Method for obtaining insulin out of natural source and insulin

FIELD: medicine.

SUBSTANCE: the present innovation deals with insulin preparations applied in therapy of diabetes mellitus. This innovation could be applied in medicinal industry for insulin manufacturing. To obtain insulin one should apply reindeer's pancreas to be homogenized in solution of hydrochloric acid ethanol followed by extraction with subsequent clarification of solution and obtaining the supernatant which then should undergo ion-exchange chromatography and isoelectric deposition by obtaining insulin. The latter should be purified due to high-performance reversed-phase liquid chromatography. Insulin obtained is competitive for the bond with insulin receptor at concentration of above 100 ng/ml due to causing high increase of receptor binding, moreover, it has higher hydrophoby against standard insulins, thus, it has certain differences in the structure of its molecule.

EFFECT: higher efficiency of insulin manufacturing.

2 cl, 2 dwg, 2 ex

 

The present invention relates to medicine, in particular the production used in the treatment of diabetes insulin preparations. The invention can be used in the medical industry for the manufacture of insulin.

Currently, for the treatment of diabetes is a wide range of insulin preparations derived from natural sources - islets of the pancreas of cattle and pigs. Bovine and porcine insulins, which are most similar to human insulin in structure and amino acid sequence are in the human body activity, comparable to human insulin. Bovine insulin differs in amino acids in three positions, so he has high immunogenicity and currently rarely used. Molecule of porcine insulin, which is produced by extraction from the pancreas of pigs (Great medical encyclopedia, article “Insulin”), differs from human insulin by only one amino acid in the chain (instead of a threonine in the 30 position is alanine) and thanks to modern methods of cleaning are used extensively. However, after long-term use in the body begin to build up antibodies to bovine insulin, thereby nullifying its effect. In addition, the fastest is acting insulin reaches after injection of its maximum activity over a sufficiently long period of time: from 2 to 8 hours.

But due to the increase in the incidence of diabetes mellitus material of the substrate, the pancreas of a pig) soon will be missed. Therefore, despite the undoubted quality of multicomponent pork insulins, which have been successfully used in the future should strive to move to alternative sources of insulin.

The technical result of the present invention is to expand the Arsenal of natural sources to generate insulin, capable in a dose-dependent manner to compete for the communication with the insulin receptor in a concentration above 100 ng/ml to cause a sharp increase in receptor binding, which are higher than the standard insulin, hydrophobicity, and hence some differences in the structure of its molecules.

This technical result is achieved in that in the method of producing insulin by separating it from the pancreas natural source according to the invention using the pancreas of reindeer, which homogenized in a solution of hydrochloric acid ethanol, the extraction is carried out with subsequent bleaching solution and obtaining the supernatant, which is subjected to ion-exchange chromatography and isoelectric precipitation with obtaining insulin, treatment which is carried out by high-performance obetovannoi zhidkosti the th chromatography.

Further, the invention is illustrated specific embodiments thereof and the accompanying drawings, in which according to the invention:

Figure 1 - depicts ortofoto liquid chromatography of a mixture of standard insulin bull, pig and pancreatic insulin reindeer;

Figure 2 - curves of competitive exclusion labeled125I insulin pancreas reindeer associated with homologous receptor plasma membranes of rat liver.

The proposed method of producing insulin as follows.

Pancreas reindeer, containing, along with the endocrine tissue of the large number of fatty layers, homogenized in a solution of hydrochloric acid pattern in the ratio of 1:15. The extraction is performed on suchtelen-apparatus for 2 hours at room temperature, after which the solution is lighten by centrifugation to obtain supernatant (70, 3000 rpm, 60 min, 4°).

Ion-exchange chromatography

The supernatant of the pancreas is subjected to ion-exchange chromatography. For this purpose, the supernatant applied to a column (5×50 cm)filled microporous sulfonic cation exchanger KU-23 in H+form. Subsequent cleaning steps consist of degreasing protein-Katina 70% ethanol, washing from the ballast proteins (0.5 M solution of acetic AMM is tion, pH 5.0), elution of insulin (or 0.2 N solution of ammonium buffer, pH 9,4). Control of the composition of the eluates carried out on a spectrophotometer at a wavelength of 280 nm. The protein peak detected in the same conditions as the standard insulin, collect and after reduction to pH 2.3 spend his rechromatography.

Isoelectric precipitation

Accompanying the main substance of the proteins precipitated by successive casts obtained after ion-exchange chromatography of the eluate to pH 2.0 and after adding acetone (20% by volume solution) to pH 4.0. The mixture is left for 1 hour at +20°and over night at +4°C. the Solution is centrifuged (sediment freeze). To supernatu add 10%Zn acetate solution (2% by volume solution) and adjusted pH to 5.95. The solution is kept at +20°C for one hour, then placed for 72 hours in the refrigerator. The precipitate was separated by centrifugation (23, 4000 rpm or K-70, 3000 rpm, 60 min, 4°).

Highly ortofena liquid chromatography (HPLC)

HPLC purification obtained from the pancreas of reindeer insulin perform using the system Knauer (Germany) column Diaster From 18.5 microns×4,0×250 mm (ELZIKO”, Russia), using buffer mixtures. The rate of elution 1 ml/min the Optical density of the eluate register at 226 nm. Rechromatography significant peaks is carried out in the analogues of the different fractionation conditions.

Radioligand studies

The ability of isolated from the pancreas of reindeer insulin to contact the insulin receptor check in specific hormone radioreceptor system (Rusakov et al, “Isolation and characterization of insulin in Russian sturgeon (Acipenser guldenstaedti)” J.Peptide Res. 1998. v.51. p.395-400). It includes labeled insulin, plasma membrane of the liver and standard unlabeled hormone. The analysis consists of constructing curves of competitive exclusion of pork125I insulin associated plazmaticheskimi membranes, unlabeled porcine insulin, pancreatic insulin reindeer.

Getting labeled hormone

In radioreceptor test system insulin use plasma membranes of rat liver, isolated by the method Neville (enriched membrane receptors 15-20 times).

Purification of pancreatic insulin reindeer (test preparation) carried out as follows. Sequential extraction procedure, ionoobmennoi chromatography, isoelectric precipitation allow to obtain a coarse fraction of insulin, which after lyophilization fractionary using HPLC. System buffers gives the opportunity to spend one-step fractionation of pancreatic insulin.

In the same test preparation conditions were obtained reference chromatogram CME and commercial insulin bull (peak 1) and pigs (pic No. 2), the elution profiles for which is presented in figa.

The elution profile for pancreatic insulin reindeer presented on figb (peak No. 3).

The chromatogram of the test preparation allows us to analyze the physico-chemical properties. As can be seen from figure 1 (B), the resolution of the used HPLC system allows for the separation of insulin, the structure of which differs by two amino acids, it is possible to speak about the high homogeneity of the resulting peptides.

The value of the mobility (Rf), significant fractions of the test drug was calculated relative to the standard insulin bovine and swine, are given in the table.

Table
The mobility of the individual fractions of the tested drugs
 Insulin reindeer
 The retention time on the column (min)Rf
Insulin bull9.28 are1
Insulin pigs12,200,76
Peak No. 110,810,86
Peak No. 2--
Peak No. 314,230,65
Peak No. 19,730,47

As can be seen from the table, the comparison of the retention time on the column confirmed the lack of similarity of pancreatic insulin reindeer as with standard insulin cattle (bull), and hormone pigs. Insulin deer (major fraction) is higher than the standard insulin, hydrophobicity, and hence some differences in the structure of its molecules.

Rough drug pancreatico insulin reindeer were evaluated in vivo for the ability to induce hypoglycemic effect.

The experimental group of rats received intraperitoneal injections of 50 or 100 μg of insulin deer dissolved in 1.5 ml of physiological solution. After 20 min after injection at a dose of 50 µg infant rats observed violation of coordination of movements, and at a dose of 100 μg of active seizures, which was filmed by glucose. Convulsions in rats in response to the injection of Zn-insulin reindeer in vivo and subsequent withdrawal of the drug action by intraperitoneal glucose unequivocally testify to its hypoglycemic effect. This method is a standard rapid test used for determining the biological activity of any insulin.

The ability of the individual fractions of insulin deer vzaimode ystavat with insulin receptor in plasma membranes of rat liver illustrated in figure 2.

As can be seen from figure 2, two fractions of insulin deer (peak No. 1-2 and peak No. 3 can, in a dose-dependent manner to displace associated with the receptor labeled hormone. Moreover, in the concentration range of peptide peak No. 3 from 0.1 to 100 ng/ml curves of displacement standard insulin and test fraction No. 3 is virtually the same. When the concentration of the peptides of more than 100 ng/ml was observed a sharp increase in binding of labeled hormone.

The results of radioligand analysis has shown that some peptides insulin reindeer capable differently, but in a dose-dependent manner to compete for the communication with the insulin receptor in a concentration above 100 ng/ml caused a sharp increase in receptor binding. This fact indicates the presence of all the investigated peptide fractions factor stimulating the binding of the hormone with insulin receptor.

None of the known vertebrate insulins such activating the ability is not.

Following are specific examples of how to perform the claimed method of producing insulin.

Example 1.

Prepreparatory way to get insulin from the pancreas of reindeer (Rangifer tarandus)

stage 1. Ion-exchange chromatography hydrochloric acid extracts by cation-exchanger KU-23 N'-form

1.1. The pancreas. Before treatment e is stipriaan the pancreatic tissue reindeer (1300 g) keep frozen

1.2. Homogenization. A solution of hydrochloric acid ethanol (96%ethanol: distilled water: 12 N hydrochloric acid (ratio 880:220:17). The pancreas is crushed and homogenized using a knife homogenizer in a solution of hydrochloric ethanol. The ratio of tissue : hydrochloric acid ethanol 1:15. Temperature 0-4°C.

1.3. The extraction. The obtained homogenate was shaken for shuttel-apparatus for 2 hours at a temperature of 20-25°C.

1.4. Centrifugation. The extract obtained lighten by centrifugation (4500g 4°C for 60 minutes). Centrifuge To-70.

1.5. Chromatography:

Equipment:

Glass column: 5×50 see the Amount of resin 900 cm3.

The spectrophotometer SF-26 (or detector 280 nm flow-through cuvette).

Reagents:

Ion exchange resin: Sulfonation KU-23 to N+form.

Buffer: a solution of hydrochloric acid ethanol (96%ethanol: distilled water: 12 N hydrochloric acid (ratio 880:220:17).

Buffer B: 70%ethanol.

Buffer: 0.5 M solution of ammonium acetate. pH 5.0.

Buffer D: 0.2 M solution of ammonium chloride. pH 9.4.

Application washing elution. 19.5 l clarified by centrifugation of the extract (pH 2.3) is applied to the column. The rate of application of 100 ml/h/cm2. After degreasing 70%ethanol (2 l) complex proteins-cation spend elution ballast proteins dissolve 0.5 M is ω ammonium acetate. pH 5.0, the volume of 15 L. the Elution of insulin is carried out using 0.2 M solution of ammonium chloride. pH 9.4 volume of 1.0 L. the Composition of the Eluate control spectrophotometrically at 280 nm and cause the pH to 2.3, followed by his rahmatullahi (see paragraph 1.1.-1.5.).

stage 2. Isoelectric precipitation, obtaining a complex of zinc-insulin reindeer.

Equipment: pH meter.

Centrifuge To-70. K-23.

Reagents: 10%solution of Zn acetate.

6 N hydrochloric acid.

12% ammonia solution.

The acetone.

2.1. Exemption from ballast proteins.

Obtained after ion-exchange chromatography, the eluate containing insulin, bring the pH to 2.0 (6 N hydrochloric acid), turbidity (usually within 30-60 min) solution to lighten the day at 4°C. the Solution is centrifuged at 3000 g. Within 30 minutes Centrifuge To-70. The residue is diluted 0.6% acetic acid and lyophilizers: nadeshiko add acetone (205 volume/volume). Lead to pH 4.0 with a solution of 12% ammonia and leave for 1-3 days at 4°C. Insulinotherapy the solution is centrifuged at 3000 g. Within 30 minutes Centrifuge To-70. In future work use adosados.

2.2 Getting rough Zn-insulin reindeer. Adosados quickly bring the pH to 7.0 12% solution of NH4OH then stepped acidified with 6 N Hcl to a pH of 5.95. While portions add 10% solution of zinc acetate Zn(CH3Soo)22% of the volume of adosado. Actively stirring the contents of the vessel with a glass rod for 1-1 .5 h at room temperature (20-25°). The solution is kept for 2 days in the refrigerator at 4°C, then centrifuged (Centrifuge K-23, 4000 g, 60 min, 4° (C) further use of precipitated Zn-insulin.

stage 3. Obtaining a crystal of the drug is insulin reindeer.

Equipment: pH meter.

Centrifuge To-23.

Reagents: 0.1 N citric acid

10%solution of Zn acetate.

6 N hydrochloric acid.

12% ammonia solution.

Acetone

3.1. Crystallization of insulin. The precipitate of Zn-insulin was dissolved in 25 ml of 0.1 N citric acid and 5 ml of acetone 12%ammonia solution was adjusted pH to 7.0, then stepped lead to 5.95 solution of 6 N Hcl and adding at each step a 5%zinc chloride (ZnCl2) (total=2% volume/volume). The process takes 1-1 .5 hours, during which a glass rod is conducted rubbing the walls of the vessel. Thus the initial crystallization process. A drop of the solution onto a glass slide under the microscope control the beginning of the formation of crystals. Then the solution is kept at 203 days in the refrigerator at 4°C.

3.2. Sedimentation, dewatering and drying of the drug insulin reindeer.

Suspended sediment insulin crystals centrifuged at 3000 g. Within 30 minutes Adosados obrazy is up. To the precipitate poured 1-2 ml of chilled acetone or ethanol (-18 -25°C) and transferred into a polypropylene tube (insider). The suspension is shaken, then centrifuged for 2-3 min at 3000 g. Adosados removed and the procedure of dehydration drug repeat 3-5 times. The wet precipitate is dried in a thermostat (or a vacuum chamber) at 25-30°within a few hours, determining the readiness of the drug received in descending order of weight tubes with crystalline insulin.

stage 4. Analysis of biological activity of the drug insulin reindeer.

4.1. Biotesting in vitro. Pancreatic insulin reindeer tested for the ability to bind to the insulin receptor specific for that hormone radioreceptor system that includes labeled125I insulin (160-180 µci/µg). Plasma membrane of the liver (0.4 mg/ml) and standard unlabeled insulin pigs (0.1-10000 kg/ml).

4.2. Biotesting in vitro. The purified preparation of pancreatic insulin reindeer tested for ability to cause hypoglycemic effect in vitro. Rats (weighing 60-100 g) receive intraperitoneal injections of 50 or 100 mcg of pancreatic insulin reindeer. Dissolved in 1.0 ml of saline. Rats from the control group receive the same volume of physiological solution without pancreati the definition of insulin reindeer. 20 minutes after drug injection in a dose of 50 mg in rats noted poor coordination of movement, but at the dose of 100 µg active seizures, which are removed by intraperitoneal injection of glucose solution. Convulsions in rats in response to the injection of Zn-insulin reindeer in vitro and subsequent withdrawal of action of the drug after administration of glucose, unequivocally, the hypoglycemic effect of pancreatic insulin reindeer. This method is a standard rapid test used for determining the biological activity of any insulin.

Example 2.

Analytical and preparative method of obtaining insulin from the pancreas of reindeer (Rangifer tarandus)

stage 1. Ion-exchange chromatography hydrochloric acid extracts by cation-exchanger KU-23 in H+form

1.1. The pancreas. Before treatment ekstrudirovannyy the pancreatic tissue reindeer (1300 g) keep frozen

1.2. Homogenization. A solution of hydrochloric acid ethanol (96%ethanol: distilled water: 12 N hydrochloric acid (ratio 880:220:17). The pancreas is crushed and homogenized using a knife homogenizer in a solution of hydrochloric ethanol. The ratio of tissue:hydrochloric acid ethanol 1:15. Temperature 0-4°C.

1.3. The extraction. Obtained Homo is enat shake on shuttel-apparatus for 2 hours at a temperature of 20-25° C.

1.4. Centrifugation. The extract obtained lighten by centrifugation (4000g 4°C for 60 min). Centrifuge To-70.

1.5. Ionoobmennaya chromatography:

Equipment:

Glass column: 5×50 see the Amount of resin 900 cm3.

The spectrophotometer SF-26.

Reagents:

Ion exchange resin: Sulfonation KU-23 to N+form.

Buffer: a solution of hydrochloric acid ethanol (96%ethanol:distilled water:12 N hydrochloric acid (ratio 880:220:17).

Buffer: the 70%ethanol.

Buffer: 0.5 M solution of ammonium acetate. pH 5.0.

Buffer D: 0.2 M solution of ammonium chloride. pH 9.4.

Application washing elution. 19.5 l clarified by centrifugation of the extract (pH 2.3) is applied to the column. The rate of application of 100 ml/h/cm2. After degreasing 70%ethanol (2 l) complex proteins-cation spend elution ballast proteins 0.5 m solution of ammonium acetate. pH 5.0, the volume of 15 L. the Elution of insulin is carried out using 0.2 M solution of ammonium chloride. pH 9.4 volume of 1.0 L. the Composition of the eluate control spectrophotometrically at 280 nm and cause the pH to 2.3, followed by his rahmatullahi (see paragraph 1.1.-1.5.).

stage 2. Isoelectric precipitation, obtaining a complex of zinc-insulin reindeer.

Equipment: pH meter.

Centrifuge To-23.

Reagents: 10%solution of Zn acetate.

6 N hydrochloric the acid.

12% ammonia solution.

The acetone.

2.1. Exemption from ballast proteins.

Accompanying the main substance of the proteins precipitated by successive casts, protein eluate to pH 2.0 (6 N hydrochloric acid and after the addition of acetone (20% by volume solution) to pH 4.0 with a solution of 12% ammonia. Insulinotherapy the solution is kept for 1 h at 20°C, then overnight at 4°C. In the case of turbidity, or the occurrence of sediment ballast protein solution was centrifuged (5000 g. 4°C, for 60 min). Centrifuge To-23.

2.2 Deposition of Zn-insulin reindeer. To the supernatant, add 10%solution of zinc acetate (2% of the volume of the solution) and bring the pH up to 5.95. The solution is allowed to stand 1 h at 20°and for 72 h at 4°C. the Precipitate of Zn-insulin separated by centrifugation (Centrifuge To-23. 4000 g. 60 minutes 4°).

stage 3. Highly ortofena liquid chromatography drug. Zn-insulin reindeer.

Equipment: a System for high-performance liquid chromatography (Knauer).

Chromatographic column Diaster-110-C18, 5 µm. 4.6×250 mm (ELZIKO”, Russia).

Reagents: Triperoxonane acid.

Acetonitrile.

Bidistilled water.

3.1. Purification of the preparation of Zn-insulin reindeer. The drug is obtained from the pancreas of reindeer, cleaned by means of high-performance back ofasnoi liquid chromatography (HPLC) using system Knauer column on diasorb-110-C18, 5 μm, 4.6×250 mm (ELZIKO”, Russia). The elution of the sample is carried out at a rate of 1 ml/min in the gradient of buffer A - 0.1% triperoxonane acid, H2Oh, buffer B is 0.05% Triperoxonane acid in 84% acetonitrile (CH3JV). The optical density of the eluate register at 226 nm. The resulting preparation is subjected to repeated cleaning with rechromatography in similar conditions. After carrying out cleaning the content of insulin in the preparation of at least 97%.

stage 4. Analysis of biological activity of the drug insulin reindeer.

4.1. Biotesting in vitro. Pancreatic insulin reindeer tested for the ability to bind to the insulin receptor specific for that hormone radioreceptor system that includes labeled125I insulin (160-180 µci/µg). Plasma membrane of the liver (0.4 mg/ml) and standard unlabeled insulin pigs (0.1-10000 kg/ml).

4.2. Biotesting in vitro. The purified preparation of pancreatic insulin reindeer tested for ability to cause hypoglycemic effect in vitro. Rats (weighing 60-100 g) receive intraperitoneal injections of 50 or 100 mcg of pancreatic insulin reindeer, dissolved in 1.0 ml of saline. Rats from the control group receive the same volume of physiological solution without pancreatic insulin stand the aqueous deer. 20 minutes after drug injection in a dose of 50 mg in rats noted poor coordination of movement, but at the dose of 100 µg active seizures, which are removed by intraperitoneal injection of glucose solution. Convulsions in rats in response to the injection of Zn-insulin reindeer in vitro and subsequent withdrawal of action of the drug after administration of glucose, unequivocally, the hypoglycemic effect of pancreatic insulin reindeer. This method is a standard rapid test used for determining the biological activity of any insulin.

1. A method of producing insulin, characterized by the fact that the pancreas reindeer homogenized in a solution of hydrochloric acid in ethanol ratio of 1:15, extracted for 2 h at room temperature, clarify by centrifugation, the supernatant is subjected to ion exchange chromatography using microporous sulfonic cation exchanger K-23 to N+form, conduct isoelectric precipitation by successive casts obtained after ion-exchange chromatography of the eluate to pH 2.0 and after adding acetone in an amount of 20% of the volume of the solution to pH 4.0, while the mixture is left for 1 h at +20°and over night at +4°C, the solution is centrifuged, the supernatant, add 10%solution of Zn acetate in STA shall be 2% of the volume of the solution and adjusted the pH to 5.95, then the solution is kept at +20°C for one hour, then placed 72 hours in the refrigerator and the precipitate was separated by centrifugation, receive insulin, treatment which is carried out by high-performance obetovannoi liquid chromatography.

2. Insulin, characterized by the fact that he received according to claim 1 and capable of competing for communication with the insulin receptor at concentrations over 100 ng/ml.



 

Same patents:

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to new compound: N-[2-hydroxy-3(1-piperidinyl)-propoxy]-pyridine-1-oxyde-3-carboxyimidoyl chloride, stereoisomers thereof acid additional salts useful in treatment of pathological insulin resistance.

EFFECT: new compound useful in medicine.

5 cl, 10 tbl, 10 ex

FIELD: organic chemistry, medicine.

SUBSTANCE: invention relates to new 2-aminopyridine derivatives of formula I , wherein R1 is cyano, carboxyl or carbamoyl; R2 is hydrogen, hydroxyl, C1-C6-alkoxy or phenyl; R3 and R4 are aromatic hydrocarbon such as phenyl or naphthyl, 5-14-membered 5-14-membered optionally substituted aromatic group, excepted cases, when (1) R1 is cyano, R2 is hydrogen, and R3 and R4 are simultaneously phenyl;(2) R1 is cyano, R2 is hydrogen, R3 is 4-pyridyl, and R4 is 1-pyridyl; (3) R1 is cyano, R2 is 4-methylphenyl, and R3 and R4 are simultaneously phenyl;(4) R1 is cyano, R2, R3 and R4 are simultaneously phenyl, or salts thereof. Derivatives of present invention have adenosine receptor antagonist activity and are useful in medicine for treatment of irritable bowel syndrome, constipation, and defecation stimulation.

EFFECT: 2-aminopyridine derivatives as adenosine receptor antagonists useful in medicine.

34 cl, 2 tbl, 179 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to a form with reduced size of particles of the compound (S)-2-ethoxy-3-[4-(2-{4-methanesulfonyloxyphenyl}ethoxy)phenyl]propanoic acid described by the formula (I)

or its pharmaceutically acceptable salt or any solvate. Above said compound is useful in treatment of metabolic disorders, such as insulin resistance syndrome determined as reduced sensitivity to insulin effect. Also, invention relates to pharmaceutical compositions containing form with reduced size of particles of this compound or its pharmaceutically acceptable salt or their solvate as an active component, and to methods for preparing form with reduced size of particles of this compound or its pharmaceutically acceptable salt.

EFFECT: improved preparing methods, valuable medicinal properties of compound.

12 cl, 5 ex

FIELD: medicine, cardiology, endocrinology.

SUBSTANCE: invention proposes applying false flax plant oil as a hypoglycemic agent and agent that exerts the normalizing effect on lipid fraction of alpha-lipoproteins (high density lipoproteins; HDLP) and used in treatment of cardiovascular and endocrine diseases, and a method for it applying. This agent is known early as an antioxidant and a hypolipidemic preparation. Detection of new properties allows expanding application of this agent in clinics for treatment of patients with heart ischemic disease, stenocardia, hypertension and diabetes mellitus. The preparation reduces risk for development of atherosclerosis and allows significant reducing doses of basic drugs.

EFFECT: valuable medicinal properties of agent, enhanced effectiveness of treatment.

4 cl, 6 ex

FIELD: organic chemistry, biochemistry, medicine, endocrinology.

SUBSTANCE: invention relates to a trans-olefinic activator of glucokinase representing compound taken among the group consisting of olefinic amide of the formula (I): wherein R1 and R2 mean independently of one another hydrogen, halogen atom, nitro-group, perfluoro-(lower)-alkyl, (lower)-alkylsulfonyl or (lower)-alkylsulfonylmethyl; R means -(CH2)m-R3 or lower alkyl comprising from 2 to 4 carbon atoms; R3 means cycloalkyl comprising from 3 to 8 carbon atoms; R4 means the group: or unsubstituted, or monosubstituted five- or six-membered heteroaromatic ring linked by ring carbon atom with indicated amino-group wherein this five- or six-membered heteroaromatic ring comprises from 1 to 2 heteroatoms taken among the group consisting of sulfur or nitrogen atom wherein one heteroatom being as nitrogen atom is arranged near with binding ring carbon atom, and wherein indicated monosubstituted heteroaromatic ring is substituted at ring carbon atom not adjacent with mentioned binding carbon atom with a substitute taken among the group consisting of halogen atom and group of the formula: m = 0 or 1; n = 0, 1, 2, 3 or 4; R7 means hydrogen atom or lower alkyl; Δ means trans-configuration relatively to a double bond; or its pharmaceutically acceptable salt. Also, invention relates to pharmaceutical composition, method for prophylactic or therapeutic treatment of diabetes mellitus of type II and to methods for preparing compounds of the formula (I). Invention provides preparing activators of glucokinase that enhance insulin secretion in treatment of diabetes mellitus of type II.

EFFECT: valuable medicinal properties of compounds.

25 cl, 29 ex

FIELD: pharmaceutical agents, in particular glyburide containing composition.

SUBSTANCE: claimed composition contains 5-chloro-N-[2-[4-[[(cyclohexylamino) carbonyl]amino]sulfonyl]ethyl]-2-methoxybenzamide, known under generic name as glyburide, and has the next grain-size classification (%): undersize of 3-11 mum - 25; undersize of 6-23 mum - 50 %, and undersize of 15-46 mum - 75 %. Such grain-size classification affords the ability to increase glyburide dissolution rate and provide reproducible biological availability of glyburide.

EFFECT: pharmaceutical composition useful for treatment of II- type diabetes.

11 cl, 2 tbl, 6 ex

FIELD: organic chemistry, pharmacy.

SUBSTANCE: invention relates to new derivatives of benzimidazole represented by the following formula (I) or its salt:

wherein R1 represents (lower)-alkyl group; R2 represents aromatic (lower)-alkyl group that can be substituted with one or more groups taken among halogen atom, alkyl group, halogen-(lower)-alkyl group, nitro-group, aromatic group, aromatic (lower)-alkoxy-group, (lower)-cycloalkyloxy-(lower)-alkyl group, aromatic (lower)-alkyl group, aromatic (lower)-alkenyl group, aromatic (lower)-alkynyl group, aromatic oxy-(lower)-alkyl group, (lower)-cycloalkyl-(lower)-alkoxy-group, alkenyl group, (lower)-alkoxy-group, (lower)-alkylthio-group and (lower)-alkanesulfonylcarbamoyl group; R3 represents alkyl group, hydroxy-(lower)-alkyl group, alkenyl group, aromatic group, halogenated aromatic group, (lower)-alkyl aromatic group, (lower)-alkenyl aromatic group or aromatic (lower)-alkenyl group; -X- represents cross-linking group represented by one of the following formulas: (II) , (III) , (IV) , (V) . Also, invention relates to pharmaceutical compositions eliciting activity that reduces blood glucose level based on this compound. Invention provides preparing new compounds and pharmaceutical compositions based on thereof used for prophylaxis and treatment of damaged tolerance to glucose, diabetes mellitus, insulin-resistance syndrome, vascular failures syndrome, hyperlipidemia and cardiovascular disorders.

EFFECT: valuable medicinal properties of compounds and compositions.

16 cl, 1 tbl, 86 ex

The invention relates to new derivatives and analogs 3-arylpropionic acid having the General formula (I), and their stereo and optical isomers and racemates, as well as their pharmaceutically acceptable salts, wherein in the formula And is located in the meta - or para-position and represents a

where: R represents hydrogen;

-ORawhere Rarepresents hydrogen, alkyl, phenyl or alkylphenyl;

-NRaRbwhere Raand Rbare the same or different and represent hydrogen, alkyl, phenyl, alkylphenyl, cyano;

R1represents alkyl, cyano;

-ORewhere Rerepresents alkyl, phenyl or alkylphenyl;

-O-(CH2]m-ORfwhere Rfrepresents alkyl, and m is an integer of 1-2;

-SRdwhere Rdrepresents an alkyl or phenyl;

-SO2ORawhere Rarepresents alkyl, phenyl or alkylphenyl;

-COORdwhere Rdrepresents alkyl;

R2represents hydrogen or alkyl;

R3and R4

D is situated in the ortho-, meta - or para-position and represents a

-OSO2Rdwhere Rdrepresents alkyl, phenyl or alkylphenyl;

-OCONRfRawhere Rfand Rarepresent hydrogen, alkyl, phenyl or alkylphenyl;

-NRcCOORdwhere Rcrepresents hydrogen or alkyl and Rdrepresents alkyl, phenyl or alkylphenyl;

-NRcCORawhere Rcrepresents hydrogen or alkyl, and Rarepresents hydrogen, alkyl, phenyl or alkylphenyl;

-NRcRdwhere Rcand Rdrepresent hydrogen, alkyl, phenyl or alkylphenyl;

-NRcSO2Rdwhere Rcrepresents hydrogen or alkyl, and Rdrepresents alkyl, phenyl or alkylphenyl;

-NRcCONRaRkwhere Rcrepresents hydrogen, Raand Rkare the same or different and each represents hydrogen, alkyl, phenyl or alkylphenyl;

-NRcCSNRaRkwhere Rcrepresents hydrogen, Raand Rkare the same or different and each represents hydrogen, phenyl иLASS="ptx">-SRcwhere Rcrepresents alkyl, phenyl or alkylphenyl;

-SO2ORawhere Rarepresents alkyl, phenyl or alkylphenyl;

-CN;

-CONRcRawhere Rcrepresents hydrogen or alkyl, and Rarepresents hydrogen or alkyl;

D’ is located in the meta-position and represents-ORfwhere Rfrepresents alkyl; or is located in the ortho-, meta - or para-position and represents hydrogen;

D’ is located in the ortho - or para-position and represents-NO2, -ORfwhere Rfrepresents alkyl; or is located in the ortho-, meta - or para-position and represents hydrogen;

where specified, the alkyl means a straight or branched alkyl group having from 1 to 6 carbon atoms, or cyclic alkyl having from 3 to 6 carbon atoms, with the specified alkyl may be substituted by one or more than one group of alkyl, alkoxy, halogen or phenyl; where the specified phenyl may be substituted by one or more than one group of alkyl, alkoxy, nitro, thiol, or halogen; the invention also relates to a method of their production, pharmaceutical preparations containing them, the Sabbath.

The invention relates to organic chemistry, in particular to the compounds representing amide of the formula I:

in which * denotes an asymmetric carbon atom; R1and R2independently from each other represent a hydrogen atom or halogen, amino, hydroxyamino-, nitro-, cyano-, sulfamidihappo, (ness.)alkyl, -OR5, -C(O)OR5, PERFLUORO(ness.)alkyl, (ness.)alkylthio, PERFLUORO(ness.)alkylthio, (ness.)alkylsulfonyl, PERFLUORO(ness.)alkylsulfonyl or (ness.)alkylsulfonyl; R3denotes cycloalkyl containing from 3 to 7 carbon atoms, or (ness.)alkyl containing from 2 to 4 carbon atoms; R4means (O)other40or unsubstituted or monosubstituted five - or six-membered heteroaromatic ring bound ring carbon atom of the amino group, and a five - or six-membered heteroaromatic ring contains from 1 to 3 heteroatoms selected from sulfur atoms, oxygen, and nitrogen, with one heteroatom is a nitrogen atom, which is adjacent to the connecting ring carbon atom; this is monosubstituted heteroaromatic ring monogamist on the ring angle is found (ness.)alkyl, halo-, nitro-, cyano, -(CH2)n-OR6, -(CH2)n-C(O)OR7, -(CH2)n-C(O)OTHER6, -C(O)-C(O)OR8and -(CH2)n-OTHER6or its pharmaceutically acceptable salts

The invention relates to new pharmaceutical compositions that contain the basis or agonist of the basis for the treatment of diabetes, slowing of gastric emptying or reduce food intake, and their dosage forms and methods for their introduction

FIELD: medicine.

SUBSTANCE: method involves taking lavage fluid samples from injured bronchi in preoperative period in making fiber-optic bronchoscopy examination. Microflora colonizing bronchial mucous membrane and its sensitivity to antibiotics is determined. Therapeutic dose of appropriate antibiotic and therapeutic dose of immunomodulator agent like leykinferon is introduced in endolymphatic way 40-60 min before operation. Smears are taken from outlying bronchi in doing operation. Sputum or fluid in retained pleural cavity are taken in 1-2 days after the operation. Prophylaxis effectiveness is determined on basis of bacteriological study data. Therapeutic dose of antibiotics and leykinferon are introduced in 6-8 and 20-24 h after the operation in endolymphatic way. The preparations are introduced at the same doses in endolymphatic way making pauses depending on selected antibiotic elimination half-time once or twice a day until the drains are removed mostly during 48-72 h after operation.

EFFECT: enhanced effectiveness of antibacterial protection; high reliability of antibiotic prophylaxis.

The invention relates to biotechnology and is a way of expression of polypeptides in yeast by culturing a yeast strain that does not contain a functional marker gene for resistance to the antibiotic

The invention relates to medicine, in particular to endocrinology and immunology, and for the correction of the pathological autoimmune process in diabetes mellitus

The invention relates to medicine, more specifically to insulin analogs

The invention relates to medicine, namely to concentrated water preparative forms of insulin with high physical and chemical stability

The invention relates to water-soluble unit derived insulin, which remains In 24-B30 of the b-chain derived insulin is sequence Phe-x-X-x-X-x-X, where each X independently represents any amino acid or a deletion of at least one X is Nsubstituted by a lysine residue in which the Deputy is a 5--lithocholic acid or 5--lithocholic acid, connected through the-glutamyl,-glutamyl oror-aspartyl as a linker, where the unit size is defined in the gel-filtration system, more than aldolase, and the Assembly includes at least 2 zinc ions per 6 molecules derived insulin

The invention relates to pharmacology, medicine, namely, endocrinology, and relates to pharmaceutical compositions for the prevention and treatment of diabetes mellitus type I and II

The invention relates to technology and concerns a method for design validation and application associates with increased payload and a controlled degree of Association/dissociation

The invention relates to medicine, namely to the development of new dosage forms of the biologically active peptide compounds

The invention relates to the medical industry and relates to a derivative of insulin or its physiologically acceptable salt with accelerated compared with human insulin onset of action, in which the asparagine (Asn) at position B3 chain replaced by natural basic amino acid residue and at least one amino acid residue in positions B27, B28 or W chain replaced by other natural neutral or acidic amino acid residue, and asparagine (Asn) at position A21 chain And may be substituted for Asp, Gly, Ser, Thr or Ala, and the phenylalanine (Phe) in position B1 chain and the amino acid residue in position B30 chain may be missing

FIELD: medicine, veterinary science, veterinary pharmacy.

SUBSTANCE: velvet antlers are milled or milled waste after their pharmaceutical processing is used, mixed with artificial gastric juice in the ratio = 1:10, placed in thermostat at 37-40oC for 3 days, stirred one time per 24 h for 2-3 min and filtered. Then filtrate is disinfected by boiling, preserved with resorcinol, methylcellulose is added in the concentration 3 g/100 ml and stirred up to preparing the homogenous gel consistence. Method provides the wasteless in producing and enhancing profit. Gel prepared from non-ossified deer horns accelerates healing wounds, it doesn't seal skin pores and provides penetration of medicinal substances into deep layers of skin. Invention can be used for preparing gel from velvet antlers and their waste after pharmaceutical processing.

EFFECT: improved preparing method.

Up!