Li Y.P., Pei Y.Y., Zhou Z.H. et al. // J. Control Release.- 2001. - Vol.71, No.3. - P.287-296 (prototype).
1. Antitumor agent based on nanoparticles that carry recombinant tumor necrosis factor alpha human, wherein each nanoparticle has a size of about 50 to 70 nm and contains a nucleus, consisting of the polynucleotide complex, representing double-stranded RNA inducer interferonogenesis, and covered with a layer of spermidine conjugate with polyglycine held due to ionic interactions between the negatively charged polynucleotide complex and the positively charged spermidine, and recombinant tumor necrosis factor alpha h the rights of covalently bonded carbon polyglycine.
2. Antitumor agent according to claim 1, characterized in that as a double-stranded RNA, it contains double-stranded RNA of the yeast Saccharomyces cerevisiae.
3. Antitumor agent according to claim 1, characterized in that poliglyukin activated by periodate sodium.
4. Antitumor agent according to claim 1, characterized in that the nanoparticles have a spherical shape.
5. Antitumor agent according to claim 1, characterized in that one molecule of double-stranded RNA of the yeast Saccharomyces cerevisiae have 60-80 molecules of recombinant human TNF-α with cytolytic activity is not below 106IU/mg protein, 60-80 molecules poliglyukina and 1000-1300 molecules spermidine.
SUBSTANCE: invention refers to preparation of a plant drug for treating stomach cancer. The drug for treating stomach cancer contains cod-liver oil, badger fat, plant seed blood chosen from the group: pomegranate, hot pepper, fennel and activated coal in the following ratio, wt %: cod-liver oil 28; badger fat 28; seed blood 42; activated coal the rest.
EFFECT: treatment with the declared drug involves activating body defences, resistance ensured by improved functioning of an individual's organs and systems, and targeting the lesion focus.
SUBSTANCE: invention concerns medicine, namely oncology and can be used in mesothelioma treatment. The method consists in monthly subcutaneous introduction to the patient of powdered fibrin solution recovered from the donor's blood clot in a dose 100-600 mg of dry powder per one introduction session.
EFFECT: application of the invention allows for effective cancer therapy by fibrin preparation only without administration of cytostatic agents, radioactive drugs and radioisotopes that ensures reduced complications.
3 dwg, 1 ex
SUBSTANCE: invention describes a biomarker intended for determining sensitivity of proliferative diseases, such as cancer, to mTORs inhibitor combined with a cytotoxic agent, first of all with a cytotoxic agent (CA) that damages or disturb DNA integrity. According to the invention, the biomarker designated as p21 represents cip/kip-family of cyclinkinase inhibitors. Sensitivity or response of the proliferative disease in an individual on treatment with mTOR inhibitor combined with CA is determined by the level of p21 expression after CA treatment and a combination therapy with using CA and mTOR inhibitor. Favorable treatment and sensitivity of disease to the combination therapy is predicted by the absence of expression induction reduced. Besides according to the invention, the biomarker can be used in the method to overcome the CA resistance in the patient treated with CA. That is ensured by evaluating p21 level in a sample, the increasing regulation of p21 expression following CA introduction to the patient, mTOR inhibitor is administered in combination with CA, while lowered expression regulation observed following the combination therapy ensures to continue treatment with mTOR inhibitor with simultaneous or consecutive CA introduction.
EFFECT: application of the invention allows for more accurate prediction of sensitivity of a proliferative diseases in an individual to the combination therapeutic treatment.
4 cl, 5 ex
SUBSTANCE: invention relates to novel substituted quinoline derivatives of general formula (I) in which: m is an integer from 0 to 3; R1 is selected from a group comprising an acylamino group, ester carboxylic group and an alkyl with 1-5 carbon atoms, an optionally substituted hydroxy and a halogen; R2 denotes hydrogen or an alkyl with 1-5 carbon atoms; R3 denotes - C(=X)-A, where A is selected from a group comprising aryl, heteroaryl, heterocyclyl and cycloalkyl, each of which can optionally contain from 1 to 4 substitutes selected from a group comprising an alkyl with 1-4 carbon atoms, an alkoxy with 1-4 carbon atoms, a halogen, hydroxy or nitro, and X denotes oxygen or sulphur; R4 denotes alkylene-heterocyclyl or alkylene-NR7R8, where alkylene is a linear alkylene with 1-4 carbon atoms; R7 and R8 are independently selected from a group comprising hydrogen, an alkyl with 1-4 carbon atoms, arylalkyl, heteroarylalkyl, cycloalkyl or cyclo-alkylalkyl; R5 is selected from a group comprising L-A1, where A1 is selected from a group comprising aryl, heteroaryl, heterocyclyl and cycloalkyl, each of which can optionally contain from 1 to 4 substitutes selected from a group comprising an alkyl with 1-4 carbon atoms, an alkoxy with 1-4 carbon atoms, a halogen, hydroxy and nitro, and where L is selected from a group consisting of oxygen, -NR9, where R9 denotes hydrogen or alkyl; -S(O)q-, where q equals 0, 1 or 2, and an alkylene with 1-5 carbon atoms, optionally substituted with a hydroxy, halogen or acylamino; and R6 is selected from a group comprising an alkyl with 1-5 carbon atoms, an alkenyl with 2-5 carbon atoms, an alkynyl with 2-5 carbon atoms, -CF3, an alkoxy with 1-5 carbon atoms, a halogen and a hydroxy; or its pharmaceutically acceptable salts or esters, as well as to a pharmaceutical composition having anticancer activity or inhibitory effect on mitotic kinesin based on the said compounds, to a method of treating disorders and use of these compounds for making a medicinal agent.
EFFECT: novel compounds which can be useful in treating cancer are obtained and described.
40 cl, 3 ex, 1 tbl
SUBSTANCE: invention describes novel derivatives of 2,6-diaminopyridine of formula (I), where R1 is piperidine which is optionally substituted with up to four substitutes independently selected from a group comprising: (a) hydrogen, (b) lower alkyl, (c) lower alkyl substituted with an oxo group or aryl, (d) CO2R7, (e) COR12, (f) C(O)NR13R14, and (g) S(O)nR15; R2 is phenyl which can be substituted with up to four substitutes independently selected from a group comprising: (a) lower alkyl, (b) lower alkyl substituted with a halide or OR10, (c) halide, or (d) OR12; R5 and R6 are independently selected from a group comprising (a) hydrogen and (b) lower alkyl; R7 is selected from a group comprising (a) hydrogen and (b) lower alkyl; R10 is selected from a group comprising (a) lower alkyl, (b) aryl and (c) aryl substituted with a halide or NR5R6; R12 is selected from a group comprising (a) hydrogen and (b) lower alkyl; R13 and R14 are independently selected from a group comprising (a) hydrogen and (b) lower alkyl, R15 is selected from a group comprising (a) aryl, (b) aryl substituted with a halide, CO2R12, SO2R10, COR12, lower alkyl or lower alkyl substituted with a halide, OR12, oxo group, CO2R12, C(O)NR5R6 or NR5R6, (c) heteroaryl, (d) heteroaryl substituted with a halide, CO2R12, SO2R10, COR12, lower alkyl and lower alkyl substituted with a halide, OR12, oxo group, CO2R12, C(O)NR5R6 or NR5R6, (e) NR5R6, (f) lower alkyl, (g) lower alkyl substituted with a halogen, OR12, oxo group, CO2R12, C(O)NR5R6 or NR5R6, (h) a heterocycle and (i) a heterocycle substituted with CO2R12, COR12, SO2R10, lower alkyl, C(O)NR5R6 or NR5R6; n equals 0, 1 or 2; as well as a pharmaceutical composition having inhibitory effect on cyclin-dependant kinase and a method of producing the compound of formula I.
EFFECT: novel compounds which have antiproliferative activity and can be used for treating or curbing cancer are obtained and described.
28 cl, 58 ex, 4 tbl
SUBSTANCE: invention refers to 3-oxo-28-(N-methylpiperazine)-carbonyl-lup-20(29)-ene of formula (I) which can be used in medicine as a corrective agent for paraneoplastic damages and toxic effects of cytostatic polychemotherapy. .
EFFECT: compound I shows an apparent anticancer activity, reduces severity of pathological changes in tissues caused by paraneoplastic syndromes; in cytostatic polychemotherapy, it exhibits an apparent antioxidant and cytoprotective effect in normal cells of viscera and thereby does not stimulate proliferation and dissemination of a tumour.
8 ex, 8 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: there is described an immunogen for making an immunogenic cancer composition free of DNA-binding function and all domains of a zinc finger, on the basis of polynucleotide coding a nonfunctional mutant form of a related molecule ("brother") of regulator of imprint sites (BORIS) of protein, polypeptide or peptide, containing amino acid sequence presented in the description. The immunogenic cancer composition contains aforementioned immunogen and an adjuvant chosen particularly from cytokine, chemokin, a costimulating molecule. There is described an expression vector containing polynucleotide, coding above-stated protein, e.g., in bacterial systems, mammal systems, in yeast or viral systems. The cancer vaccine under the invention contains polynucleotide (immunogen), additionally the adjuvant and, if necessary, a pharmaceutically acceptable carrier. The invention describes the method for of cancer immunisation of a mammal with using said immunogen on the basis of polynucleotide.
EFFECT: invention allows improving effectiveness of cancer prevention.
28 cl, 7 dwg, 2 tbl, 1 ex
SUBSTANCE: invention covers medical products which contain pharmacologically acceptable liquid crystals and their derivatives of general structural formula: , where X represents a group -CH=N-, while Y and Z are alkyl or alkoxyl group and are taken in a daily dose in amlount 355 mcg to 10 g a day.
EFFECT: medical products under the invention exhibit antiinflammatory, immunomodulatory, analgetic (anaesthetising) and antineoplastic action when administered in optimal doses without foreign substances.
11 cl, 18 ex
SUBSTANCE: invention relates to a method of producing 10-deacetyl-N-debenzoyl-paclitaxel (I) , involving the following stages: a) reacting 2-(2,4-dimethoxyphenyl)-3-(2-nitrobenzene sulfenyl)-4(S)-phenyl-5(R)-oxazolidine carboxylic acid (V) with 10-deacetyl-7,10-bis-trichloroacetyl baccatin III (VI) to obtain 10-deacetyl-7,10-bis-trichloroacetyl baccatin(III)-13-yl ether 2-(2,4-dimethoxyphenyl)-3-(2-nitrobenzene sulfenyl)-4(S)-phenyl-5-(R)-oxazolidine carboxylic acid (VII) , b) hydrolysis of trichloroacetyl groups in positions 7 and 10 of the compound of formula (VII) to obtain 10-deacetylbaccatin(III)-13-yl ether 2-(2,4-dimethoxyphenyl)-3-(2-nitrobenzene sulfenyl)-4(S)-phenyl-5(R)-oxazolidine carboxylic acid (VIII) , c) treatment of the compound of formula (VIII) with acid to obtain 10-deacetyl-N-debenzoylpaclitaxel (I). This compound is a synthon used in making taxanes with anti-tumour activity. The invention also relates to a method of producing docetaxel from the said compound of formula (I).
EFFECT: improved method.
10 cl, 2 dwg, 5 ex
SUBSTANCE: invention represents a combination containing VEGF Trap and 5-fluorouracil to be applied in treatment of neoplasms.
EFFECT: higher effectiveness of the combination ensured by therapeutic synergism of its components, and reduced toxicity.
4 cl, 1 ex, 1 tbl
SUBSTANCE: present invention concerns pharmacology and represents a pharmaceutical composition containing therapeutically effective protein and nondetergent sulfobetaine (NDSB) where NDSB is chosen from the group containing dimethylethyl-(3-sulfopropil)ammonium salt, 3-(1-pyridino)-1-propanesulfonate, propanesulfonate dimethylbenzylammonium, dimethyl-tert-butyl-(3-sulfopropyl)ammounium salt, 3-(1-methylpiperidine)-1-propanesulfonate and dimethyl-(2-hydroxyethyl)- (sulfopropyl)ammounium salt.
EFFECT: invention provides preparation of the stabilised pharmaceutical compositions.
8 cl, 2 ex, 3 tbl, 2 dwg
SUBSTANCE: invention concerns medicine, namely oncology, and aims at inducing the immune response in patients with higher levels of soluble receptors of tumour necrosis factor (sTNFRl, sTNFR2) and soluble receptors of interleukine-2 (sIL2R). Therefor, the patient's blood purpose circulates through a filter or a column wherein contacts to effective amount of binding partners. Said binding partners represent immobilised TNFRI, TNFR2 and IL2 or immobilised antibodies binding with sTNFRl, sTNFR2 and sIL2. The blood or plasma levels of receptors are reduced until they become below the normal. It involves at least 12 procedures. The amount of treated plasma per one procedure is approximately equal to one volume of extracellular fluid.
EFFECT: invention allows for selective removal of soluble cytokine receptors with inducing the immune response and ensuring remission of the diseases characterised by high production of sTNFRl, sTNFR2 and sIL2 including malignant tumours.
10 cl, 4 tbl, 1 ex, 5 dwg
SUBSTANCE: invention is related to the field of medicine, in particular to microbiology and immunology and may be used in development of vaccine against melioidosis. Substance of method consists in the fact that animals are immunised with surface melioidosis complex, which consists of antigen 6 (AG6) and antigen d, which is injected to animals subcutaneously, twice, with interval of 10 days, in doses of 30 mcg by protein for mice or 150 mcg for rats. Simultaneously with primary immunisation recombinant interferon-γ - ingaron is injected in doses 8 ME for mice or 120 ME for rats, and in case of secondary immunisation, recombinant interleukin-2 - roncoleukin in doses of 0.6 mcg for mice or 10 mcg for rats, besides cytokines are injected to animals subcutaneously, daily, simultaneously with immunisation and in the next 2 days. In 21 days after primary immunisation animals are infected with 4-32 LD50 of highly virulent strain 100 of melioidosis causative agent, in 30 days after infection parametres of animal lethality are identified.
EFFECT: application of invention makes it possible to increase protectivity of melioidosis antigens due to cytokine stimulation of primary and secondary immune response to these antigens.
5 tbl, 2 ex
SUBSTANCE: invention concerns medicine, particularly gynecology, and can be applied in treatment of autoimmune oophoritis of inflammatory genesis for patients of childbearing age suffering with long-term recurrent inflammatory diseases of pelvic organs. After infect eradiation, interleukin receptor antagonist is injected daily intramuscularly in 50 mg amount until circulating autoimmune antibody level in blood serum falls below 10 U/ml.
EFFECT: enhanced efficiency of treatment at different disease stages, prevented complications inherent to hormonal immunosuppressive therapy.
SUBSTANCE: invention concerns medicine, namely oncology and can be used for treatment of inflammatory breast cancer. The therapy is implemented as follows. The preoperative therapy is combined with hypodermical introduction of prescribed recombinant tumour necrosis α-factor thymosin-α1 in a dose 200000 ME on the day of chemotherapy 30 minutes prior to introduction of cytostatic agents and 4 days after chemotherapy.
EFFECT: application of the invention allows ensuring tumour regress and reducing therapeutic toxicity due to combined cytostatic agents and recombinant tumour necrosis α-factor thymosin-α1.
SUBSTANCE: invention refers to medicine, namely, to experimental cardiology and pathological physiology, and concerns correction of post-stress myocardial dysfunction. It is ensured by simulation of traumatic stress in a laboratory animal and intraperitoneal introduction of Granocyt in a dose 8.4 ME/kg of body weight immediately after trauma and daily within three days.
EFFECT: method provides effective correction of post-stress myocardial dysfunction in laboratory animals in specific experiment.
SUBSTANCE: group of inventions concerns orthopedy and traumatology and can be applicable for treatment and assistance to cartilage and-or bone formation. CXCL6 is administered to the patient requiring it, thus the specified CXCL6 represents wild type CXCL6 or its modified version keeping chemotactic activity concerning chondrocytes and chondrocytes precursor cells. The group of inventions provides complete elimination of a defect by a hyaline cartilage of normal structure.
EFFECT: complete elimination of a defect by a hyaline cartilage of normal structure.
22 cl, 3 ex, 2 tbl, 3 dwg
SUBSTANCE: extraction is done using affinity chromatography with immobilised metal. The method can be realised in native conditions. Biologically active G-CSF is obtained with purity over 95%. Two more chromatography stages are done, cation-exchange and gel filtration, to remove trace amounts of impurities. The method allows for obtaining G-CSF with high output and over 99% purity.
EFFECT: described method is especially suitable for industrial production of G-CSF.
16 cl, 5 dwg, 5 ex
SUBSTANCE: invention relates to medicine and biochemistry and concerns compositions for stabilisation of protein active ingredients in pharmaceutical preparations, which contains two following components: a) surface-active substance, preferably non-ion detergent (tenzid), and b) mixture of four amino acids: glutaminic acid (Glu), glutamine (Gln), aspartic (Asp) and asparagine (Asn).
EFFECT: synergetic effect at combined using of said four concrete amino acids.
10 cl, 11 ex, 6 dwg
SUBSTANCE: invention refers to medicine and concerns methods of neural disorder treatment with introducing hematopoietic growth factors. Substance of the invention includes G-CSF or its functionally active version 90% identical to human G-CSF sequence presented in SEQ ID NO:28 to be applied in therapy of amyotrophic lateral sclerosis.
EFFECT: benefit of the invention consists in neuroprotection of disease-related nerve cells.
2 cl, 38 ex, 1 tbl, 41 dwg
SUBSTANCE: invention refers to medicine, namely to obstetrics and gynaecology. The method for prevention of adhesive process in peritoneal cavity in obstetric-gynaecologic surgeries involving administration of chitosan compounds and differing that the compound is applied in layer-by-layer suturing of an operative wound in preperitoneal and skin structure of anterior abdominal wall. The compound represents a sponge measured 60×100×4 mm to 240×180×4 mm and containing 2% collagen acetate, 2% chitosan ascorbate of degree of deacetylation 95% and more, molecular weight 100 to 700 kDa, ascorbic acid - 1.8 g/g of dry chitosan, chondroitin-sulphuric acid - 5-100 mg/g of dry chitosan, hyaluronic acid - 10-100 mg/g of dry chitosan, heparin - 2.5-5 mg/g of dry chitosan and bovine serum growth factor "Adgelon" - 11-220 mkg/g of dry chitosan.
EFFECT: method improves effectiveness of prevention of postoperative adhesive process in small pelvis in women, prevents development of exudative processes in the operative wound tissues and accelerates the healing.
2 tbl, 1 ex