Microparticle and pharmaceutical composition thereof

FIELD: medicine, pharmaceutics.

SUBSTANCE: microparticle contains an agglomerate of particles containing a hydrophilic active substance, wherein the particle contains an amphiphilic polymer composed of a hydrophobic segment of polyhydroxy acid and a hydrophilic segment of polysaccharide or polyethylene glycol, and a hydrophilic active substance. What is also disclosed is a method of producing the agglomerated microparticles, which involves (a) a stage of preparing a reverse phase emulsion, (b) a stage of preparing a solid residue containing the hydrophilic active substance, and (c) a stage of introducing the solid residue into a liquid phase containing a surface modifier.

EFFECT: agglomerated microparticles provide the effective encapsulation of the hydrophilic active substance and the release of the hydrophilic active substance at an appropriate speed.

14 cl, 22 dwg, 4 tbl, 31 ex

 

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention relates to a microparticle comprising an agglomerate of particles containing hydrophilic active substance, and its pharmaceutical compositions. In particular, the invention relates to a microparticle and its pharmaceutical composition as a so-called system of drug delivery. More specifically, for example, the invention relates to a microparticle, effectively containing protein, peptide drugs, drugs based on nucleic acids and similar, having a hydrophilic property and a large molecular weight, and its pharmaceutical compositions.

The LEVEL of TECHNOLOGY

Currently developing medications in the form of particles containing drugs, prisoners in fine particles, called nanoparticle, a microparticle, a nano scale, a microsphere or microcapsule, and attempts are being made of their use as agents for slow release of drugs.

Dispersed drugs using polymeric compounds as bases include fine particles consisting of biodegradable polylactic acid or a copolymer of lactic and glycolic acids. In the data dispersed drugs it is difficult to conclude protein or peptide Lakers the public means, having a hydrophilic property and a large molecular weight, at the same time retaining biological activity. In addition, when introduced into the human body it is known that the drug being released within a short time, and this phenomenon is called the initial release.

As fine particles consisting of a polymer is covalently linked saccharide and polyhydroxybutyrate, patent document 1 describes a microcapsule for delivery of pharmacologically active substances, consisting of a reaction product of a polyol and polylactic acid. In this way polysaccharides are not used, and nothing is specified regarding the inclusion of the peptide or protein. The microcapsule produced by a method of spray drying, released 62% of the encapsulated drug within 24 hours. This rate of release is too fast, and a microcapsule can hardly be used as a slow release agent for medicines.

Patent document 2 and non-patent document 1 describes a nanoparticle or nanoparticle consisting of a material having a biodegradable polymer grafted to the polysaccharide, but in this document, nothing is specified about the microparticles are composed of nanoparticles. Patent document 2 describes nab the emer, the double emulsion, already cited in other sources, as a method of making microparticles to encapsulate hydrophilic active substances, but there is no specific description, and the inclusion of the drug in the particle or the release of drug from the particles are not carried out. Non-patent document 1 describes a particle that encapsulates the albumin produced by a method of double emulsions, but the efficiency of encapsulation in relation to the amount of albumin is 53% or less, and low efficiency of encapsulation of hydrophilic active substance has a problem of cost of production.

Patent document 3 describes a fine particle containing amphiphilic polymer consisting of polysaccharides and aliphatic polyester, more specifically, fine particle, consisting of an inner core of polysaccharides, hydrophobic outer layer of the aliphatic polyester and the surface modifier associated with a hydrophobic outer layer. This fine particle is not agglomerated structure of the fine particles, and specific examples do not demonstrate that the diameter of the particles is in the micrometer range. The efficiency of encapsulation of hydrophilic substances is 50% or less, and this is low the efficiency of encapsulation is the problem, similar to the above case.

Patent document 4 describes the nanoparticles with an average particle diameter less than 300 nm, consisting of a obtained from a natural source polymer dextran, but specific examples are not shown. It is not agglomerated structure of the fine particles, the average particle diameter is hundreds of nanometers, drug, probably diffuses from the site of introduction, and it is not preferred as a means of slow release.

As the polymer for forming particles, patent document 5 and patent document 6 describe and propose the use of amphiphilic block polymer having a hydrophilic part, such as a polyethylene glycol and a hydrophobic part, such as the copolymer of lactic and glycolic acids. Particle micelles using an amphiphilic block polymer, are generally hydrophobic inside and hydrophilic external layer, and they are suitable for retention of hydrophobic low-molecular-weight drugs, but are not suitable for retention of hydrophilic active substances, such as protein or peptide.

Patent document 7 and non-patent document 2 describe attempts to include protein in the particle using amphiphilic block polymer, but the amount contained medicines is minor, is whether the initial peak allocation is significant, and to date, the technology of producing particles having properties suitable for the introduction of hydrophilic drugs with a slow release, not yet implemented.

Patent document 1: Publication of the patent application of Japan No. 8-19226

Patent document 2: Japanese translation of international publication of PCT application No. 2004-521152

Patent document 3: WO 2006/095668

Patent document 4: Japanese translation of international publication of PCT application No. 10-511957

Patent document 5: Japanese translation of international publication of PCT application No. 2004-513154

Patent document 6: Japanese translation of international publication of PCT application No. 2004-514734

Patent document 7: Japanese translation of international publication of PCT application No. 2000-501084

Non-patent document 1: Yuichi Oya and 3 co-author, “Encapsulation and/or Release Behavior of Bovine Serum Albumin within and from Polylactide-Grafted Dextran Microspheres” (Macromolecular Bioscience, 2004, vol. 4, pp. 458-463).

Non-patent document 2: Anshu Yang and 5 co-authors, “Tumor necrosis factor alpha blocking peptide loaded PEG-PLGA nanopeptides: Preparation and in vitro evaluation” (International Journal of Pharmaceutics, 2007, vol. 331, pp. 123-132).

Description of the INVENTION

PROBLEMS THAT MUST be SOLVED by the INVENTION

As described above, were developed microparticles using the polymer, and therefore, the main objective of the invention is to provide microparticles are able to effectively encapsulate the guide is opornoe active substance, and, more specifically, microparticles, is capable of releasing the encapsulated drug at an appropriate speed, without causing significant initial release.

Resolving PROBLEMS

The authors of the present invention have performed intensive research to solve these problems and ultimately made the invention.

The invention relates to a microparticle comprising an agglomerate of particles containing hydrophilic active substance, and the particle includes amphiphilic polymer comprising a hydrophobic segment of PHAs and the hydrophilic segment of polysaccharides or of polyethylene glycol, and a hydrophilic active substance, or, more specifically, to a microparticle comprising an agglomerate of particles containing hydrophilic active substance, and this particle has a hydrophilic segment of amphiphilic polymer in the inner part and has an outer layer of hydrophilic segment of amphiphilic polymer, its preparation and its pharmaceutical compositions.

The EFFECTS of the INVENTION

The microparticle according to the invention is able to effectively encapsulate hydrophilic active substance and release hydrophilic active substance at an appropriate speed in the human body and, therefore, is applicable as a new DDS drug.

The QUICK DESCRIPTION of the DRAWINGS

Figure 1 shows the release of drug from microparticles with encapsulated human growth hormone.

Figure 2 shows the release of drug from microparticles dextran-PLGA encapsulated with human insulin.

Figure 3 shows the SEM image of the microparticles dextran-PLGA.

Figure 4 shows the SEM image of the micro-particles of the polyethylene glycol-poly(Epsilon-caprolactone).

Figure 5 shows the dynamics of changes in the concentration of the drug in the blood of mice, which was subcutaneously injected particles with encapsulated human growth hormone.

6 shows the dynamics of changes in the concentration of the drug in the blood of mice, which was subcutaneously injected microparticles with encapsulated human growth hormone.

Fig.7 shows the changes in the body weight of the mouse, which was subcutaneously injected microparticles with encapsulated human growth hormone.

Fig shows the dynamics of changes in the concentration of IGF-1 in the blood of mice, which was subcutaneously injected microparticles with encapsulated human growth hormone.

Fig.9 shows the release of the drug in buffer solution of microparticles with encapsulated Basis 4.

Figure 10 shows the dynamics of changes in the concentration of the drug in the blood of mice, which was subcutaneously injected microparticles with encapsul is included in the Basis 4.

11 shows the release of drug from microparticles consisting of associated particles with encapsulated human growth hormone.

Fig shows the relationship between the particle diameter and the number of dimethylcarbonate added during preparation of the emulsion type S/O/W.

Fig shows the results for the efficiency of entrainment for microparticles with encapsulated FD40.

Fig shows the SEM image of the powder of microparticles prepared from polymer PEG-PLGA (5k-10k).

Fig shows the SEM image of the powder of microparticles prepared from polymer PEG-PLGA (5k-61k).

Fig shows the nature of the release FD40 of microparticles with encapsulated FD40.

Fig shows the nature of the release of drug from microparticles with encapsulated human insulin.

Fig shows the dynamics of changes in the concentration of the drug in the blood of mice, which was subcutaneously injected microparticles with encapsulated human growth hormone.

Fig shows the dynamics of changes in the concentration of the drug in the blood of mice, which was subcutaneously injected microparticles with encapsulated human growth hormone.

Fig shows the dynamics of changes in the concentration of IGF-1 in the blood of mice, which was subcutaneously injected microparticles with encapsulated gormo the om human growth.

Fig shows the dynamics of changes of pharmacokinetics in the blood of mice, which was subcutaneously injected microparticles with encapsulated Basis 4.

Fig shows the relationship between the particle diameter and the number of dimethylcarbonate added during preparation of the emulsion type S/O/W.

The BEST WAY of carrying out the INVENTION

The invention is characterized by the formation of micro-particles by aggregation of particles containing hydrophilic active substance, the particle comprises an amphiphilic polymer and a hydrophilic active substance. In the present description, aggregation is the binding of two or more particles through the interaction force between particles or other substances, and the formation of the group. The force of interaction between particles is not specifically indicated, but acceptable examples include hydrophobic interaction, hydrogen bond force and van der Waals forces. Aggregation is not limited to the condition of joint contact of the particles, but the particles may contain a substance having affinity to particles, or the particles can be distributed in the matrix. As substances having an affinity for the particles, or matrix, it is preferable for the polymer. In the invention, aggregation of particles containing hydrophilic active substance, compared with the single particle reaches the I effect consisting in the fact that the efficiency of encapsulation of hydrophilic active substances above. The diameter of the particles containing hydrophilic active substance, which should be associated, is a variable parameter.

Microparticles are particles having a diameter in the range from sub-micron to sub-millimeter. In the invention, the average diameter of the microparticles is not specifically limited, but, in the case of the introduction of microparticles by injection into the body, the more the average particle diameter, the more the needle of the syringe, and increases the burden on the patient and, therefore, from the viewpoint of reducing the load on the patient, it is preferable that he was in the range from 1 μm to 50 μm. The average diameter of the microparticles can be determined by image analysis using a scanning electron microscope.

The number of agglomerates of particles containing hydrophilic active substance for the preparation of microparticles, preferably, is in the range from 10 to 10 in the seventh degree, more preferably in the range from 10 in the fifth degree to 10 in the seventh degree. The number of agglomerates calculated from the average diameter of the particles containing hydrophilic active substance, and the mean diameter of the microparticles.

In the invention, the amphiphilic polymer comprises a hydrophobic segment PHAs and hydrof the high segment of polysaccharides or polyethylene glycol. In the present description amphiphilic property is the condition of having both hydrophilic and hydrophobic properties, and as for the hydrophilic properties when the solubility in water is higher in a particular segment than in the other segments, they say that this segment is hydrophilic. Preferably, the hydrophilic segment is soluble in water, but if it is it is soluble, it is hydrophilic, if its solubility in water is higher than in other segments. A certain segment called hydrophobic, if its solubility in water is lower than the other parts. The hydrophobic segment, preferably, insoluble in water, but if it is soluble, it can be hydrophobic if its solubility in water is lower than the other segments.

Specific examples of PHAs amphiphilic polymer include polyglycolic acid, polylactic acid, poly(2-hydroxybutiric acid), poly(2-hydroxyvaleric acid), poly(2-hydroxypropranolol acid), poly(2-hydroxyamino acid), polyblock acid and derivatives and copolymers data of high-molecular compounds. However, since it is desirable that the microparticles according to the invention had no significant actions during the introduction to the human body, polyhydroxybutyrate amphiphilic polymer also preferably is of Biosafety is a diversified high-molecular polymer. Biocompatible high molecular weight polymer is a substance that do not have significant effects on the human body with the introduction and, more specifically, sub-lethal dose LD50 is preferably 2000 mg/kg or more in oral introduction of high-molecular polymer rat.

As PHAs biocompatible high molecular weight polymer, preferred is a copolymer of polylactic acid and polyglycolic acid or poly(lactic acid-glycolic acid). When polyhydroxybutyrate is a copolymer of polylactic acid and polyglycolic acid, the ratio of the components of the copolymer of polylactic acid and polyglycolic acid (mol/mol%) is not specifically limited, provided that the objectives are achieved inventions, but this ratio preferably ranges from 10/0 to 30/70, or more preferably from 60/40 to 40/60.

When the hydrophilic segment of amphiphilic polymer is a polysaccharide, examples of polysaccharides may include cellulose, chitin, chitosan, Gellan gum, alginic acid, hyaluronic acid, pullulan or dextran, and dextran is preferred.

Amphiphilic polymer, preferably, get grafted polymerization graft(s) chain(s) of PHAs on the main circuit p is lisamaria. In the present description, the average molecular weight main chain of the polysaccharide, preferably from 1,000 to 100,000, or more preferably from 2,000 to 50,000, and the average molecular weight of the PHA, preferably from 500 to 100,000, or more preferably, from 1000 to 10000. The ratio of the average molecular weight of PHAs to medium molecular weight polysaccharides, preferably ranges from 0.01 to 100, preferably from 0.02 to 10, or, most preferably, from 0.02 to 1.

The number of grafted chains PHAs associated with the main chain of the polysaccharides, preferably, ranges from 2 to 50. The number of grafted chains can be determined from the average molecular weight amphiphilic polymer graft type, the main chain of the polysaccharides and grafted chain PHAs.

When the hydrophilic segment of amphiphilic polymer is a polyethylene glycol, an amphiphilic polymer, preferably a block polymer of polyethylene glycol and PHAs. In the invention, the term "block" refers to a portion of a polymer molecule consisting of at least five or more Monomeric units, and which is excellent in chemical structure or configuration between this part of the segment and the other adjacent part of the segment, and a polymer, education is ing two or more blocks, directly, called block polymer. Each block forming the block polymer may include two or more Monomeric level, i.e. can be formed statistical, alternating or gradient polymer. When the hydrophilic segment of amphiphilic polymer is a polyethylene glycol, an amphiphilic polymer, preferably, is a block-polymer linking blocks of polyethylene glycol and PHAs.

When the hydrophilic segment of amphiphilic polymer is a polyethylene glycol, specific examples of the glycol to be used include polyethylene glycol with a straight or branched chain or its derivatives, a preferred example of a derivative of polyethylene glycol is monoalkyl ether of polyethylene glycol. Alkyl group monoalkyl ether of polyethylene glycol is an alkyl group with straight or branched chain, containing from 1 to 10 carbon atoms, and more preferred is a branched alkyl group containing from 1 to 4 carbon atoms, and particularly preferred are methyl, ethyl, sawn and isopropyl group.

The average molecular weight of polyethylene glycol is not specifically limited, but, preferably, it is from 2,000 to 15,000, more is preferable, from 2000 to 12000, more preferably from 4000 to 12000 and, particularly preferably, from 5000 to 12000.

When the hydrophilic segment of amphiphilic polymer is a polyethylene glycol, average molecular weight of the PHA is not specifically limited, but, preferably, it ranges from 5000 to 200000, more preferably from 15,000 to 150,000 or even more preferably from 20000 to 100000. The ratio of the average molecular weight of the PHA to the average molecular weight of the polyethylene glycol preferably is 1.0 or more, more preferably 2 or more, most preferably 4 or more and, particularly preferably, from 4 or more to 25 or less.

In this description, the average molecular weight refers to srednekamennogo molecular weight, unless stated otherwise, and srednekislye molecular weight is an average molecular weight calculated by the method, not taking into account the weighting of the absolute value of the molecule and an average molecular weight of the amphiphilic polymer, polysaccharides and polyethylene glycol can be obtained in the form of a molecular weight converted to polystyrene or pullulan measured by gel permeation chromatography (GPC). The average molecular weight of PHAs can be determined from the peak integral values to the core balance and peak integral values, non-end balance measured by the method of nuclear magnetic resonance (1H-NMR).

Amphiphilic polymer consisting of polysaccharides and PHAs used in the invention can be synthesized by any known method, and provided that may be formed emulsion with reversed phase synthesis method is not precisely specified, and it can be made, for example, any of the following methods (1), (2) and (3).

(1) In the presence of a tin catalyst, a monomer, an activating gidrokshikislotu add to polysaccharides for the implementation of the polymerization reaction, then add polyhydroxybutyrate, and get amphiphilic polymer grafted type [Macromolecules, 31, 1032-1039 (1998)].

(2) a Hydroxyl group of polysaccharides with partially removed for copyright protection, the majority of the hydroxyl groups of which are protected by the Deputy, activate the base, add the monomer, activating gidrokshikislotu to form grafted(s) chain(s)consisting of the PHA and, finally, remove the protective group, and get amphiphilic polymer graft type Polymer, 44, 3927-3933 (2003)].

(3) In polysaccharides add a copolymer of PHAs to carry out the condensation reaction using a dehydrating agent and/or functional activating agent, and get amphiphilic polymer grafted type [Macromolecules, 33, 368-3685 (2000)].

Amphiphilic polymer consisting of polyethylene glycol and PHAs used in the invention can be synthesized by any known method and, provided that it is possible to obtain an emulsion with reversed phase synthesis method is not specified and, for example, in the presence of a tin catalyst, a monomer, an activating gidrokshikislotu added to the glycol for the implementation of the polymerization reaction to obtain polyhydroxybutyrate, and made amphiphilic block polymer [Journal of Controlled Release, 71, 203-211 (2001)].

Structure containing hydrophilic active substance particles comprising amphiphilic polymer and hydrophilic biologically active substance is not specifically limited, but since the particle containing hydrophilic active substance, has a hydrophilic segment of amphiphilic polymer in the inner part and has an outer layer of hydrophobic segment of amphiphilic polymer, it is preferred, as contained hydrophilic active substance can be kept more stable.

When a particle containing hydrophilic active substance is a particle having a hydrophilic segment of amphiphilic polymer in the inner part and having an outer layer of hydrophobic segment of amphiphilic polymer, one of the preferred embodiments which is, if the surface modifier is associated with the outer layer of PHAs. In the present invention, the binding can be either non-covalent binding or covalent binding. Non-covalent binding, preferably, is a hydrophobic interaction, but may include electrostatic interactions, hydrogen bonds or forces van der Waals forces, or combinations thereof. When non-covalent binding of hydrophobic outer layer of fine particles containing amphiphilic polymer, and the hydrophobic portion of the surface modifier, described below, preferably, can be connected to each other by hydrophobic interaction. In this case, the dispersant fine particles, especially preferably represents water, buffer solution, physiological saline, an aqueous solution of a surface modifier or a dispersant fine particles on the basis of the hydrophilic solvent.

The surface modifier preferably is a compound capable of stabilizing the division surface water-in-oil emulsion type S/O/W, or surface section-oil emulsion oil emulsion type S/O1/O2 and, more preferably, a compound which has properties to enhance the colloidal stability of the particles. The surface modifier can be one the type or mixture of many types. In the present description the property to increase the colloidal stability means the prevention or deceleration of the aggregation of microparticles in a solvent.

In the present invention, the surface modifier preferably is an amphiphilic compound or a hydrophilic polymer.

Hydrophilic polymer surface modifier according to the invention, preferably selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyethylenimine, polyacrylic acid, polymethacrylic acid, poly-1,3-dioxolane, polymer with 2-methacryloyloxyethyl, poly-1,3,6-trioxane, polyaminoamide, peptide, protein, sugars and their analogues.

Analogs of the hydrophilic polymer may include surface-active agent containing a hydrophilic polymer, a partially modified hydrophobic group, such as alkyl long chain, but they do not specifically limited to this.

As polietilenglikoli analogue of the surface modifier according to the invention preferably Pluronic (registered trademark)sold by BASF, or its equivalent.

As polyaminoamide of the surface modifier according to the invention can preferably be used Polisario acid, polyglutamic acid or their analogues. Similar, in which ASI alkyl group with a long chain in part Polisario acid or polyglutamic acid, it is especially preferred.

As the peptide of the surface modifier according to the invention it is possible to use a basic peptide.

As the protein surface modifier according to the invention, preferred for improving the characteristics of the dispersion particles is gelatin, casein or albumin. As a protein, one of the preferred examples is an antibody.

As the saccharides of the surface modifier according to the invention are preferred monosaccharides, oligosaccharides and polysaccharides. As preferred polysaccharides are cellulose, chitin, chitosan, Gellan gum, alginic acid, hyaluronic acid, pullulan and dextran. In particular, cholesteryl pullulan is preferred, taking from the point of view of better dispersive ability of the pigment particles. Preferred are analogs of any substance selected from the group consisting of cellulose, chitin, chitosan, Gellan gum, alginic acid, hyaluronic acid, pullulan and dextran.

As the surface modifier data examples of peptide, protein and sugars, especially preferred are analogs, partially modifying the hydrophobic group of the alkyl long chain, or analogs, modifying hydrophilic polymer or an amphiphilic compound.

The surface modifier is t according to the invention the amphiphilic compound comprises a lipid as one of preferred examples.

The surface modifier according to the invention the amphiphilic compound comprises a surfactant as one of the preferred examples. Preferred examples of surfactants include non-ionic active substance, such as a copolymer of polyoxyethylene and polypropylenglycol, ester of sucrose and a fatty acid ester of polyethylene glycol and fatty acid, ester of polyoxyethylenesorbitan and monobasic fatty acid, ester of polyoxyethylenesorbitan and dibasic fatty acid ester of polyoxyethyleneglycol and monobasic fatty acid, ester of polyoxyethyleneglycol and dibasic fatty acids, polyglycerol fatty acid, polyoxyethylene castor oil, polyoxyethylene utverjdenie castor oil; alkyl sulphates, such as sodium laurylsulfate, ammonium laurylsulfate, sodium sterilant; or lecithin.

In the invention the hydrophilic active substance is illustrated by the low molecular weight compound, protein, peptide, DNA, RNA, or modifying nucleic acid. In the microparticle according to the invention it is possible to conclude even hydrophobic drugs, when to make them hydrophilic, using solubilizers agent. Solubilizers agent in the present invention preferably includes cyclade the valleys and its analogs.

Protein or peptide used in the invention as hydrophilic active substance is not specifically limited, but preferred is a bioactive protein or bioactive peptide. Bioactive protein or bioactive peptide comprises a peptide hormone, cytokine, enzyme, protein or antibody. And specific examples include a peptide antagonist of GLP-1 receptor, such as on the basis of 4, parathyroid hormone (PTH), calcitonin, insulin, insulin-like growth factor, angiotensin, glucagon, GLP-1; bombezin, motilin, gastrin, growth hormone, prolactin (luteotropic hormone), gonadotropin (gonadotropic hormone), thyrotropic hormone, adrenocorticotropic hormone (ACTH), derived ACTH (aberated), melanocyte-stimulating hormone, folliculo-stimulating hormone (FSH), sermorelin, vasopressin, oxytocin, protirelin, luteinizing hormone (GnRH), corticotropin, secretin, somatropin, thyrotropin (thyroid stimulating hormone), somatostatin, gonadotropin releasing hormone (GnRH), G-CSF, erythropoietin (EPO), thrombopoietin (SRW), potentiator megakaryocytes, a growth factor for hepatocytes, EGF, endothelial growth factor vascular, interferon-α, interferon-β, interferon-γ, interleukins, FGF (growth factor fibroblast), BMP (bone morphogenetic protein), humoral thymic factor (THF), serum thymic factor (FTS), superoxide is mustasa (SOD), urokinase, lysozyme, tissue plasminogen activator, asparaginase, kallickrein, ghrelin, adiponectin, leptin, atrial natriuretic peptide, atrial natriuretic factor, brain natriuretic peptide (BNP), conotoxin G, dynorphin, endorphin, kyotorphin, enkephalin, neurotensin, angioten, bradykinin, substance P, Kaledin, hemoglobin, protein C, factor VIIa, glucocerebrosidase, streptokinase, staphylokinase, thymosin, pancreozymin, cholecystokinin, placental lactogenic human tumor necrosis factor (TNF), polymyxin b, colistin, gramicidin, bacitracin, thymopoietin, bombesin, cerulein thymostimulin, secretin, resistin, hepcidin, neuropeptide Y, neuropeptide S, cholecystokinin-pancreozymin (CCK-PZ), neurotrophic factor brain (BDNF), vaccine and similar. Data bioactive proteins or bioactive peptides can be natural proteins or peptides, or derivatives, modified in terms of their sequence, or compounds, modified chain polyethylene glycol or sugar.

When the hydrophilic active substance is a DNA, RNA or modifying nucleic acid, it can be any cationic surface-active agent, a cationic lipid, a cationic polymer, or other compounds forming complexes with their counterparts.

In the picture is hetenyi sugars, used as hydrophilic active substances include hyaluronic acid, heparin, dextran sulfate, dextran or labeled FITZ dextran (for example, FD40 and so on).

The invention also relates to a method for manufacturing microparticles are formed by aggregation of particles containing hydrophilic active substance, and the method includes:

(a) the stage of formation of the emulsion with reversed phase mixing an aqueous solvent comprising a hydrophilic active substance, and a water-immiscible organic solvent, dissolving the amphiphilic polymer

(b) a step for dry residue containing hydrophilic active substance, by removing the solvent from the emulsion with reversed phase, and

(c) the stage of introduction of solids or dispersion liquid containing the solid residue in a liquid phase containing a surface modifier.

In the method of manufacturing microparticles are formed by aggregation of particles containing hydrophilic active substance according to the invention, the emulsion with reversed phase formed by adding an aqueous solvent containing hydrophilic active substance to the water-immiscible organic solvent, dissolving an amphiphilic polymer, and mixing them. If necessary, you can use, for example, a mixing device, Taco is how, magnetic stirrer, turbine mixer, a homogenizer or a membrane emulsifying device equipped with a porous film. Water-immiscible organic solvent in the invention is an organic solvent, the solubility of which in water is 30 grams (water-immiscible organic solvent)/100 ml (water) or less, and other organic solvents, the solubility of which in water is higher than the specified value, are characterized as miscible with water and organic solvents.

As the aqueous solution in stage (a) use water or an aqueous solution containing a water-soluble substance. Water-soluble substance may be any substance from the group consisting of inorganic salts, saccharides, organic salts, amino acids and similar.

The property of water-immiscible organic solvent in stage (a) is not specifically limited, but, preferably, it is a solvent capable of dissolving polyhydroxylated as the hydrophobic segment of amphiphilic polymer, and difficult to dissolve or not to dissolve the hydrophilic segment. Water-immiscible organic solvent, preferably, scatters and removed freeze-drying or similar and preferred is entrusted, 0.1 g (water-immiscible organic solvent)/100 ml (water) or less. Specific examples of water-immiscible organic solvent include ethyl acetate, isopropylacetate, butyl acetate, dimethylcarbonate, diethylcarbamyl, methylene chloride and chloroform. The ratio of water-immiscible organic solvent to aqueous solvent is preferably from 1000:1 to 1:1, more preferably from 100:3 to 3:1. The concentration of the amphiphilic polymer in a water-immiscible organic solvent varies on the type of water-immiscible organic solvent or amphiphilic polymer, but preferably is from 0.01 to 90% (wt./mass.), more preferably from 0.1 to 50% (wt./mass.) or, even more preferably from 1 to 20% (wt./mass.).

At stage (a) in the process of formation of the emulsion with reversed phase aqueous solvent containing hydrophilic active substance, and a water-immiscible organic solvent, dissolving an amphiphilic polymer, depending on pharmacological targets emulsion with reversed phase can be obtained by using water-immiscible organic solvent, dissolving two or more types of amphiphilic polymer.

At stage (a) in the process of formation of the emulsion with reversed phase aqueous solvent, terrasim hydrophilic active substance, and water-immiscible organic solvent, dissolving the amphiphilic polymer to facilitate the formation of an emulsion with reversed phase and to form a uniform and thin emulsion with reversed phase, you can add an auxiliary agent. Such auxiliary agent, preferably, may be a compound selected from the group consisting of Olkiluoto alcohol containing from 3 to 6 carbon atoms, alkylamine containing from 3 to 6 carbon atoms, and alkilani carboxylic acid containing from 3 to 6 carbon atoms. The structure of the alkyl chain data auxiliary agents are not specifically specified, and can be applied either structure with a straight chain or branched structure, or you can use a saturated alkyl or unsaturated alkyl. In particular, the invention is preferred as the auxiliary agent is tert-butanol, Isobutanol and pentanol.

The average particle diameter of the emulsion with reversed phase to phase (a) varies with the desired particle diameter of the microparticles according to the invention and is not specifically limited, but for the manufacture of microparticles for pharmaceutical drug that is one of the areas of the use of microparticles according to the invention, the upper limit of the average particle diameter of, preferably, 50 μm, more preferably, 5 m, even more preferably, 500 nm, particularly preferably 150 nm and, most preferably, 100 nm. The lower limit of the average particle diameter of the emulsion with reversed phase preferably is 10 nm or, more preferably, 50 nm.

Further, in the method for manufacturing microparticles are important to include in stage (b) to obtain a dry residue containing hydrophilic active substance, removing the solvent from the emulsion with reversed phase obtained in stage (a).

At the stage (b) removal of the solvent from the emulsion with reversed phase is not specifically limited, but may include, for example, heating, drying in vacuum, dialysis, drying by freezing, surgery centrifugation, filtration, re-deposition, and combinations thereof.

Among these techniques, the removal of solvent from the emulsion with reversed phase especially preferred is drying by freezing, because it leads to minor structural changes due to the Association of the particles in the emulsion with reversed phase, and capable of avoiding degradation of the hydrophilic active substances due to the high temperature. Condition and apparatus for freeze-drying include the process of freezing and drying under reduced pressure, and particularly preferably, the method comprises the preliminary stage of freezing in the quality of the ve conventional method of freeze-drying, stage of primary drying under reduced pressure and a low temperature stage and a secondary drying under reduced pressure. For example, cooling and freezing below the melting temperature of an aqueous solution and water-immiscible organic solvent for the preparation of the emulsion with reversed phase, and then drying under reduced pressure, get subjected to drying by freezing the dry residue containing hydrophilic active substance. The pre-freezing temperature can appropriately be determined by experiment, taking into account the composition of the solvent and, as a rule, preferably, it is -20°C or less. The degree of reduced pressure in the drying process can properly be determined by experiment, taking into account the composition of the solvent and, as a rule, preferably, it is 3000 PA or less or, more preferably 500 PA or less, to reduce drying time. For freeze-drying, it is preferable to use a laboratory device for freeze-drying, with a cold trap and attached to the vacuum pump or vacuum device for freeze-drying rack of the type used in the manufacture of pharmaceutical preparations, and after pre-freezing by liquid nitrogen or refrigerant, is apolnet drying under reduced pressure at low temperature or at room temperature, using a vacuum pump or other device to reduce pressure.

The dry residue containing hydrophilic active substance, obtained in stage (b)is obtained in the form of aggregate particles containing hydrophilic active substance, comprising amphiphilic polymer, and these units correspond to the structure of the emulsion with reversed phase. In the present description the unit is a disordered mass, nakaplivalsya fine particles by the force of interaction between particles, and it clearly differs from the microparticles according to the invention. The average diameter of the fine particles containing hydrophilic active substance, for the formation of this unit varies depending on the desired diameter of the microparticles according to the invention, and is not specifically limited, but for the manufacture of microparticles for pharmaceutical drug that is one of the areas of the use of microparticles according to the invention, the upper limit of the average particle diameter of, preferably, 50 μm, more preferably, 5 μm, most preferably, 500 nm, especially 150 nm, in particular 100 nm. The lower limit of the average diameter of the fine particles containing hydrophilic active substance is preferably 10 nm or, more preferably, 50 nm.

In the method of manufacture of the microparticles is according to the invention it is important to include stage (c) the introduction of solids, containing hydrophilic active substance, or liquid dispersion containing solid residue in the liquid phase containing surface modifier.

At the stage (c) method of introduction of solids or liquid dispersion containing solid residue in the liquid phase containing surface modifier includes, for example, a method of adding the solids in the liquid phase containing surface modifier, and a method of dispersing the dry residue immediately in the dispersion medium, and add the obtained liquid dispersion (suspension of solid substance in the oil (S/O)) in the liquid phase containing surface modifier.

When the dispersion of the dry residue containing hydrophilic active substance, directly in the dispersion medium, the dispersion medium is not specifically limited, but preferably is a solvent capable of dissolving polyhydroxybutyrate, but essentially not capable of dissolving the hydrophilic segment comprising the amphiphilic polymer, for the purpose of preserving the structure of particles containing hydrophilic active substance, consisting of an amphiphilic polymer having the structure of the emulsion with reversed phase to produce a dry residue containing hydrophilic active substance. The solvent capable of dissolving polyhydroxybutyrate, but essentially not capable of dissolving hydrofil the first segment, is a solvent in which the solubility of the hydrophilic segment is 50 mg/ml or less, preferably, 10 mg/ml or less.

The dispersion medium may be any water-immiscible organic solvent miscible with water, an organic solvent, provided that it has the above features, and more preferred is a water-immiscible organic solvent. Specific examples of water-immiscible organic solvent capable of dissolving polyhydroxylated amphiphilic polymer, but is essentially not soluble in the hydrophilic segment include ethyl acetate, isopropylacetate, butyl acetate, dimethylcarbonate, diethylcarbamyl, methylene chloride, chloroform, dioxane, toluene, and xylene.

The dispersion medium for dispersing the dry residue containing hydrophilic active substance may contain various additives, soluble in the dispersion medium, for the purpose of controlling the speed of release of hydrophilic active substances due to the decomposition or disintegration of particles containing hydrophilic active substance, for example, acid connection, the primary connection, the amphiphilic polymer or biodegradable polymer.

The liquid phase in stage (C), preferably capable of dissolving the surface modifier is resti, and has a higher boiling point than that of the dispersion medium dry residue containing hydrophilic active substance, and may include an aqueous solvent, a water-immiscible organic solvent and miscible with water, an organic solvent. In the present invention the aqueous solvent is a water or aqueous solution containing a water-soluble component and a water-soluble component includes, for example, inorganic salts, sugars, organic salts and amino acids; water-immiscible organic solvent includes, for example, silicone oil, sesame oil, soybean oil, corn oil, cottonseed oil, coconut oil, linseed oil, mineral oil, castor oil, utverjdenie castor oil, liquid paraffin, n-hexane, n-heptane, glycerin and oleic oil; and miscible with water, the organic solvent includes, for example, glycerin, acetone, ethanol, acetic acid, dipropyleneglycol, triethanolamine and triethylene glycol. In the invention the liquid phase in stage (c), preferably is an aqueous solvent or miscible with water, an organic solvent. When the liquid phase is an aqueous solvent, and the dispersion medium is a water-immiscible organic solvent, suspe the Zia, obtained in stage (c) is a so-called emulsion type solid-oil-water (S/O/W), and when the liquid phase is a water-immiscible organic solvent miscible with water, an organic solvent that is immiscible in the dispersion medium, it is an emulsion type solid-oil-oil (S/O1/O2).

The volume ratio of liquid phase to the dispersion medium for dispersing the dry residue containing hydrophilic active substance, as a rule, is from 1000:1 to 1:1000 or, preferably, from 100:1 to 1:100.

The concentration of the surface modifier in the liquid phase according to the invention varies depending on the type of surface modifier, and preferably ranges from 0.01 to 90% (wt./vol.), more preferably, from 0.1 to 50% (wt./about.) and, even more preferably from 5 to 10% (wt./vol.).

The surface modifier may be associated with polyhydroxylated outer layer of amphiphilic polymer microparticles according to the invention, and the linking number in this case, preferably, from 0.0001% to 1% by weight of the microparticles.

In the liquid phase at the stage (c), except for the surface modifier may be added various additives depending on pharmacological targets such as buffer agent, antioxidant, salt, polymer or the Achar.

At the stage (c) is also preferably in the liquid phase to add inorganic salts. Preferably, the inorganic salt is a salt of an alkali metal or alkali earth metal salt, and particularly preferred is sodium chloride. The concentration of inorganic salts in the liquid phase, preferably, ranges from 0 to 1 M, more preferably from 10 mm to 1 M or, more preferably, from 10 mm to 100 mm.

At the stage (c)to produce a smaller particle size of the formed emulsion type solid-oil-water (S/O/W) or emulsion type solid-oil-oil (S/O1/O2) can be treated with surgery emulsification. Method of emulsification is not specifically limited provided that it is possible to obtain a stable emulsion. For example, a method includes a method of mixing or a method using a homogenizer, high pressure, or high-speed mixer-homogenizer.

At the stage (c), when the liquid dispersion obtained by dispersing the dry residue containing hydrophilic active substance, once in a dispersion medium was added into the liquid phase containing surface modifier, removing the dispersion medium, get the desired suspension of the formed particles by aggregation of particles containing hydrophilic active substance. Removal on spersonal environment is not specifically limited, but it can include drying methods in fluids, dialysis, freeze-drying, the operation of centrifugation, filtration, re-deposition, and particularly preferred can be drying in the liquid and drying by freezing. At the stage (c)when the liquid phase using an aqueous solvent, in the process, get a water dispersant particles.

Removing the liquid phase from the dispersed microparticles obtained in this way, you can obtain a microparticle according to the invention. Removal of the liquid phase is not specifically limited, but, preferably, can include methods of distillation, evaporation, dialysis, freeze-drying, the operation of centrifugation and filtering.

Applications microparticles obtained in the invention are numerous and varied, and in particular it is used as a pharmaceutical preparation. When the microparticle according to the invention used as a pharmaceutical preparation, in addition to the microparticles can contain different pharmacological mineral supplements, and applicable supplements include a buffering agent, an antioxidant, salt, polymer or sugar.

When the microparticle according to the invention used as a pharmaceutical preparation, route of administration includes, for example, oral introduction and parenteral administration, and parenteral rst is giving is preferred. Parenteral administration includes subcutaneous administration, intramuscular administration, enteral introduction, inhalation introduction, local introduction (nose, skin, eyes) and the introduction into the abdominal cavity and, in particular, subcutaneous and intramuscular administration are preferred. Dose and number of times the introduction of a pharmaceutical preparation according to the invention in the patient's body may be properly selected depending on the hydrophilic active ingredients, route of administration, age and body weight of the patient, or severity of symptoms, but usually, an adult patient per day a dose of 0.1 μg to 100 mg, preferably from 1 μg to 10 mg

EXAMPLES

The following are examples, but the invention is not limited to the examples described here.

Example 1

Synthesis of dextran-polylactic acid (PLA)

1.1 Synthesis of TMS-dextran (compound 1)

Dextran (NACALAI TESQUE, INC. NAKARAI product, the appropriate standard special grade, srednekislye molecular weight: 13000, 5.0 g) was added to formamide (100 ml) and was heated to 80°C. To this solution is added dropwise within 20 minutes was added 1,1,1,3,3,3-hexamethyldisilazane (100 ml). After addition the solution was stirred for 2 hours at 80°C. After completion of the reaction, the reaction solution was cooled to room temperature, and the solution was divided into two layers using the dosing funnel. Top with the Oh was concentrated under reduced pressure, was added methanol (300 ml)and the resulting dry residue was filtered and dried, obtaining TMS-dextran (11.4 g) as a white dry residue.

1.2 Synthesis of TMS-dextran-PLA (compound 2)

Compound 1 (0.5 g) and tert-butoxylate (35 mg) were dried for 1 hour under reduced pressure, was added tetrahydrofuran (20 ml)and the mixture was stirred for 1 hour at room temperature. To this solution was added dropwise a solution of (L)-lactide (of 4.49 g) in tetrahydrofuran (20 ml)and the mixture was stirred for 5 minutes. After completion of the reaction the solvent was concentrated under reduced pressure and was purified by repeated precipitation with the system chloroform-methanol, receiving TMS-dextran-PLA (1.9 g) as a white dry residue.

1.3 Synthesis of dextran-PLA (compound 3)

To a solution of compound 2 (1.9 g) in chloroform (24 ml) was added methanol (10,8 ml) and 12 N. hydrochloric acid (1.2 ml) and was stirred for 30 minutes at room temperature. The solvent is kept under reduced pressure, the residue was dissolved in chloroform (10 ml) and was added dropwise to diethyl ether, cooled to 0°C, and precipitated product. The precipitated substance was filtered and concentrated under reduced pressure, receiving dextran-PLA (1.6 g). The mass-average molecular weight of this polymer was 48720, and srednekislye molecular weight was 43530. (GPC measurements:column Toso TSK-gel α-5000×2, the system solvent DMF, RI detector, standard product, pullulan). The average molecular weight of the grafted chains of this polymer, determined by measurement1H-NMR was 2300. The number of grafted chains ranged from 10 to 12.

Example 2

Synthesis of dextran-copolymer of lactic and glycolic acids (PLGA)

2.1 Synthesis of TMS-dextran-PLGA (compound 4, compound 5, compound 6)

Compound 1 (0.5 g) and tert-butoxylate (35 mg) were dried for 1 hour under reduced pressure, was added tetrahydrofuran (10 ml)and the mixture was stirred for 1 hour at room temperature. To this solution was added dropwise a solution of (DL)-lactide (1.12 g) and glycolide (0.9 g) in tetrahydrofuran (15 ml)and the mixture was stirred for 5 minutes. After completion of the reaction the solvent was concentrated under reduced pressure and was purified by repeated precipitation of the system chloroform-methanol, receiving TMS-dextran-PLGA (1,96 g) as a white dry residue (compound 4). In the same way, loading quantity (DL)-lactide (0,784 g) and glycolide (0,63 g) synthesized compound 5, and, loading quantity (DL)-lactide (1.12 g) and glycolide (0.9 g)was synthesized compound 6.

2.2 Synthesis of dextran-PLGA (compound 7, compound 8, compound 9)

To a solution of compound 4 (1,96 g) in chloroform (14 ml) was added methanol (6.3 ml) and 12 N. hydrochloric acid (0.7 ml) and stirred in t is within 30 minutes at room temperature. The solvent is kept under reduced pressure, the residue was dissolved in chloroform (10 ml) and was added dropwise to diethyl ether, cooled to 0°C, and precipitated product. The precipitated substance was filtered and concentrated under reduced pressure, receiving dextran-PLGA (1,25 g) (compound 7). Of compounds 5 and 6 were similarly obtained dextran-PLGA products except that used triperoxonane acid (compound 8, compound 9). The mass-average molecular weight and srednekamennogo molecular weight of the polymer compounds 7-9 were determined using GPC measurements (column Toso TSK-gel α-5000×2, the system solvent DMF, RI detector, standard product, pullulan). The average molecular weight of the grafted chains and the number of grafted chains was determined by measurement1H-NMR.

As for compound 7, the mass-average molecular weight was 43820, srednekislye molecular weight was 33422, molecular weight of grafted chains was 1900, and the number of grafted chains ranged from 7 to 10.

With regard to the connection 8, the mass-average molecular weight was 94088, srednekislye molecular weight was 81250, molecular weight of grafted chains was 3250, and the number of grafted chains was 21.

With regard to the compound (9), the mass-average molecular weight was the 137695, srednekislye molecular weight was 109630, molecular weight of grafted chains was 6442, and the number of grafted chains was 15.

Example 3

A method of producing microparticles with encapsulated human growth hormone (hGH)

5 mg dextran-polylactic acid (PLA) of example 1 (average molecular weight of the dextran is equal to 13000, average molecular weight PLA is equal to 2300, the number of grafted PLA chains is from 10 to 12, the connection 3) or dextran-copolymer of lactic and glycolic acids (PLGA) example 2 (average molecular weight of the dextran is equal to 13000, average molecular weight PLGA equal 19000, the number of grafted chains PLGA is from 7 to 10, compound 7) was dissolved in 100 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 20 ml of tert-butanol was added dropwise 20 ml of 2 mg/ml aqueous solution of hGH and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 200 μl of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 2 mladenovo solution, containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated hGH. The obtained particles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of the microparticles was equal to 4.0 microns.

Example 4

The measurement of the efficiency of encapsulation of drug microparticles with encapsulated human growth hormone (hGH)

20 mg of microparticles with encapsulated human growth hormone prepared by the method of example 3 by use of the polymer dextran-PLA (compound 3) or dextran-PLGA (compound 7), weighed, using a 1.5-ml Eppendorf tube and dissolved in 1 ml of buffer solution A (saline phosphate buffer containing 0.1% bovine serum albumin, 0.1% of Pluronic F-68 (recorded the reported trade mark of BASF) and 0.02% of sodium azide), centrifuged for 10 minutes at 18000×g and were divided into particles (sedimentation) and the supernatant. The supernatant was collected in another tube, the particles are again suspended in 1 ml buffer, and the centrifugation and separation of the particles and the supernatant liquid is again carried out under the same conditions. This cleaning operation was repeated one more time (just three operations centrifugation and the concentration of human growth hormone in each supernatant is collected by centrifugation operation was measured using a kit for enzyme-linked immunosorbent assay (ELISA) (manufactured by R&D Systems). From the loaded amount of hGH during the preparation of the particles (the mass of particles is equal to 20 mg) subtracted the total amount of hGH in three of the supernatant liquids obtained by the operations centrifugation, and the encapsulation efficiency was calculated according to the formula below.

EffeKtandinnaboutwith atbandnKandpwith aylandpaboutinandnandI(%)=(CandgR yWennabouteKaboutlandhewith atinabouthGH(ng)-aboutbyeeKaboutlandhewith atinabouthDHinnanddaboutwith aanddabouthnsxWanddKaboutwith atIx(ng))CandgpyWennabouteKaboutlandhewith atinabouthDH(ng)×100

In the microparticles dextran-PLA or microparticles dextran-PLGA encapsulation efficiency hGH was 92.6 per cent in the microparticles dextran-PLA and 85.7% in the microparticles dextran-PLGA, and it was proved that the protein drug can be encapsulated with high quality, the th efficiency in particles of either type.

Example 5

Analysis of the speed of release in vitro drug from microparticles with encapsulated human growth hormone (hGH)

Microparticles was centrifuged three times in example 4, suspended and dispersible in 1.2 ml of buffer solution A. this solution part (40 ál) was transferred into another tube and centrifuged for 10 minutes at 18000×g to precipitate particles, and 30 µl of the supernatant was collected in another tube (0-hour sample). The remaining particle suspension was placed in 1.5-ml Eppendorf tube and slowly rotated and stirred in an incubator at 37°C using a rotary device with a speed of 6 rpm./minutes From this solution, a small portion (40 μl) were dosed out at specific time intervals, and the supernatant was separated in a similar manner through the operation of centrifugation. In the sample of the supernatant liquid collected in each moment of time, measured the concentration of hGH using the kit enzyme-linked immunosorbent assay, and the released amount (%) was calculated by the formula below.

Inswith ainaboutbaboutWdennabouteKaboutlandhewith atinabout (%)=(KaboutnCentpandCandIhGHinnanddaboutwith aanddabouthnaboutyWanddKaboutwith atand(ng/ml)×1,2(ml))andnKandpwith aylandpaboutinandnnabouteKaboutlandhewith atinabouthDH(ng)in20mghandwith atandC×100

Figure 1 shows the dynamics of changes in the release of drug from microparticles produced using a polymer dextran-PLA or dextran-PLGA. The particles about the type of the initial peak was not almost observed, the drug was released linearly proportional to the flow of time, and there have been favorable profile. The time required for 50% release of the drug was approximately 1 month in the microparticle dextran-PLA and about 1 week in the microparticle dextran-PLGA, and it was hypothesized that the rate of release can be controlled by selecting the type of PHA.

Example 6

A method of producing microparticles with encapsulated human insulin

5 mg dextran-PLA (average molecular weight of the dextran is equal to 13000, average molecular weight PLA is equal to 2300, the number of grafted PLA chains is from 10 to 12, the connection 3) or dextran-PLGA (average molecular weight of the dextran is equal to 13000, average molecular weight PLGA equal 19000, the number of grafted chains PLGA is from 7 to 10, compound 7) was dissolved in 100 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 20 ml of tert-butanol was added dropwise 20 ml of 2 mg/ml an aqueous solution of human insulin and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) when temperature is round cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 200 μl of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated human insulin. The obtained particles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of the microparticles was equal to 6.4 µm in microparticles obtained from compound 3, and 5.3 μm, the microparticles obtained from compound 7.

Example 7

The measurement of the efficiency of encapsulation of drug microparticles with encapsulated human insulin

20 mg of microparticles with encapsulated human insulin, prepared by the method of example 6 by using the project for a polymer dextran-PLGA (compound 7), weighed using a 1.5-ml Eppendorf tube and dissolved in 1 ml of buffer solution A (saline phosphate buffer containing 0.1% bovine serum albumin, 0.1% of Pluronic F-68 (registered trademark of BASF) and 0.02% of sodium azide), centrifuged for 10 minutes at 18800×g and were divided into particles (sedimentation) and the supernatant. The supernatant was collected in another tube and the particles are again suspended in 1 ml buffer, and the centrifugation and separation of the particles and the supernatant liquid is again carried out under the same conditions. This cleaning operation was repeated one more time (just three operations centrifugation and the concentration of human insulin each supernatant is collected through the operation of centrifugation, was measured using a layered method of enzyme-linked immunosorbent assay. From the loaded quantities of human insulin during preparation of the particles (the mass of particles is equal to 20 mg) subtracted the total amount of human insulin in three of the supernatant liquids obtained by the operations centrifugation, and the encapsulation efficiency was calculated according to the formula below.

EffeKtandinnaboutwith atb andnKandpwith aylandpaboutinandnandI(%)=(CandgpyWennabouteKaboutlandhewith atinaboutandnwith aylandnand(ng)-aboutbyeeKaboutlandhewith atinaboutandnwith aylandnandinnanddaboutwith aanddabouthnsxWanddKaboutwith atIx(ng))CandgpyWennabouteKaboutlandhewith atinaboutandnwith aylandnand (ng)×100

In the microparticles dextran-PLA or microparticles dextran-PLGA encapsulation efficiency of human insulin 75.7%, and it was proved that the drug can be encapsulated with high efficiency.

Example 8

Analysis of the speed of release in vitro drug from microparticles with encapsulated human insulin

Microparticles was centrifuged three times in example 7, suspended and dispersible in 1.2 ml of buffer solution A. this solution part (40 ál) was transferred into another tube and centrifuged for 10 minutes at 18800×g to precipitate particles, and 30 µl of the supernatant was collected in another tube (0-hour sample). The remaining particle suspension was placed in 1.5-ml Eppendorf tube and slowly rotated and stirred in an incubator at 37°C using a rotary device with a speed of 6 rpm From this solution, a small portion (40 μl) were dosed out at specific time intervals, and the supernatant was separated in a similar manner through the operation of centrifugation. In the sample supernatant collected at each time point, the concentration of human insulin was measured by a multilayer solid-phase method is immunofermentnogo analysis, and the released amount (%) was calculated by the formula below.

Inswith ainaboutbaboutWdennabouteKaboutlandhewith atinabout(%)=(KaboutnCentpandCandIhelaboutinehewith aKaboutgaboutandnwith aylandnandinnanddaboutwith aanddabouthnaboutyWanddKaboutwith atand(ng/ml)×1,2(ml))andnKandpwith aylandpaboutinandnnabouteKaboutlandhewith atmi> inabouthelaboutinehewith aKaboutgaboutandnwith aylandnand(ng)in20mghandwith atandC×100

Figure 2 shows the dynamics of changes in the release of human insulin. The initial peak was not almost observed, the drug was released linearly proportional to the flow of time, and there have been favorable profile. The time required for 50% release of the drug was approximately 6 days.

Example 9

The dynamics of changes in the morphology of microparticles

5 mg of microparticles with encapsulated hGH, prepared in example 3 were weighed in an Eppendorf tube was dispersible in 1 ml Milli-Q and were separated by centrifugation for 30 minutes at 13000 rpm, was separated from the supernatant liquid was again dispersible in 1 ml Milli-Q and were separated by centrifugation, and the microparticles were purified. In the solution of suspension of microparticles incubated for a certain period of time, was added 1 ml of Milli-Q, the solution was separated by centrifugation for 30 minutes at 13000 rpm, tdelay from the supernatant and again dispersible in 1 ml Milli-Q, were separated by centrifugation, and the microparticles were purified. Microparticles obtained after purification was dispersible in 100 ál Milli-Q and 3 µl of the liquid dispersion of particulate matter was collected on a silicon substrate was left to stand at room temperature for 10 minutes and dried for 3 hours in desicator. Then, using an ion sputtering device (HITACHI E-1030), on the sample surface was besieged platinum (settling time of 15 seconds), and the shape and surface condition of the particles was observed by a scanning electron microscope (SEM: HITACHI, S-4800) at the acceleration voltage of 1 kV and a high probe current.

As shown in figure 3, directly after manufacturing the surface was smooth and spherical, and the particles were visibly deformed after keeping for 13 days at 37°C, was formed many long and it was proved that the particles gradually decomposed together with the progress of the release of the drug.

Example comparison 1

5 mg polyethylene glycol-poly(Epsilon-caprolactone) (average molecular weight of polyethylene glycol 5000, the average molecular weight of poly(Epsilon-caprolactone) 37000) was dissolved in 100 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 20 ml of tert-butanol was added dropwise 20 ml of 2 mg/ml aqueous solution of hGH and mixed vortex mix the structure, receiving emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 200 μl of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated hGH. The obtained microparticles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of the microparticles was equal to 8.0 µm.

5 mg of the prepared powder of microparticles with encapsulated hGH weighed using samples of the CGS Eppendorf, was dispersible in 1 ml Milli-Q and centrifuged for 30 minutes at 13,000, was separated from the supernatant liquid was again dispersible in 1 ml Milli-Q and were separated by centrifugation in the same way, and microparticles were purified. Microparticles obtained after purification was dispersible in 100 ál Milli-Q and 5 µl of the liquid dispersion of particulate matter was collected on a silicon substrate was left to stand at room temperature for 10 minutes and dried for 3 hours in desicator. Then, using an ion sputtering device (HITACHI E-1030), on the sample surface was besieged platinum (settling time of 15 seconds), and the shape and surface condition of the particles was observed by a scanning electron microscope (SEM: HITACHI, S-4800) at the acceleration voltage of 1 kV and a high probe current.

As shown in figure 4, unlike microparticles dextran-PLGA in example 9, after incubation for 21 days at 37°C, the particles hardly changed morphologically, and there was a problem in the feature release hydrophilic active substances.

Example 10

Subcutaneous administration mouse microparticles with encapsulated human growth hormone (hGH)

25 mg of dextran-polylactic acid (PLA) (average molecular weight of the dextran is equal to 13000, average molecular weight PLA is equal to 2300, the number of grafted PLA chains is from 10 to 12, the connection 3) or dextran-copolyme is as lactic and glycolic acids (PLGA) (average molecular weight of the dextran is equal to 13000, the average molecular weight PLGA equal 19000, the number of grafted chains PLGA is from 7 to 10, compound 7) was dissolved in 500 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 100 μl of tert-butanol was added dropwise 250 ál of 10 mg/ml aqueous solution of hGH and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 1 ml of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA within 24 hours and got the powder of microparticles with encapsulated hGH. The obtained microparticles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of the microparticles was equal to 4.9 μm, the microparticles obtained from compound 3, and 4.2 μm in the microparticles obtained from compound 7.

300 mg of the prepared microparticles suspended and dispersible in 3 ml of physiological phosphate buffer solution (PBS) and centrifuged for 5 minutes at 80×g for deposition of particles, and the supernatant transferred to another tube. The supernatant was again centrifuged for 5 minutes at 80×g to precipitate the remaining particles, and the supernatant was removed. Re-dispersion in 1 ml of physiological solution with phosphate buffer after the first time of deposition by centrifugation and the second deposition times by centrifugation, the same operation purification by centrifugation was repeated three times, and the growth hormone is not encapsulated in the microparticles were removed. Finally, the precipitate was again dispersible in 200 μl of physiological solution with phosphate buffer and received the solution for injection. The amount of growth hormone encapsulated in dextran-PLA a microparticle and dextran-PLGA the microcapsule was measured by a kit for an enzyme-linked immunosorbent is Lisa, determined concentration in the cleaning solution and subtracted from the loaded quantity, and determined the amount of encapsulated 300 mg particles entered one mouse, and microparticles dextran-PLA this number was 590 mg, and microparticles dextran-PLGA - 536 mcg.

This solution was administered subcutaneously at two positions in the back of a 10-week-old male Balb/C mice, and blood samples were collected at certain time intervals from the tail vein. In the blood samples was added heparin to a final concentration of 3.3 int. units/ml, the plasma was collected by centrifugal separation for 5 minutes at 5000 rpm./minutes, and the concentration of growth hormone in plasma was measured using a kit for enzyme-linked immunosorbent assay.

In order to compare the solution in the form of the protein human growth hormone (700 μg/0.2 ml) was subcutaneously injected mice, and similarly selected samples.

In order to suppress the production of antibodies, resulting from the introduction of human growth hormone, is a foreign protein for mouse, three days before the introduction of the particles were injected subcutaneously immunosuppressive drug Tacrolimus hydrate (Astellas) 26 µg/mouse and then 13 μg/mouse during the administration of medicines, and 3 days and 7 days later.

Figure 5 shows the dynamics of changes in the concentration of human growth hormone in plasma. The mouse, which introduced negatives is annulled drug, the level in the blood within one hour after administration was very high, more than 5000 ng/ml, and then sharply decreased to the level before the introduction during the day. On the other hand, in mice, which were injected drug in the form of microparticles prepared using polymer dextran-PLA, a short-term increase in blood levels immediately after injection was reduced to 200 ng/ml or less, and within seven days level in the blood was maintained at high levels. In the microparticles dextran-PLA temporary increase in concentration after injection was not observed, and about the exact concentration in the blood was maintained for seven days and observed a excellent slow release.

Example 11

Subcutaneous administration mouse microparticles with encapsulated human growth hormone (hGH) (pharmacological activity)

2 mg of dextran-copolymer of lactic and glycolic acids (PLGA) (average molecular weight of the dextran is equal to 13000, average molecular weight PLGA equal to 1900, the number of grafted chains PLGA is from 7 to 10, compound 7) was dissolved in 500 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 100 μl of tert-butanol was added dropwise 250 ál of 10 mg/ml aqueous solution of hGH and mixed vortex mixing device, the floor is th emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 1 ml of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated hGH. The obtained microparticles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of the microparticles was equal to 4.1 microns.

300 mg of the prepared microparticles suspended and dispersible in 3 ml of physiological phosphate buffer solution (PBS) and frequent the hospital was besieged by separation by centrifugation for 5 minutes at 80×g, and the supernatant transferred to another tube. The supernatant was again separated by centrifugation for 5 minutes at 80×g, the remaining particles were besieged and the supernatant was removed. The first precipitate after centrifugation and the second precipitate after centrifugation were combined and again dispersible in 1 ml PBS, and similarly conducted a third operation centrifugation, and the growth hormone is not encapsulated in the particles were removed. Finally, the precipitate was again dispersible in 200 μl PBS, getting a solution for injection.

This solution was administered subcutaneously at two positions in the back of 8-week-old mouse with the remote pituitary (Japan SLC), and blood samples were collected at certain time intervals from the tail vein. In the blood samples was added heparin to a final concentration of 3.3 int. units/ml and centrifuged for 5 minutes at 5000 rpm./minutes, plasma was collected and the concentration of growth hormone in plasma and the concentration of insulin-like growth factor (IGF-1) mice were measured using enzyme-linked immunosorbent assays.

In order to compare the solution in the form of the protein human growth hormone (700 μg/0.2 ml) was subcutaneously injected mice, and similarly selected samples.

In order to suppress the production of antibodies, resulting from the introduction of human growth hormone, is h is geronim protein for mouse three days before the introduction of the particles were injected subcutaneously immunosuppressive drug Tacrolimus hydrate (Astellas) 26 µg/mouse and then 13 μg/mouse during the administration of medicines, and 3 days and 7 days later.

6 shows the dynamics of changes in the concentration of human growth hormone in plasma. In mice, which was introduced in the form of drug level in the blood within one hour after administration was very high, and then sharply decreased to the level before the introduction within two days. On the other hand, in the microparticles dextran-PLGA, a short-term increase in blood levels immediately after injection was suppressed to a low value, and within ten days after injection, the plasma concentration was maintained at high levels. At the moment the changes in the body weight of the mouse is shown in Fig.7. In mice, which were injected only growth hormone, increase of body weight was reduced at the level of around 5%, but in mice, which were injected microparticles dextran-PLGA, body weight was increased by about 20%.

Fig shows the concentration of IGF-1 in plasma. The concentration of IGF-1 in plasma correlates with the concentration of human growth hormone in the blood, and in mice, which were injected microparticles dextran-PLGA, high levels were maintained within ten days after injection.

Example 12

Analysis of the rate of drug release medium spans the VA in a buffer solution of microparticles with encapsulated Basis 4 (agonist GLP-1 receptor)

25 mg of dextran-copolymer of lactic and glycolic acids (PLGA) (average molecular weight of the dextran is equal to 13000, average molecular weight PLGA equal 3250 (compound 8) or 6442 (compound 9), the number of grafted chains PLGA is 21 (compound 8) or 15 (compound 9)) was dissolved in 500 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 100 μl of tert-butanol was added dropwise 250 ál of 10 mg/ml of Basis 4 (synthesized on behalf of the company Sigma Genosys)and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 1 ml of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF)were mixed and were emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion ICRI the particles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated Basis 4. The obtained particles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of the microparticles was $ 4.3 microns in connection 8 and 4.5 microns in connection 9.

These microparticles three times was purified according to the method of example 4, suspended and dispersible in 1.2 ml of buffer solution A. this solution part (40 ál) was transferred into another tube and centrifuged for 10 minutes at 18000×g, precipitating particles, and 30 µl of the supernatant was collected in another tube (0-hour sample). The remaining particle suspension was placed in 1.5-ml Eppendorf tube, slowly rotated and stirred in an incubator at 37°C using a rotary device with a speed of 6 rpm./minutes From this solution, a small portion (40 μl) were dosed out at specific time intervals, and the supernatant was separated in a similar manner through the operation of centrifugation. In the sample of the supernatant liquid collected in each moment of time, by the method of enzyme-linked immunosorbent assay measured the concentration of Basis 4, and the released amount (%) was calculated by the formula below./p>

Inswith ainaboutbaboutWdennabouteKaboutlandhewith atinabout(%)=(KaboutnCentpandCandIEKwith aendandnand-4innanddaboutwith aanddabouthnaboutyWanddKaboutwith atand(ng/ml)×1,2(ml))andnKandpwith aylandpaboutinandnnabouteKaboutlandhewith atinaboutEKwith aendandnand-4 (ng)in20mghandwith atandC×100

Fig.9 shows the dynamics of changes in the release of drug from microparticles produced using each of the polymer dextran-PLGA. In those and other microparticles initial peak was not almost observed, the drug was released linearly proportional to the flow of time, and there have been favorable profile.

Example 13

Subcutaneous administration mouse microparticles with encapsulated Basis 4 (agonist GLP-1 receptor)

300 mg of microparticles prepared in example 12, suspended and dispersible in 3 ml of physiological phosphate buffer solution (PBS) and particles besieged by separation by centrifugation for 5 minutes at 80×g and the supernatant transferred to another tube. The supernatant was again separated by centrifugation for 5 minutes at 80×g, the remaining particles were besieged and the supernatant was removed. The first precipitate after centrifugation and the second precipitate after centrifugation were combined and again dispersible in 1 ml PBS, and similarly conducted a third operation center is pulirovaniya, and on the Basis of 4, not encapsulated in the particles were removed. Finally, the precipitate was again dispersible in 200 μl PBS, getting a solution for injection.

This solution was injected subcutaneously into the back of 8-week-old SCID mice (CB17/lcr-Prkdcscid/CrlCrlk) (from Crea Japan, Inc.), and blood samples were collected at certain time intervals from the tail vein. In the blood samples was added heparin to a final concentration of 3.3 int. units/ml and centrifuged for 5 minutes at 5000 rpm and the plasma was collected and the concentration of Basis-4 in plasma was measured by the method of enzyme-linked immunosorbent assay. In order to compare the solution in the form of Basis 4 (700 μg/0.2 ml) was subcutaneously injected mice, and similarly selected samples.

Figure 10 shows the dynamics of concentration changes of Basis-4 in the plasma. In mice, which was introduced in the form of drug level in the blood within 1 hour after administration was very high, and then sharply decreased to the level before the introduction. On the other hand, in the microparticles dextran-PLGA, short-term increase in concentration after injection was suppressed to a low value, and within five days after injection, the plasma concentration was maintained at high levels.

Example 14

Synthesis of dextran-copolymer of lactic and glycolic acids (PLGA)

14.1 Synthesis of TMS-dextran-PLGA (compound 10, compound 11, is connected to the e 12, compound 13)

Compound 1 (0.5 g) and tert-butoxylate (35 mg) were dried for 1 hour under reduced pressure, was added tetrahydrofuran (10 ml)and the mixture was stirred for 1 hour at room temperature. To this solution was added dropwise a solution of (DL)-lactide (0,558 g) and glycolide (0.45 g) in tetrahydrofuran (15 ml)and the mixture was stirred for 5 minutes. After completion of the reaction the solvent was concentrated under reduced pressure and was purified by repeated precipitation of the system chloroform-methanol, receiving TMS-dextran-PLGA (1,96 g) as a white dry residue (compound 10).

In the same way, loading quantity (DL)-lactide (0,67 g) and glycolide (0.54 g) was synthesized compound 11.

In the same way, loading quantity (DL)-lactide (0,781 g) and glycolide (0,629 g) synthesized compound 12.

In the same way, loading quantity (DL)-lactide (1,123 g) and glycolide (0.9 g) was synthesized compound 13.

14.2 Synthesis of dextran-PLGA (compound 14, compound 15, compound 16, compound 17)

To a solution of compound 10 in chloroform (10 ml) was added triperoxonane acid (1 ml) and was stirred for 30 minutes at room temperature. The solvent is kept under reduced pressure, the residue was dissolved in chloroform (10 ml)was added dropwise to diethyl ether, cooled to 0°C, and precipitated product. The precipitated substance hoteltravel is whether and concentrated under reduced pressure, receiving dextran-PLGA (0,44 g) (compound 14).

Of compounds 11, 12 and 13 were similarly obtained products dextran-PLGA (compound 15, compound 16, compound 17). The mass-average molecular weight and srednekamennogo molecular weight of the polymer compounds 14-17 were determined using GPC (column Toso TSK-gel α-5000×2, the system solvent DMF, RI detector, standard product, pullulan). The average molecular weight of the grafted chains and the number of grafted chains was determined by measurement1H-NMR.

As for the connection 14, the mass-average molecular weight was 99462, srednekislye molecular weight was 85101, srednekislye molecular weight of the grafted chains was 2167, and the number of grafted chains was 33.

With regard to the connection 15, the mass-average molecular weight was 107779, srednekislye molecular weight was 92134, srednekislye molecular weight of the grafted chains was 3127, and the number of grafted chains was 25.

As for the connection 16, the mass-average molecular weight was 121281, srednekislye molecular weight was 101873, srednekislye molecular weight of the grafted chains was $ 3000, and the number of grafted chains was 30.

As for the connection 17, the mass-average molecular weight was 144838, srednekislye mo is ekulama weight was 122151, srednekislye molecular weight of the grafted chains was 4864, and the number of grafted chains was 22.

Example 15

A method of producing microparticles with encapsulated human growth hormone (hGH)

5 mg of each polymer dextran-copolymer of lactic and glycolic acids (dextran-PLGA polymer compounds 14-17) of example 14 was dissolved in 100 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 20 ml of tert-butanol was added dropwise 50 μl of 1 mg/ml aqueous solution of hGH and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 200 μl of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion ICRI the particles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated hGH. The obtained particles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of the particles was in the range of 1.0 to 10 microns.

Example 16

The measurement of the efficiency of encapsulation of drug microparticles with encapsulated human growth hormone (hGH)

20 mg of microparticles with encapsulated human growth hormone prepared by the method of example 15 by using each of the polymer dextran-PLGA (connection 14-17), weighed, using a 1.5-ml Eppendorf tube and dissolved in 1 ml of buffer solution A (saline phosphate buffer containing 0.1% bovine serum albumin, 0.1% of Pluronic F-68 (registered trademark of BASF) and 0.02% of sodium azide), centrifuged for 10 minutes at 18000×g and were divided into particles (sedimentation) and the supernatant. The supernatant was collected in another tube, the particles are again suspended in 1 ml buffer, and the centrifugation and separation of the hour the Itza and the supernatant liquid is again carried out under the same conditions. This cleaning operation was repeated one more time (just three operations centrifugation and the concentration of human growth hormone each supernatant collected with centrifugation, was measured using a kit for enzyme-linked immunosorbent assay (manufactured by R&D Systems). From the loaded amount of hGH during the preparation of the particles (the mass of particles is equal to 20 mg) subtracted the total amount of hGH in three of the supernatant liquids obtained by the operations centrifugation, and the encapsulation efficiency was calculated according to the formula below.

EffeKtandinnaboutwith atbandnKandpwith aylandpaboutinandnandI(%)=(CandgpyWennabouteKaboutlandhewith atinabouthGH(ng)-aboutbyee Kaboutlandhewith atinabouthDHinnanddaboutwith aanddabouthnsxWanddKaboutwith atIx(ng))CandgpyWennabouteKaboutlandhewith atinabouthDH(ng)×100

In the microparticles dextran-PLGA encapsulation efficiency hGH was 87.5% in the microparticles of compound 14, 94.2% of the microparticles of compound 15, 95.7% in the microparticles of compound 16 and 97.5% of the microparticles of compound 17, and it was proved that the protein drug can be encapsulated with high efficiency in all microparticles.

Example 2 comparison

Manufacture of particles with encapsulated human growth hormone performance measurement and encapsulate drugs

10 mg of dextran-copolymer of lactic and glycolic acids (PLGA) (compound 14, compound 17)was dissolved in 2 ml of ethyl acetate, obtaining a polymer solution. In this polymer solution was added dropwise 100 μl of an aqueous solution containing 0.5 mg/ml hGH, and stirred. After the operation, the stirring solution was added to 20 ml of dioxane. The solvent is evaporated, the solution was concentrated to about 2 ml, and the liquid dispersion of the particles were added to water containing 500 mg of Pluronic F-68 (registered trademark of BASF). The sample was dried by freezing to 50 mg of sample was added 1 ml of water, the particles again dispersible and received particles containing non-associated hydrophilic active substance. The average particle diameter was measured by dynamic light scattering, using the device ELS-Z (manufactured by Otsuka Denshi), and the efficiency of encapsulation of drugs was determined as in example 16.

As a result, the particles of compound 14 average particle diameter amounted to 190.5 nm, and the encapsulation efficiency was 73%, and particles of compound 17 average particle diameter was 197.5 nm, and the encapsulation efficiency was 70%, and the encapsulation efficiency was lower than the microparticles of example 16.

Example 17

Analysis of the speed of release in vitro drug from microparticles with encapsulated human growth hormone (hGH)

Microparticles treated three times in example 16, suspended and Dee who were peyrovani in 1.2 ml of buffer solution A. From this part of the solution (40 ál) was transferred into another tube and centrifuged for 10 minutes at 18000×g to precipitate particles, and 30 µl of the supernatant was collected in another tube (0-hour sample). The remaining particle suspension was placed in 1.5-ml Eppendorf tube and slowly rotated and stirred in an incubator at 37°C using a rotary device with a speed of 6 rpm From this solution, a small portion (40 μl) were dosed out at specific time intervals, and the supernatant was separated in a similar manner through the operation of centrifugation. In the sample supernatant collected at each time point, the concentration of hGH was measured using a kit for enzyme-linked immunosorbent assay, and the released amount (%) was calculated by the formula below.

Inswith ainaboutbaboutWdennabouteKaboutlandhewith atinabout(%)=(KaboutnCentpandCandIhGHin nanddaboutwith aanddabouthnaboutyWanddKaboutwith atand(ng/ml)×1,2(ml))andnKandpwith aylandpaboutinandnnabouteKaboutlandhewith atinabouthDH(ng)in20mghandwith atandC×100

11 shows the dynamics of changes in the release of drug from microparticles produced in example 15. In these particles, the initial peak was not almost observed, the drug was released linearly proportional to the flow of time, and there have been favorable profile. The time required for 50% release of the drug was approximately 6 days in the microparticles of compound 14, about 9 days in m is crocheting connection 15, about 16 days in the microparticles of compound 16 and about 1 month in the microparticles of compound 17, and it was hypothesized that the rate of release can be controlled by changing the loaded amount of the lactide and glycolide during the synthesis of TMS-dextran-PLGA.

Example 18

Obtaining microparticles with encapsulated dextran labeled with fluorescein (FD40), differing in the particle diameter

5 mg dextran-copolymer of lactic and glycolic acids (PLGA) (compound 7) of example 2 was dissolved in 100 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 20 ml of tert-butanol was added dropwise 20 ml of 1 mg/ml aqueous solution FD40 and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersively 50 ál, 100 ál, 200 ál and 350 ál, 500 ál, 1 ml, 2 ml and 6 ml of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in a vortex mix the eating device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated FD40. The obtained microparticles were observed by scanning electron microscope (SEM: HITACHI, S-4800) and calculated the average particle diameter.

Fig shows the correlation between the average particle diameter and the number of dimethylcarbonate added during preparation of the emulsion type S/O/W. In the range from 50 μl to 500 μl, together with the increase in the number of dimethylcarbonate observed decrease in the average diameter of the particles. From 500 μl to 6 ml no difference in the average diameter of the particles was not observed.

Example 19

Synthesis of polymer PEG-PLGA (series PEG2k)

Onomatology ether of polyethylene glycol (produced by NOF Corp., SUNBRIGHT MEH-20H, srednekislye molecular weight: 1862, Mw/Mn=1,03), (DL)-lactide and glycolide mixed in a given composition shown in table 1, and was heated at 140°C. After stirring for 20 minutes, added activit tin (II) (0.05 wt.% relatively monomial is delovogo ether of polyethylene glycol) and was stirred for 3 hours at 180°C. The reaction solution was cooled to room temperature, dissolved in chloroform (up to concentrations of about 100 mg/ml), again besieged and purified in diethyl ether, cooled at 0°C, the obtained dry residue was filtered, resultname and dried, and the polymer PEG-PLGA was received in the form of a white or light brown dry residue. Srednekamennogo the molecular weight of this polymer was determined1H-NMR (table 1).

Table 1
The number of loaded source of the substance and results of the synthesis of the polymer PEG-PLGA (series PEG2k)
Download number
(g)
Exit
(g)
Molecular mass
(1H-NMR)
The composition of the polymer
(molecular weight of PEG) - (molecular weight PLGA)
PEG(DL)-lactideGlycolic
0,8the 1.441,164,74135002k-11,5k
0,4the 1.441,16 235602k-21,5k
0,2the 1.441,162,52527002k-50,7k

Example 20

Synthesis of polymer PEG-PLGA (series PEG5k)

Onomatology ether of polyethylene glycol (produced by NOF Corp., SUNBRIGHT MEH-20H, srednekislye molecular weight: 5128, Mw/Mn=1,02), (DL)-lactide and glycolide mixed in a given composition shown in table 2, and heated at 140°C. After stirring for 20 minutes, added activit tin (II) (0.05 wt.% relatively nanometrology ether of polyethylene glycol) and was stirred for 3 hours at 180°C. the Reaction solution was cooled to room temperature, dissolved in chloroform (up to concentrations of about 100 mg/ml), again besieged and purified in diethyl ether, cooled at 0°C, the obtained dry residue was filtered, resultname and dried, and the polymer PEG-PLGA was received in the form of a white or light brown dry residue. Srednekamennogo the molecular weight of this polymer was determined1H-NMR (table 2).

Table 2
The number of loaded source of the substance and results of the synthesis of the polymer PEG-PLGA (series PEG5k)
Download number
(g)
Exit
(g)
Molecular mass
(1H-NMR)
The composition of the polymer
(molecular weight of PEG) - (molecular weight PLGA)
PEG(DL)-lactideGlycolic
0,50,720,581,31156005k-10k
0,5the 1.441,16of 2.51284005k-23k
0,33the 1.441,162,1375005k-32,5k
0,62,882,325,5444005k-39,4k
0,27the 1.441,162,62520005k-47k
0,2the 1.441,162,52660005k-61k
0,32,161,744,07699355k-65k
0,82,161,743,79595555k-55k
0,11,150,93-1093815k-105k

Example 21

Synthesis of polymer PEG-PLGA (series PEG10k)

Onomatology ether of polyethylene glycol (produced by NOF Corp., SUNBRIGHT MEH-10H, srednekislye molecular weight: 9975, Mw/Mn=1,02), (DL)-lactide and glycolide mixed in a given composition shown in table 3, and heated at 140°C. After stirring for 20 minutes, added activit tin (II) (0.05 wt.% relatively nanometrology ether of polyethylene glycol) and was stirred for 3 hours at 180°C. the Reaction solution was cooled to room temperature, dissolved in chloroform (up to concentrations of about 100 mg/ml), again besieged and was purified in jetelova ether, chilled at 0°C, the obtained dry residue was filtered, resultname and dried, and the polymer PEG-PLGA was received in the form of a white or light brown dry residue. Srednekamennogo the molecular weight of this polymer was determined1H-NMR (table 3).

Table 3
The number of loaded source of the substance and results of the synthesis of the polymer PEG-PLGA (series PEG10k)
Download number
(g)
Exit
(g)
Molecular mass
(1H-NMR)
The composition of the polymer
(molecular weight of PEG) - (molecular weight PLGA)
PEG(DL)-lactideGlycolic
0,5the 1.441,162,34900010k-39k
0,25the 1.441,162,4810500010k-95k

Example 22

A method of producing microparticles with encapsulated FD40

5 mg poly the EPA PEG-PLGA, prepared in examples 19-21, was dissolved in 100 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 20 ml of tert-butanol was added a given amount of 10 mg/ml aqueous solution FD40, as shown in table 4, and mixed, obtaining an emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 200 μl of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, got the powder of microparticles with encapsulated D40, part of it was observed by scanning electron microscope (SEM: HITACHI, S-4800) and calculated the average diameter of the particles (table 4). Image SAM powder polymer PEG-PLGA from 5k to 10k shown in Fig, and images of SAM powder polymer PEG-PLGA from 5k up to 61k shown in Fig.

Table 4
The amount of aqueous solution FD40, which should be added to each polymer, and the average diameter of the obtained microparticles
The composition of the PEG-PLGAThe amount of aqueous solution FD40
(ál)
The average particle diameter
(µm)
5k-11,5k13-
5k-21,5k12-
5k-50,7k12-
5k-10k20-
5k-23k204,6
5k-32,5k204,3
5k-39,4k20-
5k-47k204,2
5k-61k20a 3.9
5k-65k203,2
10k-39k184,8
10k-95k154,5

Example 23

The measurement of the efficiency of encapsulation of microparticles with encapsulated FD40

Microparticles (5 mg) with encapsulated FD40, prepared by the method of example 22 using a polymer PEG-PLGA, weighed, using a 1.5-ml Eppendorf tube, dispersible in Milli-Q (1 ml), centrifuged within 30 minutes and was divided into the supernatant, containing unencapsulated public FD40, and particles with encapsulated FD40, and collected. The collected particles with encapsulated FD40 was dissolved in N,N-dimethylformamide (250 μl) and the particles disintegrated. The supernatant, containing unencapsulated public FD40, and a solution of N,N-dimethylformamide (50 μl)containing encapsulated FD40, was added to Milli-Q (3 ml) separately and intensively stirred, and FD40 quantitatively determined using a fluorescence spectrophotometer (HORIB, Fluoro MAX-3, the wavelength of the excitation 495 nm, the wavelength of 520 nm fluorescence), and expected efficiency encapsulate all the collected volume.

Fig shows the effectiveness of encapsulation FD40 in microparticles prepared from polymer PEG-PLGA. All series 2k, 5k, 10k molecular weight PEG, when the molecular weight PLG was high, the efficiency of encapsulation tended to be high. In particular, in PEG5k series, 5k-65k, the encapsulation efficiency was very high, at around 90%. The encapsulation efficiency was approximately 55% in 10k-95k (PLGA/PEG=9.5) is approximately the same ratio of molecular masses as in 5k-47k *OKGA/PEG=9,4) with high encapsulation efficiency (about 80%).

Example 3 comparison

Manufacture of particles with encapsulated FD40

10 mg of the polymer PEG-PLGA (5k-61k) was dissolved in 2 ml of ethyl acetate, obtaining a polymer solution. In this polymer solution was added dropwise 100 μl of 2 mg/ml solution of human growth hormone and stirred. After the operation, the stirring solution was added to 20 ml of dioxane. The solvent is evaporated and concentrated to about 2 ml, and the liquid dispersion of the particles were added to water containing 500 mg of Pluronic F-68 (registered trademark of BASF). The sample was dried by freezing to 50 mg of sample was added 1 ml of water, the particles again dispersible and received particles containing non-associated hydrophilic active substance. The average particle diameter was measured by dynamic light scattering, using the device ELS-Z (manufactured by Otsuka Denshi), and the efficiency of encapsulation of drugs was determined in the same way as in example 23.

As a result, the efficiency of encapsulation FD40 was 48%, the average particle diameter was 203,8 nm, and the encapsulation efficiency was lower than the microparticles of example 23.

Example 24

Analysis of the speed of release in vitro FD40 of microparticles with encapsulated FD40

In order to assess the relationship between the behavior of slow-release and long PLGA chains for making polymer particles of PEG-PLGA, evaluated the behavior of the release particles 5k-23k, 5k-32,5k, 5k-47k and 5k-61k of microparticles with encapsulated FD40 prepared in example 22.

Microparticles, immediately after cooking, kept in dried by freezing state at -30°C and warmed up to normal temperature before using. Exactly 20 mg of the powder particles was weighed and placed in 1.5-ml tube (Eppendorf tube) and add 1 ml of buffer for analysis (0,02% sodium azide, 0.1% of Pluronic F-68 (registered trademark of BASF) and 0.1% saline solution with phosphate buffer containing 0.1% bovine serum albumin), were intensively mixed in a mixing device and suspended. Then, using a high speed recording motion is astou centrifuge Hitachi (CF16RX) the solution was centrifuged for 10 minutes at 18900×g and deleted 950 ál of the supernatant fraction, containing unencapsulated public FD40 was again added to 950 μl of buffer for analysis, the particles suspended and centrifuged, and the operation of cleaning particles was repeated in total three times.

In particles, purified three times, once added to 950 μl of buffer for analysis, the particles suspended and 100 µl of each sample was divided into 1.5-ml tube. To each tube was added 900 μl of buffer for analysis, receiving a total solution volume of 1 ml, which was kept in the incubator at 37°C, at the same time rotating with a speed of 10 rpm./minutes through a torque device. Then each incubated the tube was centrifuged for 10 minutes at 18900×g and 950 µl of the supernatant were dosed out and kept at 4°C until the time of measuring the intensity of fluorescence.

The fluorescence intensities of the test solution was measured using 3 ml of the removed cell (KARTELL) and HORIBA, Fluoro MAX-3 when the wavelength of excitation 494 nm and the wavelength of fluorescence 512 nm), and the degree of slow-release was determined from the ratio of the number of FD40 used in the preparation of the particles.

Fig shows the released amount FD40 from various microparticles determined by the evaluation of the release. The abscissa indicates the incubation time, and the ordinate axis represents the ratio of the released amount of a loaded number of. The particles 5k-23k having to will oduu PLGA chain, about 40% of the loaded amount is released within 1 day of the initial period of incubation, and one month would be released almost all the number except for part of the initial peak selection. The opposite way, as the length of the PLGA chain becomes longer, the initial released amount was decreased, and the microparticles 5k-61k released amount on the first day of the initial period was 10% or less.

Example 25

The measurement of the efficiency of encapsulation of drug microparticles with encapsulated human insulin

Using the polymer PEG-PLGA (5k-61k), prepared in example 20 using the same method as in example 22 was prepared microparticles with encapsulated human insulin. The obtained microparticles (20 mg) was weighed using a 1.5-ml Eppendorf tube and dissolved in 1 ml of buffer solution A (saline phosphate buffer containing 0.1% bovine serum albumin, 0.1% of Pluronic F-68 (registered trademark of BASF) and 0.02% of sodium azide), centrifuged for 10 minutes at 18800×g and were divided into particles (sedimentation) and the supernatant. The supernatant was collected in another tube and the particles are again suspended in 1 ml of buffer solution A, and the centrifugation and separation of the particles and the supernatant LM the bone again carried out under the same conditions. This cleaning operation was repeated one more time (just three operations centrifugation and the concentration of human insulin in each supernatant is collected by centrifugation operation, measured multilayer method enzyme-linked immunosorbent assay.

Multilayer enzyme-linked immunosorbent assays were performed as follows. Anti-human monoclonal antibody to insulin (manufactured Fitzgerald, clone No. EE) was immobilized on the ELISA plate (Maxisorp from Nunc Corp.) at a concentration of 5 μg/ml, was added 50 ál of ELISA buffer solution (0.1 M Tris chlorate buffer solution containing 0.25% bovine serum albumin and 0.05% Tween 20, pH 8.0) and 50 μl of the measured sample or standard sample, diluted in dilution solution ELISA (PBS containing 0.25% bovine serum albumin and 0.05% Tween 20), and the solution was reacted at room temperature with shaking for 1 hour. The plate three times purified in a purifying solution (PBS containing 0.05% Tween 20), unreacted reagent was removed and added to 0.5 μg/ml labeled with Biotin anti-human monoclonal antibodies (manufactured Fitzgerald, clone No. D4B8) and strepto-avidin-HRP conjugate (manufactured by Zymed), and the solution was reacted at room temperature with shaking for 1 hour and 15 minutes. After each reaction, the plate is cleaned and three times in the cleaning solution (PBS, containing 0.05% Tween 20) and unreacted reagent was removed. Finally, added the substrate of HRP and HRP enzymatic activity of the United conjugate was determined by colorimetry, and on the basis of a working curve prepared by the manifestation of the colors of a standard insulin was determined by the concentration of insulin in the sample.

From the loaded quantities of human insulin during preparation of the particles (the mass of particles (20 mg) subtracted the total amount of human insulin in three of the supernatant liquids obtained by the operations centrifugation, and the encapsulation efficiency was calculated according to the formula below.

EffeKtandinnaboutwith atbandnKandpwith aylandpaboutinandnandI(%)=(CandgpyWennabouteKaboutlandhewith atinaboutandnwith aylandnand(ng) -aboutbyeeKaboutlandhewith atinaboutandnwith aylandnandinnanddaboutwith aanddabouthnsxWanddKaboutwith atIx(ng))CandgpyWennabouteKaboutlandhewith atinaboutandnwith aylandnand(ng)×100

The average diameter of the obtained particles was 4.7 μm. The efficiency of encapsulation of human insulin in the microparticles was 86,75%, and it was proved that the protein drug can be kept with high efficiency.

Example 26

Analysis of the speed of release in vitro drug from microparticles with encapsulated human insulin

Microparticles was centrifuged three times in PR the least 25, suspended and dispersible in 1.0 ml of buffer solution A. this solution samples of 0.1 ml were distributed in ten Eppendorf tubes (capacity 1.5 ml), and to each tube was added 0.9 ml of buffer solution A, and diluted 10 times. Immediately after dilution, one tube was centrifuged for 10 minutes at 18800×g to precipitate the particles, and the supernatant was collected in another tube (0-hour sample). The remaining nine test tubes were rotated and slowly stirred in an incubator at 37°C using a rotating device with a speed of 6 rpm./minutes At defined intervals, each tube was centrifuged in a similar way and the supernatant was separated. In the sample of the supernatant liquid collected in each moment of time, measured the insulin concentration, a layered method of enzyme-linked immunosorbent assay, and released insulin (%) was calculated by the formula below.

Inswith ainaboutbaboutWdennabouteKaboutlandhewith atinabout(%)=(KaboutnC entpandCandIandnwith aylandnandinnanddaboutwith aanddabouthnaboutyWanddKaboutwith atand(ng/ml)×1(ml))andnKandpwith aylandpaboutinandnnabouteKaboutlandhewith atinaboutandnwith aylandnand(ng)in20mghandwith atandC×100

Fig shows the dynamics of changes in the release of insulin. Over time the drug is released gradually, and the rate of release increased after 30 days, and the majority of medicines, visualaid who stayed for about 60 days.

Example 27

Subcutaneous administration mouse microparticles with encapsulated human growth hormone (hGH)

25 mg of polymer PEG-PLGA was dissolved in 500 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 100 μl of tert-butanol and was added dropwise to 250 ál of 10 mg/ml aqueous solution of hGH and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 1 ml of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of cooling the soup traps -45°C and a vacuum of 20 PA within 24 hours and got the powder of microparticles with encapsulated hGH. The average diameter of the obtained microparticles rate of 6.0 microns.

300 mg of the prepared microparticles suspended and dispersible in 3 ml of physiological phosphate buffer solution (PBS) and centrifuged for 5 minutes at 80×g for deposition of particles, and the supernatant transferred to another tube. The supernatant was again centrifuged for 5 minutes at 80×g to precipitate the remaining particles, and the supernatant was removed. The first precipitate from the centrifugation and the second precipitate from the centrifugation were combined and again dispersible in 1 ml of physiological solution with phosphate buffer, the same operation purification by centrifugation was repeated in total three times, and the growth hormone is not encapsulated in the microparticles were removed. Finally, the precipitate was again dispersible in 200 μl of physiological solution with phosphate buffer and received the solution for injection. The amount of growth hormone encapsulated in PEG-PLGA particles was measured by a kit for enzyme-linked immunosorbent assay and subtracted from the loaded amount, determined the amount of encapsulated 300 mg particles, introduced the mouse, and received 700 mg of microparticles PEG-PLGA.

This solution was administered subcutaneously at two positions in the back of a 10-week-old male Balb/C mice, and samples of blood which was collected at certain time intervals from the tail vein. In the blood samples was added heparin to a final concentration of 3.3 int. units/ml, the plasma was collected by centrifugal separation for 5 minutes at 5000 rpm./minutes and the concentration of growth hormone in plasma was measured using a kit for enzyme-linked immunosorbent assay.

In order to compare the solution in the form of the protein human growth hormone (700 μg/0.2 ml) was subcutaneously injected mice, and similarly selected samples.

In order to suppress the production of antibodies due to the introduction of human growth hormone, which is a foreign protein for mouse, three days before the introduction of the particles were injected subcutaneously immunosuppressive drug Tacrolimus hydrate (Astellas) 26 µg/mouse and then 13 μg/mouse during the administration of medicines, and 3 days and 7 days later.

Fig shows the dynamics of changes in the concentration of human growth hormone in plasma. In mice, which was introduced in the form of drug level in the blood within one hour after administration was very high, more than 5000 ng/ml, and then sharply decreased to the level before the introduction during the day. On the other hand, in mice, which were injected drug in the form of microparticles prepared using polymer PEG-PLGA, a short-term increase in blood levels immediately after injection was reduced to 100 ng/ml or less, and within seven days from Owen in the blood was maintained at high levels.

Example 28

Fabrication of microparticles with the addition of salt in the liquid phase in stage (C)

100 ál of 50 mg/ml solution of the polymer PEG-PLGA (5k-61k)/dimethylcarbonate was added 20 ml of tert-butanol, was added 20 μl of an aqueous solution containing 10 mg/ml FD40, and the mixture was stirred, getting solution of micelles with reversed phase (emulsion W/O). The resulting solution was pre-frozen with liquid nitrogen and subjected to drying by freezing during the night, using the device for freeze-drying, and obtained dry residue containing FD40. In the obtained dry residue containing FD40, was added 200 μl of dimethylcarbonate and was stirred for 10 seconds of vortex mixing device, receiving the suspension S/O, and it was added dropwise 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF) together with sodium chloride at given concentrations (0 M, 10 mm, 50 mm, 1 M)was stirred and emulsiable in the vortex mixing device, receiving emulsion type S/O/W From the solution obtained emulsion type S/O/W immiscible with water, the organic solvent was removed using an evaporator (rolled back to 30 hPa (30·102PA), and was pumped out and drove for 5 minutes), getting dispersed in water substance particulate matter containing FD40. Dispersed aqueous solution of microparticles containing FD40, pre Zamaraev what does liquid nitrogen and subjected to drying by freezing during the night, using the device for freeze-drying, and received the powder of microparticles containing FD40. The obtained particles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter, and at all concentrations of sodium chloride, the average diameter of the microparticles was 6.5 μm.

20 mg of the obtained powder of microparticles containing FD40, weighed and dispersible in 1 ml of physiological phosphate buffer solution (containing 0.1% Pluronic F-68 (registered trademark of BASF), 0.1% bovine serum albumin and 0.01% sodium azide), and centrifuged (14000 rpm./minutes, 10 minutes). After collecting the supernatant liquid microparticles again suspended in 1 ml of physiological phosphate buffer solution and centrifuged, and the microparticles were purified twice more. Purified microparticles again suspended in 1 ml of physiological phosphate buffer solution was divided into 900 ál in a 1.5-ml Eppendorf tubes and were added to 900 μl of physiological phosphate buffer solution, the solution was incubated at 37°C and samples were collected after 24 hours. The collected samples were centrifuged for 10 minutes at 14000 rpm./minutes and FD40 contained in the supernatant liquid was measured using a fluorescence spectrophotometer (HORIBA, Fluoro MAX-3, the wavelength of the excitation 495 nm, the wavelength of fluorescence 520 nm), and the expected high is obedienoe number. The number FD40 the supernatant is collected during the cleanup, measured similarly, and the encapsulation efficiency was calculated from the loaded number of.

The encapsulation efficiency was 73%, 97%, 84% and 82% when the sodium chloride concentrations of 0 M, 10 mm, 50 mm and 1 M Released the amount in 1 day was 14%, 7%, 15% and 11% when the sodium chloride concentrations of 0 M, 10 mm, 50 mm and 1 M, and the concentration of sodium chloride 10 mm, the encapsulation efficiency was highest and the released amount in 1 day (initial peak) was the lowest.

Example 29

Subcutaneous administration mouse microparticles with encapsulated human growth hormone (hGH) (pharmacological activity)

25 mg of each polymer PEG-PLGA (5k-55k) and PEG-PLGA (5k-105k) of example 20 was dissolved in 500 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 100 μl of tert-butanol and was added dropwise to 250 ál of 10 mg/ml aqueous solution of hGH and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. Polucheniyami the residue was dispersible in 1 ml of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated hGH. The obtained microparticles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of the obtained particles was 4.2 μm in the microparticles of the polymer PEG-PLGA (5k-55k) (microparticles 5k-55k) and 7.5 μm in the microparticles of the PEG-PLGA (5k-105k) (microparticles 5k-105k).

300 mg of microparticles of each type, prepared above, suspended and dispersible in 3 ml of physiological phosphate buffer solution (PBS) and particles besieged by centrifugal separation for 5 minutes at 80×g and the supernatant transferred to another tube. The supernatant was again divided centrifuged who eat for 5 minutes at 80×g, besieged the remaining particles and the supernatant was removed. The first sludge from sedimentation by centrifugation and the second sludge from sedimentation by centrifugation were combined and again dispersible in 1 ml of physiological solution with phosphate buffer, similarly conducted a third operation purification by centrifugation, and the growth hormone is not encapsulated in the microparticles were removed. Finally, the precipitate was again dispersible in 200 μl of physiological solution with phosphate buffer to obtain a solution for injection.

This solution was injected subcutaneously into the back of 8-week-old ICR mice with remote pituitary (Japan SLC), and blood samples were collected at certain time intervals from the tail vein. In the blood samples was added heparin to a final concentration of 3.3 int. units/ml, centrifuged for 5 minutes at 5000 rpm./minutes, plasma was collected and the concentration of growth hormone in the plasma concentration of mouse IGF-1 was measured by the method of enzyme-linked immunosorbent assay.

In order to compare the solution in the form of the protein human growth hormone (700 μg/0.2 ml) was subcutaneously injected mice, and similarly selected samples.

In order to suppress the production of antibodies due to the introduction of human growth hormone, which is a foreign protein for mouse, three days before the introduction of the particles were injected subcutaneously, immunosu the springs Tacrolimus hydrate (Astellas) 26 µg/mouse and then 13 μg/mouse during the administration of medicines and twice a week later.

Fig shows the dynamics of changes in the concentration of human growth hormone in plasma. In mice, which was introduced in the form of drug level in the blood within 1 hour after administration was very high, and then sharply decreased to the level before the introduction of in one day. On the other hand, in mice, which were injected drug in the form of microparticles prepared using polymer PEG-PLGA, a short-term increase blood concentration immediately after injection was suppressed to a low level, approximately 1/100 of the level in mice, which was introduced in the form of drug, and for more than nine consecutive days after the introduction of the level in the blood was maintained at high levels.

Fig shows the concentration of IGF-1 in plasma during this period of time. The concentration of IGF-1 in plasma increased after the introduction as in the case of 5k-55k microparticles, and 5k-105k microparticles, and high levels were maintained for 7 days for 5k-55k microparticles and for more than 14 days for 5k-105k microparticles.

Example 30

Subcutaneous administration mouse microparticles with encapsulated Basis 4 (agonist GLP-1 receptor)

25 mg of polymer PEG-PLGA (5k-61k) in example 20 was dissolved in 500 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml In the solution of polymer d is balali 100 μl of tert-butanol and was added dropwise to 250 ál of 10 mg/ml aqueous solution of Basis 4 (synthesized on behalf of the company Sigma Genosys) and stirred vortex mixing device, receiving emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersible in 1 ml of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 10 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated Basis 4. The obtained microparticles were observed by scanning electron microscope (SEM: HITACHI, S-4800), to calculate the average particle diameter and the average diameter of microparticles rate of 6.0 microns.

300 mg of the prepared microparticles suspended and dispersible in 3 ml of phosphate physical and the logical buffer solution (PBS), particles precipitated with centrifugation for 5 minutes at 80×g and the supernatant transferred to another tube. The supernatant was again centrifuged for 5 minutes at 80×g and besieged the remaining particles, and the supernatant was removed. The first precipitate from the centrifugation and the second precipitate from the centrifugation were combined and again dispersible in 1 ml of physiological solution with phosphate buffer, and similarly conducted a third operation centrifugation, and on the Basis of 4, not encapsulated in the particles were removed. Finally, the precipitate was again dispersible in 200 μl of physiological solution with phosphate buffer, receiving a solution for injection.

This solution was administered subcutaneously at two positions in the back of a 10-week-old male Balb/C mice (Japan SLC), and blood samples were collected at certain time intervals from the tail vein. In the blood samples was added heparin to a final concentration of 3.3 int. units/ml and the plasma was collected by centrifugal separation for 5 minutes at 5000 rpm./minutes, and the concentration of growth hormone in plasma was measured by the method of enzyme-linked immunosorbent assay.

In order to compare the solution in the form of Basis 4 (700 μg/0.2 ml) was subcutaneously injected mice, and similarly selected samples.

In order to suppress the production of antibodies due to the introduction of Basis 4,which is a foreign protein for mouse three days before the introduction of the particles were injected subcutaneously immunosuppressive drug Tacrolimus hydrate (Astellas) 26 µg/mouse and then 13 μg/mouse during the administration of medicines, and twice a week later.

Fig shows the dynamics of concentration changes of Basis-4 in the plasma. In mice, which was introduced in the form of drug level in the blood within 1 hour after administration was very high, and then sharply decreased to the level before the introduction during the day. On the other hand, in mice, which were injected drug in the form of microparticles prepared using polymer PEG-PLGA, a short-term increase in blood levels immediately after injection was suppressed to less than about 1/100, and the blood level is maintained high during the month.

Example 31

Obtaining microparticles with encapsulated dextran labeled with fluorescein (FD40), differing in the particle diameter

5 mg of the polymer PEG-PLGA (5k-55k) in example 20 was dissolved in 100 μl of dimethylcarbonate, obtaining a polymer solution with a concentration of 50 mg/ml of this polymer solution was added 20 ml of tert-butanol and was added dropwise 20 ml of 1 mg/ml aqueous solution FD40 and mixed vortex mixing device, receiving the emulsion with reversed phase. This emulsion with reversed phase pre-frozen with liquid nitrogen and subjected to drying is marajuana, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours. The obtained dry residue was dispersively 50 ál, 200 ál and 500 ál of dimethylcarbonate, getting the suspension S/O. This suspension S/O was added dropwise to 2 ml of an aqueous solution containing 10% Pluronic F-68 (registered trademark of BASF), and was mixed and was emulsiable in the vortex mixing device, receiving emulsion type S/O/W Of the emulsion type S/O/W / dryer in the liquid were removed water-immiscible organic solvent and obtained liquid dispersion of microparticles. Liquid dispersion of microparticles was pre-frozen with liquid nitrogen and subjected to drying by freezing, using the device for freeze-drying (EYELA, FREEZE DRYER FD-1000) at a temperature of the cooling trap -45°C and a vacuum of 20 PA for 24 hours, and received the powder of microparticles with encapsulated FD40. The obtained microparticles were observed by scanning electron microscope (SEM: HITACHI, S-4800) and calculated the average particle diameter.

Fig shows the correlation between the average particle diameter and the number of dimethylcarbonate added during preparation of the emulsion type S/O/W. In the range from 50 μl to 500 μl, with the increase in the number of dimethylcarbonate observed decrease in the average diameter of the particles.

APPLY THE FEATURE INDUSTRY

The microparticle according to the invention releases a hydrophilic active substance at a suitable speed in the human body and apply as DDS pharmaceutical drug.

1. The microparticle comprising the agglomerate particles containing hydrophilic active substance, the particle comprises an amphiphilic polymer comprising a hydrophobic segment of PHAs and the hydrophilic segment of the polysaccharide or polyethylene glycol, and a hydrophilic active substance.

2. The microparticle according to claim 1, where the particle containing hydrophilic active substance, has a hydrophilic segment of amphiphilic polymer in the inner part and has an outer layer of hydrophobic segment of amphiphilic polymer.

3. The microparticle according to claim 1 or 2, where the amphiphilic polymer is an amphiphilic polymer graft type, consisting of a main chain of the polysaccharide and grafted(s) chain(s) of the PHA.

4. The microparticle according to claim 3, where the main chain of the polysaccharide is a dextran.

5. The microparticle according to claim 1 or 2, where the amphiphilic polymer is a block polymer consisting of a polyethylene glycol and PHAs.

6. The microparticle according to claim 5, where the average molecular weight of polyethylene glycol is from 2,000 to 15,000.

7. The microparticle according to claim 5, where the ratio of the average molecular weight of PHAs to Wed the days of the molecular weight of the polyethylene glycol is 4 or more.

8. The microparticle according to claim 1 or 2, where polyhydroxybutyrate is a copolymer of lactic acid and glycolic acid.

9. The microparticle according to claim 1 or 2, where the average particle diameter is from 1 to 50 microns.

10. The microparticle according to claim 1 or 2, where the hydrophilic active substance is a peptide or a protein.

11. A method of making microparticles according to any one of claims 1 to 10, including:
(a) a step for emulsion with reversed phase mixing an aqueous solvent containing hydrophilic active substance, and is not miscible with water, an organic solvent, dissolving amphiphilic polymer comprising a hydrophobic segment of PHAs and the hydrophilic segment of the polysaccharide or poly (ethylene glycol),
(b) a step for solid residue containing hydrophilic active substance, by removing the solvent from the emulsion with reversed phase and
(c) the stage of introduction of solids or liquid dispersion containing solid residue in the liquid phase containing surface modifier.

12. The method according to claim 11, in which the solvent is removed from the emulsion with reversed phase method of freeze-drying.

13. The method according to claim 11, in which the dispersion medium liquid dispersion containing solid residue, is a solvent capable of dissolving polyhydroxybutyrate and ladydi ability to dissolve hydrophilic segment for the preparation of amphiphilic polymer, equal to 10 mg/ml or less.

14. The method according to claim 11, in which the liquid phase is an aqueous solvent or miscible with water, an organic solvent.



 

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2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to tetrahydroimidazo[1,5-a]pyrazine derivatives of formula I or to their pharmaceutically acceptable salts (I), wherein: Ar represents phenyl, wherein phenyl is additionally substituted by 1-3 substitutes independently specified in halogen; R1 represents trifluoromethyl; R2 is specified in a group consisting of hydrohyl, alkyl having 1 to 4 carbon atoms, alkoxyl having having 1 to 4 carbon atoms, cycloalkyl representing a 5-6-member monocyclic ring group consisting of carbon completely, and -NR4R5, wherein each alkoxyl is optionally substituted by one group specified in a group consisting of phenyl and -OC(O)OR8; R3 is specified in a group consisting of a hydrogen atom and alkyl having 1 to 4 carbon atoms; each of R4 and R5 is independently specified is a group consisting of a hydrogen atom, alkyl having 1 to 4 carbon atoms, cycloalkyl representing a 3-8-member monocyclic ring group consisting of carbon completely, phenyl and pyridinyl, wherein each alkyl or phenyl is optionally substituted by one or more group specified in a group consisting of halogen, a cyano group, -SO2R7, -NR4R5 and -C(=O)OCH3; or R4 and R5 together with an atom, whereto attached form a 5-6-member heterocycle wherein the 5-6-member heterocycle optionally contains one or more N, O or S atom, and each 5-6-member heterocycle is optionally substituted by one or more groups consisting of halogen, hydroxyl, an amino group, alkyl having 1 to 4 carbon atoms, hydroxyalkyl 1 to 4 carbon atoms, -SO2R7, -C(O)NR4R5, -C(O)R7, =O; R7 represents alkyl 1 to 4 carbon atoms; and R8 is specified in a group consisting of alkyl 1 to 4 carbon atoms, and cycloalkyl representing a 5-6-member monocyclic ring group consisting of carbon completely. The invention also refers to methods for preparing them, a pharmaceutical composition having dipeptidyl peptidase IV inhibitory activity and containing said derivatives.

EFFECT: there are produced new compounds and composition on their basis which can find application in medicine for treating type 2 diabetes mellitus or hyperglycemia.

17 cl

FIELD: medicine, pharmaceutics.

SUBSTANCE: pharmaceutical composition contains as a first active agent, 6β, 7β; 15β, 16β-dimethylenene-oxo-17α-pregn-4-ene-21,17-carbolactone (drospirenone) in an amount according to a daily dose after the administration of the composition and making approximately 2 to approximately 4 mg, and as a second active agent, 17a-ethinylestradiol (ethinylestradiol) in an amount according to a daily dose and making approximately 0.01 mg to approximately 0.05 mg together with one or more pharmaceutically acceptable carriers or additives. The composition contains drospirenone applied on inert carrier particles. A method for preparing a pharmaceutical composition involves spraying of the drospirenone and ethinylestradiol solution on the inert carrier particles. The pharmaceutical preparation according to the invention contains a number of separately packed and individually taken daily dosage units of the described compositions in a single package used for oral administration for at least 21 days running with said daily dosage units containing the combination of drospirenone and ethinylestradiol. The composition may additionally contain 7 and less daily dosage units containing no active agent, or containing ethinylestradiol only.

EFFECT: invention provides higher oral bioavailability of drospirenone.

20 cl, 5 dwg, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biochemistry. What is presented is a crystalline form I and III of cortexolone-17α-propionate. The crystalline form I is characterised by a powder X-ray pattern as presented on Fig. 1, and/or by differential scanning calorimetry as presented on Fig. 2, and/or by an IR spectrum as presented on Fig. 3. The crystalline form III is characterised by a powder X-ray pattern as presented on Fig. 7, 10 or 13, and/or by differential scanning calorimetry as presented on Fig. 8, 11 or 14, and/or by an IR spectrum as presented on Fig. 9, 12 or 15. There are also presented methods for preparing the crystalline form I and III of cortexolone-17α-propionate. The method for preparing the crystalline form I involves crystallisation of cortexolone-17α-propionate for tert-butylmethyl ester. The method for preparing the crystalline form III of cortexolone-17α-propionate involves crystallisation of cortexolone-17α-propionate from a solvent representing a mixture of dichloromethane/n-hexane or a mixture of acetone/n-hexane, or a mixture of ethanol/water.

EFFECT: these crystalline forms in combination with at least one physiologically acceptable excipient are used in pharmaceutical compositions for treating urogenital, endocrine skin and/or and cutaneous appendage pathologies, particularly for treating acne, seborrheic dermatitis, androgenetic alopecia, hirsutism, benign prostatic hyperplasia, prostate cancers, male contraception, polycystic ovarian syndrome, previous puberty syndrome and control of aggressive or aberrant sexual behaviour.

21 cl, 30 dwg, 3 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to combinations of peptides in each case with the same sequence length (SEQL) which can be prepared in a stable reproducible quality and quantity of a mixture (A) containing a number of x amino acid with protected acid groups or a number of z peptides with the acid groups protected by the protective groups and the activated amino groups, with the amino acids in the mixture (A) found in a specific molar ratio, and a mixture (B), containing a number of y amino acids with the amino groups protected by the protective groups, with a molar ratio of the amino acids of the mixture (B) being the same as the molar ratio of the amino acids of the mixture (A), and the number x=y, and x is a figure from 11 to 18.

EFFECT: new combinations of the peptides are presented.

13 cl, 2 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a new lipid compound of general formula , wherein n=0; R1 and R2 are identical or different, and may be specified in a group of substitutes consisting of a hydrogen atom, a C1-C7alkyl group, a halogen atom and a C1-C7alkoxy group; X represents COR3 or CH2OR4, wherein R3 is specified in a group consisting of hydrogen, hydroxy, C1-C7alkoxy and amino; and R4 is specified in a group consisting of hydrogen, C1-C7alkyl or C1-C7acyl, Y represents C9-C21 alkene with one or more double bonds in E- or Z-configurations with the chain Y being unsubstituted and containing a double bond in the ω-3 position; provided R1 and R2 cannot simultaneously represent a hydrogen atom.

EFFECT: invention refers to pharmaceutical compositions containing the lipid compounds which are used for treating and/or preventing the conditions related to high NFkB functions, treating and/or preventing an inflammatory disease or a condition, lower plasma insulin and/or blood glucose levels, treating insulin resistance, treating and/or preventing peripheral tissue insulin resistance and/or diabetic condition, eg type 2 diabetes mellitus.

45 cl, 1 tbl, 1 dwg, 31 ex

Organic compounds // 2489439

FIELD: biotechnologies.

SUBSTANCE: invention is related to a compound of a formula

, where R' is the below formula, and R" is hydrogen, in free form or in the form of a pharmaceutically acceptable acid-additive salt. Proposed compounds of the formula (IA) inhibit activity of DPP-IV (dipeptidylpeptidase-IV).

EFFECT: compounds are justified for application as medicinal agents designed for inhibition of DPP-IV and for treatment of conditions mediated by DPP-IV, such as achrestic diabetes.

11 cl, 2 ex, 2 dwg, 7 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmaceutics and medicine and concerns a pharmaceutical combination for treating or delaying the progression or onset of diabetes, containing a compound which is an SGLT2 inhibitor of formula

,

and at least one of an antidiabetic drug other than the SGLT2 inhibitor for treating obesity, and/or an anitihypertensive drug.

EFFECT: what is presented is a new pharmaceutical combination for treating or delaying the progression or onset of diabetes.

18 cl, 16 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: disclosed is (carboxylalkylenephenyl)phenyloxamides of formula , where R3 denotes H, F, Cl, Br, CF3, phenyl, COOH, COO-(C1-C6)-alkyl, (C1-C6)-alkyl, (C3-C8)-cycloalkyl, wherein the phenyl can be monosubstituted with: R12; R1, R5 independently denote H, F, Cl, Br, CF3, phenyl, COOH, COO-(C1-C6)-alkyl, (C1-C6)-alkyl; R2, R4 independently denote H, F, Cl, Br, CF3; R7, R8, independently denote H or (C1-C6)-alkyl; X denotes (C2-C3)-alkylene, which can be monosubstituted or multisubstituted with R11; m equals 0; R6 denotes OH, F, Cl, Br, CN, OCH3; OCF3, CH3, CF3, (C1-C6)-alkyl or O-(C1-C6)-alkyl, and the alkyl can be monosubstituted or multisubstituted with OH, F, Cl, Br or CN; R11 denotes (C2-C6)-alkynyl; R12 denotes (C1-C6)-alkyl; and physiologically acceptable salts thereof.

EFFECT: disclosed compounds have activity towards the GPR40 receptor and are suitable for preparing a medicinal agent for reducing blood sugar level, treating diabetes, increasing insulin release, treating diseases of the central nervous system, treating schizophrenia and Alzheimer's disease.

19 cl, 2 tbl, 35 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to compounds of formula (I) or pharmaceutically acceptable salts thereof wherein A, R1, R2, R3 and m are specified in the patent claim. The present invention also refers to the number of specific compounds, and to a pharmaceutical composition containing the above compounds effective for inhibition of kinases, such as glycogen synthase kinase 3 (GSK-3), Rho kinase (ROCK), Janus kinase (JAK), AKT, PAK4, PLK, CK2, KDR, MK2, JNK1, aurora, pim 1 and nek 2.

EFFECT: preparing the specific compounds and pharmaceutical composition containing the above compounds effective for kinase inhibition.

18 cl, 393 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present group of inventions refers to medicine, namely therapy and endocrinology, and concerns treating metabolic syndrome and diabetes. That is ensured by administering an effective amount of a composition comprising an activator of adenosine-5'-monophosphate-activated protein kinase (AMPK), and an anti-inflammatory agent having serotonergic activity.

EFFECT: administering the given composition provides the effective treatment of metabolic syndrome and diabetes by improving lipid metabolism due to inducing the enhanced oxidation of fatty acids by the ingredients of the composition.

29 cl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to ophthalmology and can be used for treatment of diabetic diffuse macular edema. For this purpose 0.5 ml of 1% dexason solution and 0.5 ml of emoxipin are successively introduced in injection into parabulbar space and magnetic stimulation of optic analyser is performed. Magnetic stimulation is performed by pulse alternating magnetic field with growing strength within 6-12 mT with 50 Hz frequency and 10 min duration. Course of treatment constitutes 10 sessions.

EFFECT: method ensures efficient non-traumatic treatment of the last stage of proliferative diabetic retinopathy.

24 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: described are new triazolopyrimidine derivatives of general formula (I): wherein R1 means hydrogen or alkyl; R2 means hydrogen; R3 means optionally substituted C3-8cycloalkyl, optionally substituted bridged monocyclic C6-cycloalkyl, optionally substituted phenyl, optionally substituted 6-member heterocyclyl containing one or two heteroatoms specified in O and N, etc., a pharmaceutical composition containing them, and using the compounds and composition for inhibiting the enzyme type 1 diacylglycerol-O-acyltransferase (DGAT) for treating type 2 diabetes mellitus, obesity, high plasma triglycerides, metabolic syndrome, etc.

EFFECT: there are presented new triazolopyrimidine derivatives.

11 cl, 73 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a selective thiazolidinedione analogue to be used in treating and preventing diabetes and dyslipidemia, representing 5-(4-(2-(3-methoxyphenyl)-2-oxoethoxy)benzyl)thiazolidine-2,4-dione or a pharmaceutically acceptable salt thereof. Also, the invention refers to a pharmaceutical composition for diabetes and dyslipidemia, containing an effective amount of 5-(4-(2-(3-methoxyphenyl)-2-oxoethoxy)benzyl)thiazolidine-2,4-dione or the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. .

EFFECT: what is produced is the selective thiazolidinedione analogue decreasing binding and activating of the PPARγ nuclear transcription factor.

4 cl, 2 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely a drug preparation for treating diabetes. A method for preparing the drug preparation for diabetes mellitus with the drug preparation containing extracts prepared of the following herbal raw materials: Radix Trichosanthis (trichosanthes root), Radix Bupleuri (thoroughwax root), Fructus Aurantii Immaturus (immature bitter orange fruit), Radix et Rhizoma Rhei (rhubarb root and rhizomes), Rhizoma Pinelliae (pinellia rhizomes), Radix Scutellariae (Baical skullcap root), Rhizoma Coptidis (coptis chinensis rhizomes), Radix Paeoniae Alba (white peony root) and Fructus Mume (wild plums) taken in certain proportions, and additionally may contain pharmaceutically acceptable adjuvants (versions). The drug preparation for diabetes mellitus.

EFFECT: drug preparation is effective for diabetes mellitus.

24 cl, 1 tbl, 36 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are presented: agent applicable for treating or preventing diseases involving the enzyme dipeptidyl peptidase-IV (DPP-IV) representing Limiglidol (the same 9-diethylaminoethyl-2,3-dihydroimidazo[1,2-a]benzimidazole dihydrochloride, the same diabenol), a pharmaceutical composition thereof for the same application, using Limiglidol for preparing the pharmaceutical composition for the same application and using Limiglidol for normalising blood incretins (e.g., GLP-1, GIP decomposed under action of DPP-IV). What is shown is a high selectivity of Limiglidol in relation of DPP-IV.

EFFECT: invention enables using Limiglidol for preventing, delaying progression and/or treating conditions the pathogenesis of which involves the enzyme DPP-IV, including sterility, polycystic ovary syndrome, growth disorder, asthenia, arthritis, allograft rejection, autoimmune diseases (sclerodermia and multiple sclerosis), a variety of immunomodulatory diseases (lupus erythematosus and psoriasis), AIDS, intestinal diseases (necrotic enteritis, microvilli involving diseases or abdominal diseases), intestinal inflammatory syndrome, atrophy, etc.

10 cl, 1 dwg, 2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and represents gel-forming mixed dextran esters containing phosphate and carbamate groups of general formula: {C6H7O2(OH)3-x-y{[(OP(O)ONa)mONa)]xl[(O2P(O)ONa)k]x2}x(OCONH2)y}n, wherein x=x1+x2 is a degree of substitution in phosphate groups (mono- and diesters), x=0.47-1.09; X1 is a degree of substitution in monoesters, X1=0.01-0.48; m is a number of phosphates in monoesters, m=1-2; x2 is a degree of substitution in diesters, x2=0.01-1.09; k is a number of phosphates in diesters, k=1-2; y is a degree of substitution in carbamate groups, y=0.39-1.23; n is a degree of polymerisation, 20≥n≤1000.

EFFECT: invention provides producing low-toxic low- and high-substituted dextran phosphates in the form of hydrogels containing additionally carbamate groups and possessing antiproliferative activity with respect to cancer cells.

2 cl, 3 dwg, 14 ex

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