The method of obtaining insulinaemia polymer hydrogels

 

(57) Abstract:

The invention relates to the field of polymer chemistry, medicine and biochemistry, and in particular to method of obtaining insulinaemia polymer hydrogels.

The proposed method of producing insulinaemia polymer hydrogels by immobilization of insulin in the amount of crosslinked polymer modified inhibitor of proteolytic enzymes, is used as an inhibitor of proteolytic enzymes ovomucoid. The concentration of ovomucoid crosslinked polymer equal to 0.2-25 mg/g of swollen hydrogel in water.

Data insulinaemia polymer hydrogels can be used as a medicine in the treatment of patients with diabetes mellitus. 1 C.p. f-crystals.

The present invention relates to the field of polymer chemistry, medicine and biochemistry, and in particular to method of obtaining insulinaemia polymer hydrogels used as drugs in the treatment of patients with diabetes mellitus.

Insulin is a polypeptide hormone with a molecular weight of about 6000. It affects all types of metabolism in the body: it increases the penetration of glucose into the tissues of organic the intensity of protein synthesis, etc.

The main method of administering insulin in the human body are subcutaneous or intramuscular injection. Attempts to introduce insulin is the most physiological and convenient for patients by oral (through the mouth) have been unsuccessful, because insulin is easily hydrolyzed by digestive enzymes with loss of activity.

A method of obtaining insulinaemia polymers, including polymer hydrogels by immobilization of insulin on the polymer using cross-linking agents [1] as the polymer used dextran, starch, polyoxyethylene, polyvinylpyrrolidone, cross-linked collagen, proteins and their derivatives.

The disadvantage of this method is the low stability of the synthesized insulinaemia polymers to the action of enzymes of the digestive tract and, consequently, the impossibility of their oral administration.

A method of obtaining insulinaemia polymers insulin-gelatin capsule with subsequent immobilization in the amount of a copolymer of styrene with hydroxyethylmethacrylate made esasterawan derived from divinylbenzene [2] the oral application is cross-linked copolymer of microorganisms to the

The disadvantage of this method is the low stability of the synthesized polymers to the action of digestive enzymes, resulting in a low activity in penetrating the blood insulin. So, when administered orally synthesized cross-linked copolymer to rats in amounts of 1-40 mg (insulin) maximum reduction of glucose concentration in the blood of animals was on average 25% (384 mg/100 ml to 287 mg/100 ml) and was observed 3-4 hours after drug administration.

The closest (prototype) to the claimed technical essence is a method of obtaining insulinaemia polymer hydrogels by immobilization of insulin in the amount of crosslinked polymer modified inhibitor of proteolytic enzymes [3] as a cross-linked polymer used acrylic or methacrylic acid, crosslinked, triethyleneglycol(meth)acrylate, and as an inhibitor of proteolytic enzymes used Aprotinin pancreatic trypsin inhibitor.

The disadvantage of this method is the low stability of the synthesized polymer hydrogels to the action of digestive enzymes, resulting in a low activity in penetrating the blood insulin is m, the corresponding 50 units of insulin, the glucose concentration in the blood is not only not reduced, but increased from 380 to 460 mg/100 ml after 300 minutes after administration. Oral administration of unmodified insulinosoderzhaschego hydrogel (50 units of insulin) is accompanied by approximately 23% decrease in the concentration of glucose (310 240 mg/100 ml after 300 minutes). At the same time subcutaneous injection the rabbits just to 0.23 units of insulin leads to a decrease in the concentration of glucose in the blood from 330 to 120 mg/100 ml (74%) after 150 minutes after injection.

The task of the invention is to improve the stability insulinaemia polymer hydrogels to the action of proteolytic enzymes, providing the possibility of oral administration of the drug.

The solution of this problem is achieved in that in the method of obtaining insulinaemia polymer hydrogels by immobilization of insulin in the amount of crosslinked polymer modified inhibitor of proteolytic enzymes as an inhibitor of proteolytic enzymes use ovomucoid.

Cross-linked polymer-modified ovomucoid, obtained by chemical modification of polymer hydrogels by ovomucoid, the number of catorce [4]

Immobilization is carried out by immersing stitched modified polymer in an aqueous solution of insulin with a concentration of 0.01-5 mg/ml to full swelling of the polymer. The modified polymer is used in an amount of 0.01 to 1.0 per 1 ml solution of insulin.

Example 1. 0.1 g of pre-dried cross-linked polyacrylamide, modified by ovomucoid, placed in 1 ml of an aqueous solution of insulin (insulin activity equal to 25 u/mg) with a concentration of 1.0 mg/ml for 1 hour at room temperature. During this time the polymer is fully swells in water and is ready to use. The synthesized hydrogel is administered to rabbits orally by means of a catheter in an amount corresponding to 5 units of insulin per 1 kg of weight of animal. Blood samples taken after 30, 60, 90 and 120 minutes. The results are shown in the table.

Examples 2 to 10. The process is conducted according to example 1, using various crosslinked modified polymers, different concentrations of a solution of insulin, different numbers sewn modified polymer and different amounts of ovomucoid. The composition and properties of the resulting hydrogels are given in table.

Example 11 (control). The process is conducted according to example 1, using unmodified polinaut on white Wistar rats, weighing 180 g by injecting intraperitoneally of streptozotocin at the rate of 70 mg per 1 kg of body weight. The synthesized polymers begin to enter after the glucose level exceeds 400mg% the Results are shown in the table.

Example 14 (control). The process is conducted according to example 12, but animals injected subcutaneously equal to the amount of insulin in solution. The results are shown in the table.

Examples 15 to 17 (control). Rabbits orally administered solution of insulin in the amount of 7 u/kg of body weight of the animal, a mixture of ovomucoid (2.5 mg/kg) and insulin (8 u/kg) or a modified hydrogel (200 mg/kg of body weight). The results are shown in the table.

Comparative analysis of the prototype shows that the stated object different chemical nature of the used inhibitor of proteolytic enzymes, i.e. the claimed solution meets the criterion of "novelty". It also meets the criterion of "inventive step" as the ability of the polymeric hydrogel modified with ovomucoid, to prevent hydrolysis of insulin under the action of proteolytic enzymes of the digestive tract and to ensure penetration of insulin into the bloodstream when oral administration is an unknown function and first discovered in this invention. Limiting the number of ovomucoid NCLI and insulin is destroyed by proteolytic enzymes. Using ovomucoid in concentrations above 25 mg per 1 ml of gel is impractical because this amount is quite sufficient to protect therapeutic dose of insulin. Comparative analysis of examples of the preparation insulinaemia hydrogels according to the claimed method, the method prototype and test cases shows that the claimed method provides for obtaining hydrogels, insulin activity in which the oral application is on average 60-70% of the activity of the parent insulin at its hypodermic use. The possibility of oral administration of insulin is not only a great convenience for diabetics, but also allows to improve the treatment strategy for this disease.

1. The method of obtaining insulinaemia polymer hydrogels by immobilization of insulite in the amount of crosslinked polymer modified inhibitor of proteolytic enzymes, characterized in that as an inhibitor of proteolytic enzymes use ovomucoid.

2. The method according to p. 1, characterized in that the concentration of ovomucoid crosslinked polymer is equal to 0.2 to 25 mg/g of swollen hydrogel in water.

 

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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.

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

FIELD: pharmaceutical industry, medicine.

SUBSTANCE: invention relates to human insulin drug with activity of 100 IU/ml, including cartridge forms. Drug contains active ingredient, glycerol as isotonic agent, conserving agent and water, wherein it contains human insulin substance of high purity with residual proteolysis activity not more than 0.005 adsorption units, sodium chloride as additional isotonic agent, m-cresol as conserving agent, and additionally sodium dihydrogenphosphate dihydrate or disodium hydrogenphosphate heptahydrate as substance with buffer capacity and pH 6.9-7.8.

EFFECT: human insulin drug of short action with increased physiological activity and physical and chemical storage stability.

6 ex, 1 tbl

FIELD: pharmaceutical industry, medicine.

SUBSTANCE: invention relates to human insulin drug of durable action. Drug contains human insulin substance of high purity, protamine sulfate, zinc chloride, glycerol, m-cresol, phenol, sodium dihydrogenphosphate dihydrate or disodium hydrogenphosphate heptahydrate, sodium chloride, and water and has residual proteolysis activity not more than 0.005 adsorption units.

EFFECT: human insulin drug of durable action with increased physiological activity and physical and chemical storage stability.

4 ex, 1 tbl

FIELD: pharmaceutical industry, in particular high quality insulin drug of durable action.

SUBSTANCE: claimed method includes providing of human insulin ester by transpeptization of porcine insulin at molar excess of threonine di-tert-butyl ester in aqueous/organic medium in presence of trypsin; acidifying for reaction inhibiting; chromatography purifying of obtained human insulin; deblocking of protective groups with trifluotoacetic acid and purifying of obtained crude human insulin; with subsequent dissolution in diluted acid, addition of acid solutions of zinc ions and protamine sulfate, mixing with buffered solution of m-cresol, phenol and glycerol; and solution conditioning to form crystals. Weight ratio of trypsin and porcine insulin is 1:300-1000. Before acidifying of reaction media reaction is additionally inhibited by mixture dilution with water by 2-3 times. Human insulin ester is purified using HPLC followed by deposition of ester derivatives fractions in presence of zinc ions and protective group deblocking to produce crystals of crude human insulin, which is purified again by HPLC, wherein both purifying processes are carried out using sorbent DIASOGEL ODS (C18) with particle size of 15 mum and pore size of 120 A as stationary phase, and as mobile phase in the first step 0.06 M-glycine HCl buffer, containing 0.015 M of ammonium sulfate and 20-30 % of propan-2-ol, having pH 2.5 is used, and in the second one 0.05 M acetate buffer containing 10-20 % of propan-2-ol with pH 2.5 is used. Then purified human insulin crystals are step-by-step dissolved in diluted acid to obtain fine dispersed suspension of insulin crystals in water; then diluted acid is added thereto, and mixture obtained after blending with buffered solution of cresol, phenol and glycerol, is held at 18-21°C for 20-22 h.

EFFECT: effective and economical method for insulin production with increased yield and purity; insulin of durable action and low immunological properties.

12 ex

FIELD: medicine, endocrinology, chemical-pharmaceutical industry, hormones.

SUBSTANCE: method involves preparing human insulin ester by transpeptidation reaction of porcine insulin in the molar excess of threonine di-tert.-butyl ester in an aqueous-organic medium in the presence of trypsin, inhibition of reaction by acidification, purification of preparing human insulin ester by chromatography, removal of protecting groups with trifluoroacetic acid and purification of prepared human crude insulin, its following dissolving in diluted acid and mixing with preserving agent solutions, isotonic agent and substances with buffer capacity. Preparing human insulin ester is carried out in the weight ratio trypsin to porcine insulin = 1:(300-1000) followed by additional inhibition of reaction by dilution of the reaction mixture with water by 2-3 times before acidifying. Purification of human insulin ester is carried out by HPLC method followed by precipitation of ester derivative fractions in the presence of zinc ions, removal of protecting groups and preparing human crude insulin crystals that is purified by repeated carrying out HPLC method. Both processes of purification are carried out by using sorbent DIASOGEL ODS (C18) as immobile phase with particles size from 15 mcm and pores size from 100 to 150 . At the first stage 0.06 M glycine - HCl buffer containing 0.015 M of ammonium sulfate and propanol-2 in the concentration from 20% to 35%, pH 2.5 is used as a mobile phase, and 0.05 M acetate buffer with the content of propanol-2 from 15% to 25%, pH 2.5 is used at the second stage. The purified human insulin is dissolved in diluted acid by stages wherein finely dispersed suspension of insulin crystals in water is prepared followed by addition a diluted acid to it. Invention provides the development of effective, economy method for preparing the ready medicinal formulation of insulin with short effect and with low immunological properties that provides reducing loss of insulin in the process of its preparing. Invention can be used in manufacturing ready insulin formulations of high quality and with short effect.

EFFECT: improved preparing method.

6 ex

FIELD: chemical-pharmaceutical industry, pharmacy.

SUBSTANCE: method involves preparing human insulin ester by transpeptidation reaction of porcine insulin in the excess of threonine di-tert.-butyl ester in an aqueous-organic medium in the presence of trypsin, inhibition of reaction by acidification, purification of prepared insulin ester by chromatography method, removal of protecting groups with trifluoroacetic acid and purification of prepared human crude insulin. Preparing part of the combined preparation of short effect by dissolving the prepared insulin with concentrations 15%, 25% or 50% in diluted acid and mixing with preserving agent solutions, isotonic agent and substances with buffer capacity. Preparing part of the combined preparation of prolonged effect by dissolving remaining insulin in diluted acid also followed by addition of zinc ions acid solutions and protamine sulfate, mixing with buffered solutions of m-cresol, phenol and glycerol, keeping the solution up to formation of crystals and combination of prepared parts of the combined insulin preparation. The transpeptidation reaction is carried out in the weight ratio trypsin to porcine insulin = 1:(300-1000) and before acidification of reaction medium additional inhibition of reaction is carried out by dilution of the reaction mixture with water by 2-3 times. Insulin ester is purified by HPLC method followed by precipitation of fractions with ester derivative in the presence of zinc ions, removal of protecting groups and preparing crystals of crude insulin that are purified by repeated HPLC method. Both purifying processes are carried out by using sorbent DIASOGEL ODS (C18) as immobile phase with particles size from 15 mcm and pores size 120 , and at the first stage 0.06 M glycine-HCl buffer with 0.015 M of ammonium sulfate and propanol-2 with concentration from 20% to 35% at pH 2.5 is used as mobile phase, and at the second stage 0.05 M acetate buffer with the content of propanol-2 from 10% to 20% at pH 2.5 is used. In preparing both parts of the combined preparation crystals of insulin are dissolved in diluted acid by stage wherein firstly finely dispersed suspension of insulin crystals in water is prepared followed by addition of diluted acid to it. In preparing the combined preparation of prolonged effect a mixture obtained after mixing with buffered solutions of m-cresol, phenol and glycerol is kept at temperature 18-21°C for 20-22 h. Invention provides the development of effective, economy method for preparing the combined preparation of human insulin with low immunological properties that allows enhancing yield of insulin in the process of its manufacturing.

EFFECT: improved preparing method.

13 ex

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SUBSTANCE: it is necessary to perform rinsing with oral remedy named "Corsodil", then comes moisturizing oral mucosa with insulin solution. Additionally, one should apply "Fluorovit" preparation onto mucous membrane of patient's tongue, cheeks and hyoid area by applying spray 30-40 min before meals 3-4 times daily. Moreover, one should apply gel named "Metrogil Denta" onto gingival mucosa once daily. The above-mentioned course consists of 10 seances, this course should be carried out once or twice annually. The innovation enables to decrease clinical signs of xerostomia and the risk for the development of oral mucosal inflammatory diseases in patients suffering diabetes mellitus.

EFFECT: higher efficiency of therapy.

3 ex

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2 ex

FIELD: medicine, oncology.

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EFFECT: improved treatment method.

2 ex

FIELD: pharmaceutical chemistry.

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EFFECT: increased chemical stability and solid state stability as compared to amorphous forms of melagatran.

14 cl, 4 dwg, 3 tbl, 9 ex

FIELD: medicine, pharmaceutics, pharmacology.

SUBSTANCE: one should apply mammalian anti-HBP-antibodies. The ways are being suggested to identify monoclonal antibody bound, at least, with one epitope upon native HBP (heparin-binding protein) and methods to detect whether a mammal produces HBR being bound with a monoclonal antibody and, also, the kits for the above-mentioned purpose. The present innovation provides the opportunity to apply the mentioned antibodies in preventing and treating disorders associated with bradykinin releasing.

EFFECT: higher efficiency of application.

25 cl, 11 dwg, 3 ex, 1 tbl

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