Method for preparing pharmaceutical composition for inducing angiogenesis in tissues, pharmaceutical composition prepared by this method, and method of treating individual's tissue and/or organ ischemia

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, specifically to compositions for inducing angiogenesis in tissues, and can be used in medicine. The method provides transforming the strain E. coli TOP10 by the pCMV-VEGF165 plasmid, culturing the strain in an environment acceptable for the biomass collection followed by separating the superhelical pCMV-VEGF165 plasmid that is followed by the pCMV-VEGF165 plasmid lyophilisation, which is conducted in the necessary presence in the lyophilised solution of a cryoprotectant, a pH stabiliser, an antioxidant and other substances enabling producing normal saline for injections with the pure plasmid concentration of 0.1 to 10 mg/ml and pH 7.0 to 9.0 and providing t the superhelical form of the pCMV-VEGF165 plasmid storage retention. The method of treating an individual's tissue and/or organ ischemia consists in administering an effective amount of the prepared pharmaceutical composition intramuscularly into the individual.

EFFECT: invention enables preparing the therapeutically applicable pharmaceutical composition of the pCMV-VEGF165 plasmid DNA with preserving properties of the main substance at long-term storage at +2-+8°C.

3 cl, 2 dwg, 4 tbl, 5 ex

 

The technical field

The invention relates to biotechnology, namely the strain-producer of plasmid DNA and pharmaceutical compositions based on it having the ability to induce the growth of blood vessels (vascularization) in the introduction and its application in complex therapy for revascularization ischemia of the lower extremities of atherosclerotic Genesis, and also in the treatment of wounds and ulcers of various origins.

Prior art

Gene therapy using free plasmid DNA (naked DNA) for the purposes of the treatment of diseases or vaccination against pathogens or antigens of cancer cells involves the creation of finished dosage forms of therapeutic DNA, which can be stored, transported and used in adverse conditions, in particular in the positive or elevated temperature. Physical and chemical stability of plasmid DNA during storage is largely determined by the composition of excipients, its concentration or content in the finished dosage form and storage conditions (Schleef M, Schmidt T. Animal-free production of ccc-supercoiled plasmids for research and clinical applications. J Gene Med. 2004; 6 Suppl 1:845-53). The main process that changes the pharmaceutically active form of plasmid DNA during storage, is the collapse of the DNA chain, in which is formed an annular Rel is sirovina form of the plasmid and, further, the linear double-stranded form. It is known that the storage of the frozen solution of plasmid DNA at a temperature below minus 80°With the collapse of the supercoiled form is practically not observed (Walther W, Stein U, Voss S, Schmidt T, Schleef M, Schlag PM. Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer. Anal Biochem. 2003; 318(2):230-5). Such a method of storage of plasmid DNA may not be widely used in clinical practice because of health facilities and pharmacies in most cases do not have the necessary refrigeration equipment. Transportation of finished dosage forms with maintaining such a low temperature the product is also extremely difficult. When selecting the proper composition and concentration of excipients can be obtained solutions of plasmid DNA, stable for 12 months when stored at 4°C or more than three years when stored at a temperature of minus 20°C (Przybylowski M, Bartido S, Borquez-Ojeda Oh, Sadelain M, Riviere I. Production of clinical-grade plasmid DNA for human Phase I clinical trials and large animal clinical studies. Vaccine. 2007; 25(27):5013-24).

The most common way to create a more stable finished dosage forms of macromolecules is lyophilization. As a rule, liofilizovannye dosage forms stable when stored at 4°C for several years, in some cases it is possible to store the product at room temp is the temperature for several months or even two years. Lyophilization also allows you to change the concentration of the active substance - vials can be filled with a concentrated solution of a small amount, and when the dissolution of the product before using the volume can be increased to the necessary. It is also possible to lyophilization hypotonic solutions and the subsequent dissolution of the lyophilized saline solution or normal osmolality (Anchordoquy TJ, Armstrong TK, Molina MC. Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes. J Pharm Sci. 2005; 94(6):1226-36).

When freeze-drying solutions of plasmid DNA is typically expected to increase the stability of the product during subsequent storage, however, the procedure of freezing and sublimation in vacuum can significantly damage the supercoiled plasmid DNA (Anchordoquy TJ, Armstrong TK, Molina MC, Allison SD, Zhang Y, Patel MM, et al. Physical stabilization of plasmid DNA-based therapeutics during freezing and drying. In: Costantino HR, Pikal MJ, editors. Lyophilization of biopharmaceuticals. AAPS press; 2004. pp.605-41). In particular, the lyophilization DNA from frozen aqueous solution that does not contain auxiliary substances, causes the removal of coordinated water molecules, i.e. the hydration shell of DNA molecules, which leads to loss of structural integrity of the DNA molecule (Poxon SW, Hughes JA. The effect of lyophilization on plasmid DNA activity. Pharm Dev Technol. 2000; 5(1):115-22). Such violations conformation of the DNA molecule, decay complementary what's the relations between the nitrogenous bases and partial violation of the stacking lead to undesirable practical consequences - the fall of the biological activity of plasmid DNA up to 25% of the original (Anchordoquy TJ, Armstrong TC, Molina MC. Low molecular weight dextrans stabilize nonviral vectors during lyophilization at low osmolalities: concentrating suspensions by rehydration to reduced volumes. J Pharm Sci. 2005; 94(6): 1226-36).

It is known that the loss of coordinated water molecules removed by sublimation, can be compensated by the introduction of liofilizatsii solution of non-volatile hydrophilic substances, which typically use sugars and polyols (Maitani Y, Aso Y, Yamada A, Yoshioka S. Effect of sugars on storage stability of lyophilized liposome/DNA complexes with high transfection efficiency. Int J Pharm. 2008; 356(1-2):69-75). Most of the known technical solutions to stabilize DNA during lyophilization relates to the field of production of lyophilized liposomes containing DNA (U.S. Patent 7,323,297), thus the applicability of these solutions to the solution of "naked" plasmid DNA to be unobvious. (Quaak S, Haanen J, Beijnen J, B. Nuijen Naked Plasmid DNA Formulation: Effect of Different Disaccharides on Stability after Lyophilisation. AAPS PharmSciTech, Vol.11, No.1, March 2010) have investigated the influence of several disaccharides on the process of lyophilization of plasmid DNA and it was found that sucrose used in a mass ratio of 20:1 with DNA gives the most stable during storage of the finished dosage form. However, in this work not considered the impact of pH and salt composition of the solution on the stability of plasmid DNA have not been studied to the position, giving isotonic DNA at a concentration of less than 5 mg/ml and were not investigated monosaccharides potentially suitable for obtaining a stable liofilizovannyh drugs macromolecules.

Description of Figures:

1. The Figure 1 shows the map of the expression plasmid pCMV-VEGF165 (length 4859 base pairs). The following symbols are used: "CMV early promoter/enhancer region" is a region of the promoter/enhancer early gene of cytomegalovirus; enhancer region - area enhancer; "CMV promoter" is a region of the promoter of the cytomegalovirus; "TATA box" - TATA-element; "reduced start - the start point of transcription; "Kozak" sequence Kozak; "start codon" - start-codon is the first codon of the open reading frames VEGF165; "VEGF165" - open reading frame (ORF) of the polypeptide vascular endothelial growth factor human 165; "stop codon" is a stop codon; "SV40 RA term" polyadenylation signal and terminator of SV40 virus; "CMV forward primer - annealing standard primer CMV forward; "M13 forward20 primer - annealing standard primer M13 fbrward20; "M13 pUC fwd primer - annealing standard primer M13 pUC front wheel drive; "pBR322ori" - the region of the beginning of replication of plasmids pBR322; f1 origin" - the site of initiation of replication of bacteriophage f1; "AmpR promoter" is a prokaryotic promoter of the gene bla; "NTPII (neomycin phosphotransferase; KanR" sequence encoding an aminoglycoside-3'-phosphotransferase, providing resistance, smear the s to kanamycin. Arrows indicate the directions of transcription of the genes in parentheses are the numbers of the first and the last nucleotide fragments. Identifies the recognition sites of restriction endonucleases in parentheses are the numbers of nucleotides at the point of cutting.

2. Figure 2. Angiograms of a patient, 74 years of age with chronic ischemia of lower extremities. The diagnosis of atherosclerosis, femoral-popliteal occlusion on both sides IIB-III century (pain at rest). Indicators at admission: the ankle-brachial index of 0.48 (right), 0,32 (left); transcutaneous oxygen tension - 61 mm RT.article The patient received FC in the standard combination therapy (dextrans, disaggregants). The figures in 90 days: the painless walking distance of 130 m; ankle-brachial index of 0.5 (right) 0,57 (left); transcutaneous oxygen tension - 78 mm RT.article A, B - angiograms before treatment: at the level of the upper and middle third of the thigh (A); at the level of the tibia (B); In, Mr angiograms after 90 days after use of the drug "Neovasculgen" in complex therapy: for the upper and middle third of the thigh (In); at the level of the lower legs (G). Arrows indicate satisfactorily filled with blood vessels, including those developed collaterals.

A brief description of the present invention

The technical problem solved by the authors, was the creation of producer strain plasmid DNA and physiologically receiving the emnd pharmaceutical composition, ensuring the stability of the finished dosage form of plasmid DNA in a long time and is suitable for gene therapy.

The pharmaceutical composition comprises a purified plasmid DNA encoding vascular endothelial growth factor human (VEGF) under the control of functional genetic elements ensuring the expression of the gene in human cells, and pharmaceutically acceptable excipients in the form of at least one of cryoprotectant with the properties of the filler and stabilizer pH, in effective amounts providing isotonic solution for injection to a concentration of the purified plasmid DNA from 0.1 to 10 mg/ml and a pH of from 7.0 to 9.0.

The pharmaceutical composition can be used to obtain a solution for injection for intramuscular, intravenous or intra-arterial, subcutaneous, and intradermal. Can also be applied to cutaneous injection in a gel base; for implanting a resorbable or preservision media.

Farmcampsite is designed to make ex tempore or otherwise solution for intramuscular or other internal ways of introducing the patient (the patient, the injured person, animal) for the induction of development in the tissues of the blood vessels.

One of the indications for application is July of farbkomposition can serve chronic ischemic condition of various etiologies, including coronary heart disease, chronic obliteratus vascular diseases of the lower extremities; the situation needing correction in the induction of reparative processes in tissues, such as extensive, long-term healing wounds (ulcers), including burns, local injury, including fractures and bone defects.

The method of using the pharmaceutical composition is in the introduction to her person (or animal) in a manner and in such quantity that will provide a therapeutic effect depending on nosological forms and medical indications.

Detailed description of the present invention

Plasmid DNA encoding vascular endothelial growth factor may be obtained by ligating the field open reading frame (ORF) cDNA VAR person with plasmid vector, which provides the expression of encoded genes in human cells and replication of the plasmid in the cells of Esherichia coli. Examples of such vectors are plasmids family pcDNA, pCMV - Script and pBK-CMV (source - electronic database http://addgene.org/vector-database/). A necessary component of such a vector is a eukaryotic promoter, preferably selected from the group of immediate early promoter of cytomegalovirus (CMV), elongation factor broadcast 1 alpha man (EFIa), ubiquitin (Ubc), aqualicious what about the monkey virus (SV40), phosphoglycerides 1 mouse (PGK), beta-actin man, but not limited to this group. A more preferred group of vectors for gene expression WAR in human cells contains the immediate early promoter of cytomegalovirus (hereinafter CMV), a gene of resistance to the action of antibiotics and the plot early replication (origin of replication), medium or high copy number selected from the group of original replication ColE1, pUC, pBR322, p15A. A more preferred group of vectors includes the gene of resistance to the antibiotic kanamycin, encoding neomycin-historystorage (NPT II), which eliminates the use of antibiotics ampicillinbuy group in the production process plasmids. As vectors to obtain the target plasmid DNA can also be used plasmids encoding ORFS of other genes. The target plasmid DNA can be obtained from such plasmid DNA by removing the area of ORS foreign gene restriction, highlight the "acceptor" of the DNA fragment and subsequent ligation with him "donor" DNA fragment encoding ORFS cDNA VAR person. An example of such "acceptor" plasmid is a plasmid pEGFP-N2.

Region open reading frame cDNA VAR person may be selected from the four known splice variants, encoding isoforms VAR length amino acid chain 121, 145, 165, 189. P is edocfile isoform WAR to obtain plasmid DNA is a splice variant of 165 amino acids. Region ORS cDNA VAR person can be obtained from total cDNA of human tissues or synthesized from overlapping oligonucleotides. In the synthetic area LFS cDNA VAR person part or all of the codons can be replaced by synonymous, this synthetic fragment of the cDNA can substitute natural, if it provides comparable with the natural lifetime of the cDNA and comparable with the natural efficiency of translation of a target gene. Used to retrieve the target plasmid cDNA VAR may contain known polymorphisms (replacement of a single nucleotide), do not change the amino acid composition of the encoded polypeptide. Using cDNA VAR containing such SNPs, does not change the properties of the obtained target plasmids.

The most preferable variant plasmids, coding WAR and intended for medical use, is the product of ligation of the area LFS WAR isoforms 165 amino acids and acceptor fragment of plasmid pBK-CMV.

This plasmid DNA, according to the present invention referred to as pCMV-VEGF165, length 4859 base pairs, presented in the sequence Listing under the number SEQ ID NO: 1. The structure of the plasmid pCMV-VEGF165 shown in Fig.1.

The indicated plasmid size 4859 p. O. consists of:

1. Plot 1-361; 968-4859 vector pBK-CMV, including

1.1. the elements provide the appropriate expression of a target gene in mammalian cells: promoter/enhancer early gene of cytomegalovirus (4626-355), including the enhancer region (4684-231) and the region of the promoter of cytomegalovirus (274-355); TATA-element (320-326); the start point of transcription (349); the polyadenylation signal and the terminator of the SV40 virus (1306-1531);

1.2. the elements that ensure the maintenance of the plasmid in a bacterial cell, the site of initiation of replication of bacteriophage f1 (1583-1995); the prokaryotic promoter of the gene bla (2058-2086); a sequence encoding an aminoglycoside-3'-phosphotransferase providing bacterial resistance to kanamycin (2519-3313); the beginning of replication of plasmids pBR322 (3907-4526);

2. Plot 362-967, including the Kozak sequence (380-391), which is located around the start codon of the target gene and promotes translation initiation of mRNA of a target gene, an open reading frame of the gene VEGF165 (392-964), stop codon (965-967).

The indicated plasmid contains a unique recognition sites of the restriction endonucleases: NdeI (1); BamHI (364); EcoRI (373); BsrGI (854); the XmaI (969); Acc651 (978); Bell (1307); NarI (2650); ApaLI (4254); PciI (4568). The structure of the plasmids shown in Fig.1.

A plasmid was obtained using standard genetic engineering techniques, commercially available plasmids sites and cloned human cDNA (Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. 2nd ed. New York, NY: Cold Spring Harbor Laboratory Press; 1989).

Elements plasmids are listed in order of their location. The mutual arrangement of the functional elements of t is aetsa essential for the efficient operation of the plasmids.

As recombinant plasmids according to the present invention may be used various plasmids containing VEGF165 gene under the control of a eukaryotic promoter.

DNA fragments that encode essentially the same regulatory elements can be obtained, for example, by modifying the nucleotide sequence of the DNA fragment (SEQ ID NO:1), for example, using the method of site-directed mutagenesis, so that one or more nucleotides at a particular site will be delegated, substituted, inserted or added. The DNA fragments, modified as described above, can be obtained using traditional processing methods with the aim of obtaining mutations.

To obtain the producer strain plasmid DNA pCMV-VEGF165 can be introduced (transformed) into a bacterial cell, preferably a bacterium belonging to the genus Escherichia, receptive to such transformation specified by the plasmid. The choice of a particular cell is not critical, because the methodology and methods of transformation are well known to the person skilled in the art. Although depending on the type of cells and culture conditions the received transformant chopinot and the total content of the plasmids pCMV-VEGF165 in bacterial suspension may vary, the fact of the presence of target plasmids will take place when the service is under the successful transformation of cells recipient.

"Transforming cells with plasmid" means the introduction of plasmids into the cell using methods well known to the person skilled in the art. Methods of transformation include any of the standard methods known to a person skilled in the technical field, for example the method described in Jac A. Nickoloff, Electroporation Protocols for Microorganisms (Methods in Molecular Biology) // Humana Press; 1st edition (August 15, 1995).

According to the present invention, the bacterial cell producing the plasmid pCMV-VEGF165" means a bacterial cell with the ability to maintain, replication and accumulation of plasmids pCMV-VEGF165 according to the present invention, when the bacterial cell according to the present invention are grown in a specified medium. Used herein, the term "bacterial cell producing the plasmid pCMV-VEGF165" also means a cell which is capable of accumulating plasmid pCMV-VEGF165 in the amount of not less than 1 mg/l (or 1 µg/109cells), more preferably not less than 10 mg/l Indicated plasmid pCMV-VEGF165 accumulates in the specified cell is preferably circular supercoiled form.

Preferably using bacteria belonging to the genus Escherichia, for transformation with plasmid pCMV-VEGF165, coding WAR under control of the CMV promoter.

The term "bacterium belonging to the genus Escherichia can mean that the bacterium from OSISA to the genus Escherichia according to the classification well-known specialist in the field of Microbiology. As examples of the microorganism belonging to the genus Escherichia can be mentioned Escherichia coli (E. coli).

The range of bacteria belonging to the genus Escherichia, is not limited in any way, however, for example, bacteria, described in the book Neidhardt, F. C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D. C., 1208, table 1), can be given as examples.

Preferred variants of the strain of the recipient to obtain the product of the plasmid pCMV-VEGF165 are strains of E. coli derived from non-pathogenic strains K12 and containing an inactivated gene system DNA repair recAl, a vaccine, a gene endonuclease end A1. Examples of such strains are DH5alpha, DH10B, XL-lBlue, LAY.

A concrete example of the strain of the recipient to obtain the product of the plasmid pCMV-VEGF165 according to the present invention is, but is not limited to, Escherichia coli LAY. The strain Escherichia coli LAY characterized by the following cultural-morphological, physiological and biochemical characteristics and genetic traits.

Cultural and morphological characteristics of strain: gram-negative rods to form filaments; on agar medium - gloss translucent convex middle colonies with smooth edges. The strain is stored in the following conditions: the environment Lurie-Bertrand, 1% glucose, 10% glits is Rina. Strain propagated in the following conditions - environment Lurie-Bertrand.

Genetic strain. The genotype of strain - Δ(Aha?-leu)7697, [araD139]B/r, Δ(codB-lacI)3, φp80dlacZ58(M15), galK0, mcrA0, galU-, recA1, endA1, nupG-, rpsL-(strR), Δ(mcrC-mrr)715.

Transformation of Escherichia coli strain TOP 10 plasmid pCMV-VEGF165 results of the producer strain TOP10/pCMV-VEGF165, which provides the biosynthesis of plasmids pCMV-VEGF165 in the amount of 5-20 mg/l of culture under cultivation in stirred flasks for 12-20 hours in an environment Lurie-Bertrand with the addition of kanamycin and 30 μg/ml, at least 70% of the plasmids pCMV-VEGF165 is in supercoiled form.

The method of obtaining high purity plasmid pCMV-VEGF165 according to the present invention includes culturing the above-described bacteria in a nutrient medium suitable for the cultivation of these prokaryotic cells, biomass harvesting cells, re-suspension of cells, alkaline lysis, selective renaturation plasmid DNA acidic solution, the sludge separation, concentration by ultrafiltration, separation of impurities and RNA gel-filtration in high salt solution, separating the residual genomic DNA, endotoxin and related impurities affine (teofilina) chromatography, final purification by anion-exchange chromatography and subsequent concentration and desalting of the solution of purified plasmid pCMV-VEGF165 p and using the ultrafiltration/diafiltration. Get the drug plasmids pCMV-VEGF165 suitable for pharmaceutical compositions and dosage forms, characterized by the following main features.

1) Share related impurities (relaksirano circular and linear forms of the plasmid) - not more than 5% (hereinafter - the fraction of the concentration of the basic substance).

2) the Proportion of the genomic DNA of E. coli - not more than 1%.

3) the Proportion of RNA - not more than 1%.

4) the percentage of the total protein is not more than 0.1%.

5) the Content of endotoxin - not more than 50 EE 1 mg of the basic substance.

The solution of plasmid DNA pCMV-VEGF165 suitable for pharmaceutical compositions and dosage forms may be obtained by using other standard methods of extraction and purification of DNA, known to the person skilled in the technical field, for example, the method of thermal lysis of bacteria in the presence of detergent, method of selective precipitation of RNA by means of calcium chloride, the method of separation of supercoiled and relaksirano forms plasmids using gradient elution and a number of other methods described in (D. M. F. Prazeres, "Plasmid Biopharmaceuticals: Basics, Applications and Manufacturing", John Wiley & Sons, Inc. (2011) ISBN: 978-0-470-23292-7).

The finished dosage form of plasmid DNA pCMV-VEGF165 must be suitable for intramuscular injection and should not lead to significant changes in the properties of the main substances during prolonged storage. Possible finished dosage form of the plasmid pCMV-VEGF165 can be selected from the group of the frozen solution, the liquid solution, freeze-dried, i.e., lyophilized solution, an amorphous film, but is not limited to them.

Preferred variants of the finished dosage form is a liquid solution or lyophilized, as they can be stored at positive temperature, i.e., in a standard pharmaceutical refrigerators, and do not require significant time to prepare for injection.

The most preferred option of the finished dosage form is a freeze-dried, as the lack of water potentially slows down the chemical reaction of the breakdown of DNA chains, leading to the conversion of supercoiled plasmid DNA in relacionado annular shape. In addition, when the storage of liquid dosage forms of plasmid DNA are possible prolonged contact of the solution with the material of the rubber tube, containing the extracted transition metal ions, accelerating the breakdown of DNA chains by catalysis of the formation of hydroxyl radicals.

Obtaining freeze-dried, i.e. amorphous or microcrystalline porous, requires the presence in liofilizatom the solution of auxiliary substances, performing the functions of cryoprotectant, pH stabilizer, chelating agent, antioxida is the filler, etc. the Minimum possible set of auxiliary substances may include at least one, cryoprotectant with the properties of the filler and stabilizer pH. Auxiliary substance, which cryoprotectants and filler may be selected from the group comprising mono - and disaccharides, polyols and polymers, such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, raffinose, polyvinylpyrrolidone, or a combination thereof. The pH stabilizer may be selected from the group comprising sodium citrate, sodium phosphate, Tris-HCl, Tris-acetate, glycine and other amino acids.

When researching the various pharmaceutical compositions of plasmid DNA pCMV-VEGF165 we have unexpectedly discovered that the best stability when stored at a temperature of +2-+8°C provides a combination of excipients glucose and phosphate pH=7,8.

While maintaining the same volume of solution before and after lyophilization, the best composition of the solution, ensuring the preservation of the properties of plasmid DNA pCMV-VEGF165 in the process of freeze-drying and subsequent storage represents:

1. Plasmid DNA from 0.1 to 10 mg/ml, preferably from 0.5 to 4 mg/ml, more preferably from 0.8 to 1.2 mg/ml

2. Glucose (dextrose) from 200 to 400 mm, preferably from 250 to 350 mm, more preferably from 280 mm to 320 mm.

3. Phosphate n is Tria (mixture trisemester, disubstituted and one-deputizing phosphate sodium) at a concentration of from 3 to 30 mm, preferably from 5 to 20 mm, more preferably from 8 to 12 mm.

4. the solution pH from 7.0 to 9.0, preferably from 7.2 to 8.5, more preferably from 7.4 to 8.2.

Features of the plasmids used to create producer strain and pharmaceutical compositions shown in Figure 1.

The present invention will be described in more detail below with reference to the following not limiting the present invention to the Examples.

Example 1.

Obtaining a solution of purified plasmid DNA pCMV-VEGF165

Preparation of inoculum

Carry out the recovery capacity of canned producer strain TOP10/pCMV-VEGF165 from the work of the Bank, growing the inoculum in liquid medium in a volume of 50 ml.

Biosynthesis pdnc

Prepare to work fermenter with a 10 l flask, sterilized flask with medium, aseptically mounted supply and exhaust, calibrated Kipp, inoculant fermenter and lead cultivation 8 hours to achieve a constant concentration of dissolved oxygen (stationary phase of culture growth) at a fixed speed mixer of 1000 rpm, then stop aeration and cooled flask. A sample of the culture suspension is passed to the analysis of culture density.

Obtaining biomass

Biomass, i.e. sediment cells separated from the culture is hidcote in outdoor high speed centrifuge periodic action with bactrim rotor. Supernatant culture fluid is passed for decontamination by autoclaving. The resulting biomass is stored in centrifuge cups low-temperature refrigerator. The sample obtained biomass is passed to the analysis of the content and authenticity of the target substance.

The suspension of biomass lysis and neutralization

Thawed biomass is transferred into a tank for lysis and suspended verhneprivodnaya stirrer in the solution for suspension. Spend lysis of the cells with a solution of sodium hydroxide and sodium dodecyl sulfate under stirring verhneprivodnaya stirrer for 5 minutes. With stirring a solution of potassium acetate to neutralize and simultaneous receipt of sediment cellular debris, genomic DNA associated with histones and proteins. The precipitate is due to the transition of sodium dodecyl sulfate in the insoluble potassium salt and coagulation of the micelles. Simultaneously with the neutralization solution is resaturate plasmid DNA.

Obtaining a clarified lysate

The resulting suspension is transferred into a centrifuge cups and separate the precipitate in outdoor high speed centrifuge periodic action with bactrim rotor. The sediment in the glasses passed on disinfection by autoclaving. The clarified solution of plasmid DNA, optionally containing related is soedineniya - relacionado and linear form of plasmid DNA, as well as impurities - residual genomic DNA, RNA, proteins and LAL-endotoxin gather in the supply tank ultrafiltration installation.

Ultrafiltration

Conduct the concentration of the solution of plasmid DNA by ultrafiltration in a tangential flow using a hollow fiber cartridge with a cutoff of 500 kDa. In the ultrafiltration process is additionally 9-fold remove all molecules smaller than 300 kDa, i.e. protein, transfer RNA, LAL-endotoxin, short fragments of genomic DNA. Prescribed concentration degree 9 times. Concentrated solution of plasmid collected in a glass container, washed with ultrafiltration installation solution for gel filtration and combined with a concentrated solution of plasmid DNA.

Gel filtration

The first process of deep chromatographic purification is carried out using gel filtration on a porous dextranomer sorbent Sepharose 6 Fast Flow, GE Lifesciences". Use a high salt solution containing 2.1 M ammonium sulfate and 10 mm EDTA-Na, which allows for the simultaneous separation of molecules by size to hold impurities RNA and LAL-endotoxin adsorbent due to nonspecific hydrophobic interactions. The solution impurities and wash solutions are collected to wtru and disposed of in the prescribed manner. A solution of procedendo plasmid DNA containing 2.1 M ammonium sulfate, collected in a glass bottle.

Affinity chromatography

A second chromatographic purification process of deep lead method teofilina (pseudoephrine) chromatography sorbent PlasmidSelect Xtra "GE Lifesciences". For washing of the column using a solution containing 2.0 M ammonium sulfate, paleoceanic solution onto the column at a concentration of ammonium sulfate to 2.1 M In the coating solution for all forms of DNA and part of the residual RNA adsorbed on the sorbent, the residual proteins and endotoxin are not adsorbed. After application the column was washed with a solution with a concentration of ammonium sulfate 2.0 M with elution of residual RNA, genomic DNA and related compounds plasmid DNA. Base material HSCI-01 elute solution with a concentration of ammonium sulfate of 1.7 M Column regenerate and purify the sodium hydroxide solution of 0.1 M Solution of impurities and wash solutions are collected in containers and disposed of in the prescribed manner. A solution of procedendo plasmid DNA containing 1.7 M ammonium sulfate, collected in a glass bottle, dilute with water for injection at a ratio of 1:2.

Anionoobmennoi chromatography

The third process of deep chromatographic purification lead method anionoobmennoi chromatography sorbent SOURCE 30Q "GE Lifesciences" base material adsorb on the column and hold the rinse solution, containing 0.4 M sodium chloride. When washing the column is replaced cation of the basic substance with ammonium ion to sodium ion and elution of residual endotoxin. Base material elute solution of 1 M sodium chloride. The solution impurities and wash solutions are collected in containers and disposed of in the prescribed manner. The solution of purified plasmid DNA is collected in a glass bottle.

Ultrafiltration and diafiltration

The solution of purified plasmid DNA concentrated by ultrafiltration on an installation with a hollow fiber module with a threshold cut-off membrane 300 kDa. Upon reaching a concentration of 0.25% setting transfer mode diafiltration and conduct a comprehensive replacement of the buffer in water for injection. The filtrate is collected in containers and disposed of in the prescribed manner. The concentrated solution is poured in a glass bottle, determine the concentration of supercoiled plasmid DNA and stored at a temperature below minus 70°C.

Example 2. Obtaining variants of lyophilized for stability studies

To obtain variants of the pharmaceutical composition used desalted solution of plasmid DNA pCMV-VEGF 165 with a concentration of about 2.5 mg/ml and residual Tris-HCl pH=7.5 and NaCl 1 mm. As cryoprotectants investigated glucose, sucrose and lactose quality is not below the requirements of the European f is makopi. As the pH stabilizer used a solution of sodium phosphate with a pH in the range from 7.2 to 9.0. The saccharides used at a final concentration of 300 mm, sodium phosphate at a final concentration of 10 mm. Such concentrations of excipients provide isotonicity solution for internal administration. The final concentration of DNA in the resulting pharmaceutical compositions comprised of 1 mg/ml. the Lyophilization was performed in glass vials of 5 ml, equipped with semi-open lyophilization rubber plugs and filled in portions of 1.2 ml of the investigated solutions. The mode of freeze drying for all studied samples are shown in table 1.

Table 1
The mode of freeze drying
The stage nameDuration, minTemperature, °C
1) freezing the solution:
a)30-50
b)300-50
2) Lyophilization:
a)60-30
b)930-30
in)60-10
g)480-10
d)6020
e)36020
3) final drying:
a)6040
b)60040

The pressure in the working chamber during the drying 60 mcbar, the product temperature at the end of step freezing should be no higher than minus 45°C, it is two degrees below the phase transition temperature (glass transition temperature) for glucose and more than 10 degrees lower than for the other sugars. The temperature of the product after lyophilization should not be below minus 15°C. the temperature Measurement products was performed using t is Mopar, frozen vials with imitators product containing all auxiliary substances, but do not contain DNA. At the end of the final were sealed vials in a sterile atmosphere of dry air compression shelves. Uploaded corked bottles with aluminum caps. For comparison, the stability during storage at elevated temperature was used a solution of sodium phosphate pH=7,8, which is obtained by mixing solutions of disubstituted phosphate and one-deputizing phosphate in a molar ratio of 91.5:8,5. Liofilizovannye sealed vials were stored at a temperature of +37°C in dry air thermostat, 1 time per month took another vials, dissolved lyophilized and identified the proportion relaksirano plasmid DNA using analytical ion exchange chromatography. The results of the measurements are presented in table 2.

Table 2
Share relaksirano plasmid DNA during storage at elevated temperatures
Storage time, months
Cryoprotectant012 3
Glucose2,2%2,8%5,3%10,0%
Sucrose2,1%6,2%8,4%10,4%
Maltose2,3%7,0%8,8%14,6%
Lactose2,2%9,0%9,6%18,5%

It was found that the use of glucose as cryoprotectant gives the smallest decay rate of supercoiled plasmid DNA at a mass ratio of saccharide:DNA 1:50 and pH=7,8.

For pharmaceutical compositions based on glucose, which investigated the dependence of the stability of supercoiled plasmid DNA from the solution pH and concentration of sodium phosphate (pH 7,8). It was found that the stability of supercoiled plasmid DNA does not change significantly with the variation of concentration of sodium phosphate from 5 to 20 mm (data not shown). By varying the pH of the solution was found (table 3) that in the pH range from 7.8 to 90 significant changes in stability during storage is not observed, but at pH 7.2 stability supercoiled plasmids decreases.

Table 3
Share relaksirano plasmid DNA during storage at elevated temperature, the variation of pH
pH0 months1 month2 months3 months
7,22,2%4.09 to%7,63%15,17%
7,82,2%2,80%5,30%10,00%
8,42,3%was 2.76%3,34%10,69%
92,1%of 2.51%2,97%8,39%

Thus, according to accelerated storage optimal pharmaceutical composition may be selected for a range of pH from 7.8 to 9.0 and the concentration of sodium phosphate from 5 to 20 mm. Since the concentration of IO is ishemic phosphate groups of DNA at a DNA concentration of 1 mg/ml is about 3 mm, and the concentration of buffer salts should substantially exceed the total concentration inisheer groups of the basic substance, the optimum concentration of sodium phosphate was chosen as 10 mm. The optimum pH of the solution was chosen as 7,8, because this value is closest to the physiological pH at 7.2 to 7.4). Note, however, that pH, on the one hand, should be as close as possible to 7.2 and on the other hand, the higher it is, the higher the stability of the plasmids (up to 9.0). It is likely that at a storage temperature of +2-+8°C at pH 7,0-9,0 plasmid DNA will remain stable enough to ganotherapy.

Example 3.

The formulation of the solution of purified plasmid DNA and production of finished dosage forms

Lead the process of obtaining plasmid DNA as described in Example 1 until the completion of stage anion-exchange chromatography. The following stages were carried out as described below.

Ultrafiltration and diafiltration

The solution of purified plasmid DNA concentrated by ultrafiltration on an installation with a hollow fiber module with a threshold cut-off membrane 300 kDa. Upon reaching a concentration of 0.15% setting transfer mode diafiltration and conduct a comprehensive replacement of the buffer solution of 10 mm sodium phosphate, pH of 7.8 and 4.4% (300 mm) glucose in water for injection. The filtrate is collected in containers and disposed of in the prescribed manner. Scancenter the bath formulated solution is poured in a glass bottle, determine the concentration of supercoiled plasmid DNA and bring the solution to a final concentration of 0.1%.

Sterile filtering

The ready solution of the substance is passed into the pure area of the class and hold a sterile filtration using a circular membrane filters in depyrogenation sterile bottles for infusion solution (250 ml GOST 19808-86. Carry out sampling for transfer to the OCC, close the bottles with rubber stoppers, sealed with aluminum caps and freeze in the quarantine area of the warehouse.

Defrost formulated solution of the substance pdnc and passed into the pure area of the class A. Open the bottle and spend sterile filtration using a circular membrane filters in depyrogenation sterile bottles for infusion solution (250 ml GOST 19808-86. Spend bottling in sterile "insulin" vials 5 ml GOST 19808-86. Close the vials with rubber stoppers on TO.006.269-90 and passed to the stage of freeze drying.

The vials are placed on the shelves of freeze drying, frozen at -45°C and lead a three-stage vacuum drying. Close the tube, remove the vials and sealed with aluminum caps according to GOST R 51314-99.

Example 4.

Check the stability of the finished dosage form pCMV-VEGF165

Vials with sterile lyophilized plasmid DNA pCMV-VEGF165 storage is whether pharmaceutical refrigerator at a temperature of +4°C for 2.5 years and conducted analysis of the proportion of related impurities using ion-exchange chromatography once in 6 months. The results of the analysis are presented in table 4.

Table 4
Stability of the finished dosage form when stored at a temperature of +4°C
NormaAt the time of issue6 months storage12 months storage18 months storage24 months storage30 months of storage
Relacionada and linear plasmid DNA in less than 5% by ion-exchange HPLC3,0%3,0%3,3%3,7%4,1%4,0%

Thus, the dosage form of plasmid DNA pCMV-VEGF165 stable during storage for two years.

Example 5.

Check the efficiency of use of the pharmaceutical composition

When farbkomposition, including in addition to plasmid construction excipients, in patients with chronic ischemia of the lower extremities were the results obtained confirm its effectiveness, demonstrating the advantages before the introduction of t is like only plasmids. This fact is established in a study involving 75 patients with 2A-3 tbsp. chronic ischemia (Century A. Pokrovsky-Fontaine). As criteria are analyzed: the length of painless walking, transcutaneous defined voltage oxygen, ankle-brachial index and the linear velocity of blood flow in the posterior tibial artery.

- Length of painless walk. In the treatment of patients clinical HCD group after 3 months of follow-up was 236,49±193,49 m (50 to 1200 m), and 6 months - 284,73±242,02 m (20 to 1500 m). The increase of the average values of the path that the patient could walk without pain in the study group was 149,47 m, the median value increased by 127,5 m, the differences were statistically significant (P=0.006).

In the control group the average distance traveled by the patient, decreased by 1.42 m, the median value increased by 35,00 m, the difference between the indices were not statistically significant. Differences in the dynamics of the index between the groups (+150,89 on the average value and +92,5 the median value) were statistically significant (P=0.001).

- Transcutaneous-defined voltage oxygen. In the clinical group were observed continuous tendency to increase the average value of the characteristic tcnq with 76,69±9,96 mm RT.article at the first visit to 85,42±10,87 mm RT.article in the fourth. In the control group was noted opposite di is Amica: the reduction of the average figure with 76,89±55,76 mm RT.article to 75,37±61,57 mm RT.article during the same period. Marked differences between the characteristic values in the clinical group were reliable, and in the control group statistically significant (P=0,096), i.e., the rate of these patients has remained almost unchanged in the course of standard treatment. Fixed differences in the growth of the average figure tcnq between groups (+of 10.25) and median (+8,00) were significant (0,0001). The relative growth in the group amounted to: in the clinical group +12,40±17,69% and in the control -2,12±to 4.38% (P=0.001).

- The ankle-brachial index ABI, measured on the target limb, patients clinical group had a tendency to increase, and among the patients of the control group - to decrease. So, in the clinical group with initial value 0,513±of 0.182 index rose on a background of application of the drug on 0,057 and totaled six months of 0.57. Marked differences in the characteristic value between the fourth and first visits in the clinical group were statistically significant (P=0.001). In the control index for the six months decreased by 0.02 units with 0,458±of 0.182 to 0,438±0,187. However, these changes were not statistically significant.

The difference between growth in average LPI between groups was +0,077 (median +0,065), marked differences were statistically significant.

Linear blood flow velocity (according to ultras okoboi Doppler rear polyamerous artery), Patients clinical group this index tended to increase throughout the study period: the average increased by 8,24 cm/s, median - 5.0 cm/s - level 14,95±10,19 cm/s to 23,19±12,71 cm/s in six months.

Patients in the control group values increased by 1,30 cm/s (17,60±6,60 cm/sec. In the beginning of the study, to 18,90±6,77 cm/s - six months), the median value remained unchanged, at the level of 20.0 cm/s during the whole study differences in values between groups were statistically significant (P=0.005).

The growth rate in the clinical group was greater than in controls the mean (+6,94 cm/s) and median (+5,00 cm/s), these differences were statistically significant (P=0.005).

Thus, according to the criterion of efficiency "painless walking distance in patients clinical group found a statistically significant increase in the rate from 135,3 m at the time of the first visit to 248,7 m six months (plus 110,5%), which were statistically significantly different from the trends identified patients in the control group (P=0.001).

Other performance criteria:

- ABI - growth rate of 11.11% (P=0.001);

- Tcnq - growth is 11,38% (P=0.001);

- BFV - growth is 55,12% (P=0.001).

While this invention is described in detail with reference to Examples, for a specialist in a specified field is ti technology obviously, what can be done various changes and produced equivalent replacement, and such modifications and substitutions are within the scope of the present invention.

1. A method of obtaining a pharmaceutical composition for the induction of the development of blood vessels in the tissues, providing for the transformation of Escherichia coli strain TOP10 with plasmid pCMV-VEGF165, characterized by SEQ ID NO:1, culturing the strain in suitable for biomass growth conditions followed by separation of the plasmid pCMV-VEGF165 in supercoiled form and subsequent lyophilization of plasmid pCMV-VEGF165, which is carried out with obligatory presence in liofilizirovannom solution of excipients, namely cryoprotectant, pH stabilizer, antioxidant and other substances, allowing to obtain an isotonic solution for injection to a concentration of the purified plasmid from 0.1 to 10 mg/ml and a pH of from 7.0 to 9.0 and ensuring the preservation of supercoiled form of the plasmid pCMV-VEGF165 during subsequent storage.

2. Pharmaceutical composition for the induction of the development of blood vessels in the tissue obtained by the method according to p. 1 and containing purified plasmid pCMV-VEGF165, characterized by the sequence SEQ ID NO:1, which provides the expression of VEGF165 gene in human cells, and pharmaceutically acceptable excipients in the form of at least one of cryoprotectant with with the properties of the filler, and pH stabilizer, in effective amounts providing isotonic solution for injection to a concentration of the purified plasmid from 0.1 to 10 mg/ml and a pH of from 7.0 to 9.0.

3. A method of treating ischemia of tissues and/or organs, which consists in the introduction intramuscularly human pharmaceutical composition, described in paragraph 2, in an effective amount to provide a therapeutic effect.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology.

EFFECT: bispecific anti-human vascular endothelial growth factor VEGF and human angiopoietin-2 ANG-2 antibodies, methods for producing them, pharmaceutical compositions containing the above antibodies, and using them are described.

13 cl, 26 dwg, 15 tbl, 19 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of organic chemistry, namely to heterocyclic compounds of formula I

and to their pharmaceutically acceptable salts, where A is selected from CH or N; R1 is selected from the group, consisting of C3-6-cycloalkyl, C3-6-cycloalkyl-C1-7-alkyl, C1-7-alkoxy-C1-7-alkyl, halogen-C1-7-alkyl; R2 and R6 independently on each other represent hydrogen of halogen; R3 and R5 independently on each other are selected from the group, consisting of hydrogen, C1-7-alkyl and halogen; R4 is selected from the group, consisting of hydrogen, C1-7-alkyl, halogen and amino; R7 is selected from the group, consisting of C1-7-alkyl, C1-7alkoxy-C1-7-alkyl, C1-7-alkoxyimino-C1-7-alkyl, 4-6-membered heterocyclyl, containing one heteroatom O, phenyl, with said phenyl being non-substituted or substituted with one hydroxy group, and 5-10-membered heteroaryl, containing 1-3 heteroatoms, selected from N, S and O, said heteroaryl is not substituted or is substituted with one or two groups, selected from the group, consisting of C1-7-alkyl, hydroxy, C1-7-alkoxy, cyano, C1-7-alkylaminocarbonyl and halogen. Invention also relates to pharmaceutical composition based on formula I compound and to method of obtaining formula I compound.

EFFECT: obtained are novel heterocyclic compounds, which are agents, increasing level of LDLP.

17 cl, 2 tbl, 89 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to paediatric cardiology and paediatric infectious diseases, and can be used for evaluation of indications for cardiometabolic therapy in case of infectious affection of myocardium in children. For this purpose quantitative evaluation of clinical, electrocardiographic, biochemical and echocardiographic indices is determined and realised. As clinical indices auscultative symptomatic: sonority of tones, presence of noises, parameters of arterial pressure are evaluated. As biochemical indices evaluated are: activity of cardiospeciphic enzymes: MB-fraction of creatine phosphokinase, α-hydroxybutyrate dehydrogenase, aspartic transaminase, alanine transaminase and cardiospecific troponin I protein. Echocardiographic examination is realised with application of Dopplerography for evaluation of diastolic ventricular function. Each of indices is evaluated by from 1 to 3 points. Points are summed up and obtained result is used to evaluate indications for cardiometabolic therapy. If the total sum is lower than 3 points, cardiometabolic therapy is not indicated. If the total sum is from 3 points to 7 point including, peroral introduction of cardiometabolic preparations is carried out. If the total sum is from 8 points and higher, parenteral introduction of cardiometabolic preparations is realised.

EFFECT: method provides possibility of determining presence of indications to administering cardiometabolic therapy objectively in minimal terms, including situations, when part of results of additional examination is absent because of some reasons, and of evaluating its efficiency in differential way.

1 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the chemical-pharmaceutical industry and represents a pharmaceutical composition used for treating or preventing a cardiovascular disease in an individual in need thereof and containing at least 95% ethyleicosapentaenoate (ethyl-EPA) encased in a capsule containing gelatine, glycerol, sorbitol, maltitol and purified water, wherein the composition has an initial peroxide number of no more than 5 mEq/kg and the second peroxide number of no more than 8 mEq/kg if stored at 25°C and relative humidity (RH) of 60% for 6 months.

EFFECT: invention refers to a method of treating or preventing the cardiovascular diseases involving administering a therapeutically effective amount of this composition into the individual in need thereof.

18 cl, 4 ex, 4 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: according to a known method of treating the liver disease accompanying type 2 diabetes mellitus involving the baseline therapy of diabetes mellitus and prescribed hepatoprotectors, the above hepatoprotector is presented by Mexicor in a daily therapeutically effective dose of not less than 16 weeks. The therapeutically effective dose of Mexicor makes 100 mg 4 times a day.

EFFECT: higher clinical effectiveness ensured by eliminating the liver disease more prominently, reducing the length of treatment, normalising the liver function test results over a short period of time, and avoiding any side effects.

2 cl, 2 tbl

Pcsk9 vaccine // 2538162

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology and biotechnology. Presented is an immunogen for inducing an immune response to the PCSK9 protein, containing the PCSK9 antigen peptide specified in a group consisting of the disclosed SEQ ID NO: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 314, 318 and 319, containing the amino acid sequence TRFHRQ, and SEQ ID NO: 182, 183, 184, 185, 186, 187, 188, 317, 401, 402 and 403, containing the amino acid sequence SIPWNLE, wherein the above PCSK9 peptide is conjugated with an immunogenic carrier specified in CRM197 or a Qbeta viral-like particle (VLP). Described is a composition for inducing the immune response to the PCSK9 protein containing at least two immunogens in immunologically effective amounts, wherein the first immunogen represents the above immunogen. Disclosed is a composition for inducing the immune response to the PCSK9 protein containing at least two immunogens in the immunologically effective amounts, wherein the first immunogen is specified in a group of immunogens containing the amino acid sequence SIPWNLE, and wherein the second immunogen is specified in a group of immunogens containing the amino acid sequence TRFHRQ; and wherein the composition can additionally contain at least one adjuvant. What is also presented is a pharmaceutical composition for preventing, treating or relieving PCSK9-related disorders containing: the above immunogen in a therapeutically effective amount, or one of the above compositions, and a pharmaceutically acceptable excipient.

EFFECT: invention enables preparing the effective vaccine for the disorders related to a reaction of the PCSK9 protein and its LDLR receptor.

24 cl, 19 dwg, 6 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pyrazolopyridine derivatives of formula (I) , a based pharmaceutical composition, and using them for treating and/or preventing disorders or conditions related to nictonamide adenine dinucleotide phosphatoxidase (NADPH-oxidase), as well as to a method for preparing them and an intermediate of formula (VIII) . In general formula (I), G1 is specified in H; and optionally substituted heteroaryl-C1-C6-alkyl; G2 is specified in H; optionally substituted C1-C6-alkyl; optionally substituted C2-C6-alkenyl; optionally substituted C2-C6-alkinyl; optionally substituted aryl; optionally substituted C1-C6-alkylaryl; optionally substituted aryl-C1-C6-alkyl; optionally substituted heteroaryl; optionally substituted C1-C6-alkylheteroaryl; optionally substituted heteroaryl-C1-C6-alkyl; optionally substituted C2-C6-alkenylaryl; optionally substituted aryl-C2-C6-alkenyl; optionally substituted C2-C6-alkenylheteroaryl; optionally substituted heteroaryl-C2-C6-alkenyl; optionally substituted C3-C8-cycloalkyl; optionally substituted C3-C8-heterocycloalkyl; optionally substituted C1-C6-alkyl-C3-C8-cycloalkyl; optionally substituted C3-C8-cycloalkyl-C1-C6-alkyl; optionally substituted C1-C6-alkyl-C3-C8-heterocycloalkyl and optionally substituted C3-C8-heterocycloalkyl-C1-C6-alkyl; G3 is specified in H; optionally substituted amino; optionally substituted aminoalkyl; optionally substituted aminocarbonyl; optionally substituted alkoxy, optionally substituted alkoxy-C1-C6-alkyl; optionally substituted carbonyl; optionally substituted C1-C6-alkyl; optionally substituted C2-C6-alkenyl; optionally substituted C2-C6-alkinyl; optionally substituted aryl; optionally substituted aryl-C1-C6-alkyl; optionally substituted C1-C6-alkylaryl: optionally substituted heteroaryl; optionally substituted C1-C6-alkylheteroaryl; optionally substituted heteroaryl-C1-C6-alkyl; optionally substituted C2-C6-alkenylaryl; optionally substituted aryl-C2-C6-alkenyl; optionally substituted C2-C6-alkenylheteroaryl; optionally substituted heteroaryl-C2-C6-alkenyl; optionally substituted C3-C8-cycloalkyl; optionally substituted C3-C8-heterocycloalkyl; optionally substituted C1-C6-alkyl-C3-C8-cycloalkyl; optionally substituted C3-C8-cycloalkyl-C1-C6-alkyl; optionally substituted C1-C6-alkyl-C3-C8-hterocycloalkyl and optionally substituted C3-C8-heterocycloalkyl-C1-C6-alkyl; G4 is specified in -NR2-C(O)-R1 and -(CHR3)m-(CH2)n-R4, G5 represents H.

EFFECT: preparing the pharmaceutical composition for treating and/or preventing the disorders and conditions related to nictonamide adenine dinucleotide phosphatoxidase.

16 cl, 3 tbl, 87 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, namely to endocrinology, and can be used for treating non-alcoholic liver disease accompanying type 2 diabetes mellitus. The declared preparation Mexicor provides reducing manifestations of cytolysis and cholestasis, decreasing the steatosis index, enables improving metabolic lipid and glycaemic values and reducing insulin resistance. Mexicor is applied in a daily therapeutically effective dose of 100 mg 4 times a day for at least 16 weeks.

EFFECT: high pharmacological activity of Mexicor has been shown by achieving the pronounced and stable elimination of fatty liver disease that enables reducing the length of treatment with no side effects.

2 cl, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: pharmaceutical composition contains aleglitezar or its sodium salt in a dose of 0.01 to 0.9 mg.

EFFECT: pharmaceutical composition of aleglitezar is used for treating or preventing type II diabetes mellitus or cardiovascular diseases.

32 cl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology. Characterised is application of therapeutically efficient quantity of peptide GGF2, which contains domain, similar to epidermal growth factor (EGF-like), in treatment or prevention of heart failure in mammal by injection of said peptide every 48 hours in dose, which constitutes from approximately 0.001 mg/kg to approximately 10 mg/kg.

EFFECT: invention improves therapeutic effect with introduction of neuroregulin with minimisation of any potential side effects.

14 cl, 15 dwg, 11 tbl

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, in particular to tumour-specific promoters, and can be used in the anti-cancer therapy. There are constructed the broad-spectrum tumour-specific promoters providing the therapeutic gene expression inside a cancer cell. The invention also involves expression cassettes, expression vectors, pharmaceutical compositions, methods of treating cancer and using the expression cassettes and vectors.

EFFECT: promoters of the present invention provide a high expression level of the operatively linked therapeutic gene in the cancer cells of different origin, wherein the normal cell expression is absent or low.

29 cl, 19 dwg, 4 tbl, 20 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to biotechnology and represents molecular conjugates, capable of binding with nucleic acids (DNA or RNA) for their delivery into cells of mammals, expressing transferring receptors. The said molecular conjugates consist of a polycationic sequence, represented by a modified signal of nuclear localisation of the virus SV40 T-antigen, and a ligand. One of two sequences HAIYPRH or THRPPMWSPVWP is used as ligands of cell receptors. Complexes of the molecular conjugate and nucleic acid are used for obtaining medications for genetic therapy or diagnostics of various diseases. To obtain the said complexes solutions of nucleic acid and the molecular conjugate are poured together with a ratio of charges in the reaction medium from 1:0.63 and lower and used for complex formation of solutions with ionic power less than 300 mM.

EFFECT: claimed invention makes it possible to increase the efficiency of delivering genetic constructions into cells.

12 cl, 8 dwg, 1 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: method comprises scaling of diploid cells of line M-20 from cryobank of Institute of Poliomyelitis and Viral Encephalitis n.a. MP Chumakov of RAMS from the ampoule of the seed cell bank of 7 passage with obtaining the working cell bank of 16 passage. At that the cells of 20-33 passages, suitable for use in therapeutic and/or diagnostic purposes are produced by culturing in a nutrient medium containing 10% of human fibrinolytic active plasma (FAP), comprising platelet-derived growth factor PDGF in a concentration of from 155 to 342 pg/ml.

EFFECT: invention enables to increase proliferative activity of human fibroblast diploid cells.

2 cl, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular biology and genetic engineering. What is presented is a RNAi molecule for suppressing the thymidilate synthase expression by the action of RNAi, containing a double-stranded RNA domain consisting of a sense chain consisting of a nucleotide sequence presented by SEQ ID NO: 1 hybridised with an anti-sense chain hybridized in the demanding conditions with the sense chain.

EFFECT: molecule can substantially potentiate the antineoplastic action of 5-FU-antineoplastic agent for which reason it can be used in medicine as a part of the antineoplastic therapy.

15 cl, 2 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biotechnology and oncology. A method provides: a) isolating postnatal tissue-specific pluripotent autologous stem cells (ASCs) and/or autologous progenitor cells (APCs) for the following proteome and complete transcriptomic analysis; b) isolating ASCs and/or APCs and/or pluripotent allogenic HLA-haploidentical stem cells (HLA-SCs) for remodelling of their proteome profile; c) isolating cancerous stem cells from patient's tumour; d) carrying out ASC and/or APC and CSC proteome analysis; e) carrying out ASC and/or APC and CSC complete transcriptomic analysis; f) recognising a protein complement each of which is found in the proteome profiles both of ASCs and/or APCs, and of CSCs; g) analysing the recognising protein complement for identifying intracellular signalling pathways in CSCs not subject to the neoplastic transformation as a result of carcinogenesis, and recognising target proteins defined as membrane acceptors of identified signalling pathways; h) analysing the complete transcriptomic profile of the CSC gene expression and confirming the preservation and functional significance of the structural components of the identified signalling pathways in CSCs; i) recognising ligand proteins able to activate the target proteins; j) carrying out a comparative analysis of the ASC and/or APC complete transcriptomic profiles to the transcriptomic profiles contained in the known data bases of transcriptomes for recognising perturbagens able to modify the gene expression profile of ASCs and/or APCs and/or HLA-SCs isolated for remodelling their proteome profile in the line of secreting the pre-recognised ligand proteins; k) remodelling the ASC and/or APC and/or HLA-SC proteome profile with perturbagens to produce the modified transcriptome profile of various cell systems able to have a regulatory influence on patient's CSCs.

EFFECT: preparation produced according to the method includes all the individual peculiarities of the patient's genome and proteome modifications, and has the regulatory influence on patient's cancerous stem cells (CSCs) and malignant cells.

8 cl, 4 dwg, 11 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: population of mononuclear cells or non-germ stem cells, which is saturated with cells of monocytic lineage containing promonocytes, is used for treatment of ischemia with a patient.

EFFECT: invention allows effective treatment of ischemia with a patient by injection of the above population of therapeutical cells to ischemic tissue of the patient.

13 cl, 8 dwg, 5 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine. What is described is an active immunostimulating vaccine containing at least one RNA, preferentially iRNA coding at least two antigens evoking the immune response in a mammal and used for treating lung cancer, first non-small cells lung cancer (NSCLC), preferentially specified among three primary subtypes, squamous cell carcinoma, adenocarcinoma and large-cell lung carcinoma, or NSCLS-related disorders.

EFFECT: there are produced kits, first containing the active immunostimulating vaccine.

21 cl, 34 dwg, 1 tbl, 8 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to an agent for treatment of ischemic lesions of tissues, which is a mixture with the ratio of 1.5-3 of two cultures of mesenchymal stem cells, one of which is modified by the genetic structure based on the viral vector which provides hyperproduction of vascular endothelial growth factor, and the other is modified by the genetic structure based on the viral vector which provides hyperproduction of angiopoietin, and a method of treatment of ischemic lesions of tissues by puncture of ischemic tissue, and can be used in medicine.

EFFECT: invention enables to achieve effective vascularisation and repair of ischemic tissue.

4 cl, 4 dwg, 3 ex

FIELD: biotechnology.

SUBSTANCE: method comprises isolation of mononuclear cells (MNC) from peripheral blood of a patient, separation of cells to adherent and non-adherent fractions, addition of the adherent fraction to the MNC of growth factors, loading of the dendritic cell with antigens of tumour lysate in vitro, the stimulation of maturation of dendritic cells for the next day. At that, the obtained immature DCs are added to lysate-autologous tumour cells at a dose of 100 mcg/ml, and after 48 hours within the subsequent 24 hours the rf-tumour necrosis factor-alpha is applied at a dose of 25 ng/ml. Then, the co-culture is carried out of mature dendritic cells activated with lysate and the non-adherent fraction of MNC at a ratio of 1:10 in the presence of recombinant human interleukin-12 at a dose of 10 ng/ml and the recombinant human interleukin-18 at a dose of 100 ng/ml.

EFFECT: invention enables to improve the level of cytotoxic and interferon-producing activity of antigen-activated dendritic cells while reducing the duration of their culture.

4 tbl

FIELD: medicine.

SUBSTANCE: what is presented is a method for stimulating the post-traumatic spinal cord regeneration consisting in a single-stage transplantation of human umbilical cord blood mononuclear cells pre-transduced with recombinant adenovirus with a cloned gene of glial derived neurotrophic factor (gdnf), in the damage area.

EFFECT: using the invention enables providing a better outcome of the post-traumatic spinal cord regeneration, reduced length of staying in hospital of patients suffering from a spinal cord injury and improving the patients' quality of life.

1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to methods of treating cystitis of various origins. Increasing the clinical effectiveness and reducing a probability of recurrences are ensured by administering therapeutically active and gel preparations possessing a high adhesion to urothelium walls. The method involves instilling sodium alginate in the form of Colegel gel containing the therapeutically active preparations dioxidine, lidocaine, deoxyribonucleate, sodium hyaluronate in a therapeutically effective amount with the instillation procedure suggested to be performed according to the schedule specified depending on the cystitis origin. If observing chronic bacterial recurrent cystitis, the following schedule is presented: instillations of Colegel-ADL from the 1st to 10th therapeutic day daily, instillations of Colegel-DNK-L from the 11th to 20th therapeutic day every second day, 2-month instillation of Colegel-Hyal once a week. Treating radiation and interstitial cystitis requires the following therapeutic schedule: instillations of Colegel-ADL from the 1st to 20th day, while Colegel-DNK-L and Colegel-Hyal are instilled once twice a week from the 21st to 56th day alternated every second day. The presented therapeutic schedules using the given therapeutically active preparations enabling relieving pain, reducing a rate and a length of recurrent cystitis.

EFFECT: using these therapeutic schedules is high-efficient and safe.

1 ex

Up!