RU2185152C1 - Method of preparing pharmaceutical composition suspension based on substance of genetic engineering (recombinant) human insulin - Google Patents

Abstract

FIELD: medicine, endocrinology, pharmacy. SUBSTANCE: invention relates to preparing insulin preparations of prolonged action used in therapy of diabetes mellitus. Preparation showing stability at prolonged storage is prepared by mixing solutions of sodium-phosphate buffer, pH 7.4-7.5, containing preserving agents m-cresol and phenol and solution, pH 3.0-3.4, containing preserving agents m-cresol and phenol, protamine sulfate, insulin, zinc chloride and glycerol being mixing is carried out in the definite sequence. Indicated solutions are subjected for sterilizing filtration and suspension of components formed after final mixing is poured into cartridges or flasks followed by crystallization for 48 h at 15-30 C. Invention can be used in medicinal industry for preparing suspension of domestic genetic engineering (recombinant) human protamine-insulin in cartridges and/or flasks. Method provides preparing the preparation showing stability at storage in accordance with requirements of Pharmacopoeia article. EFFECT: improved method of preparing, expanded arsenal of antidiabetic agents. 3 cl, 2 tbl

Description

 The invention relates to medicine, in particular to the production of prolonged-acting insulin preparations used in the treatment of diabetes mellitus. The invention can be used in the medical industry for the manufacture of a suspension of domestic genetically engineered (recombinant) human protamine insulin in cartridges and vials.

 A known method of producing a suspension of cattle insulin with an insulin content of 100 PIECES / ml in vials (1).

 The disadvantages of this method include the need for freeze drying of a solution containing insulin and phenol in acidified water, after which a mixture of protamine sulfate, glycerol and phenol in water is added to the lyophilisate. This method of obtaining a suspension of protamine-insulin ensures the stability and biological activity of the drug for only 3 months.

 Known preparations of biosynthetic human protamine-insulin with concentrations of 40 and 100 PIECES / ml in bottles and cartridges (2), as well as suspensions of porcine protamine-insulin with insulin content of 100 PIECES / ml, intended for filling in bottles (2).

A known method of preparing a suspension of porcine protamine-insulin with a concentration of 100 PIECES / ml of insulin, intended for filling in cartridges (3). According to this method, the preparation of a suspension of protamine-insulin prepared by mixing four solutions in injection water is poured into cartridges, namely: a solution of preservatives phenol and m-cresol; a solution of protamine sulfate and glycerol; a solution of insulin and glycerol and a phosphate buffer solution, followed by crystallization at 20-25 ^{° C.} for 72 hours and pouring a suspension of protamine-insulin into cartridges; while at the stages of preparation of the solutions they are sterilized using a prefilter with a pore diameter of 0.45 μm and a filter with a pore diameter of 0.22 μm.

 The objective of the invention is to develop a method for preparing a suspension of a pharmaceutical composition based on domestic genetic engineering (recombinant) human protamine insulin, with an insulin concentration of 100 PIECES / ml, designed for filling into cartridges and vials, which provides a more effective than the known method (3) carrying out the technological process.

The problem is solved by creating a method of preparing a suspension of genetically engineered human protamine insulin for cartridges and vials with the addition of zinc chloride and the formation of suspension crystals not before bottling, but directly in cartridges and vials after bottling, which takes 48 hours instead of 72 hours, as in the known method, wherein the formation of the suspension (crystallization) is carried out at 15-30 ^o C instead of a more limited temperature range (20-25 ^o C) in the known method. In contrast to the known, the claimed method allows at the stages of sterilizing filtration to use not two, but a single sterilization.

 The claimed method is as follows.

 Pre-prepared solutions containing per 100 IU of insulin: 2.4 mg of sodium phosphate disubstituted, 0.7-0.8 mg of m-cresol and 0.3-0.4 mg of phenol (first solution); 0.7-0.8 mg of m-cresol and 0.3-0.4 mg of phenol (second solution); 0.3-0.4 mg of protamine sulfate and 7.5-8.5 mg of glycerol (third solution); insulin, 4.0-40.0 μg of zinc chloride and 7.5-8.5 mg of glycerol (fourth solution); then the second solution is mixed with the third and fourth (fifth solution), and then the first solution is sterilely filtered in the first solution and the fifth, the resulting suspension is poured into cartridges and / or bottles, corked, caps are rolled on them.

Crystallization is carried out in run-in cartridges or vials for 48 hours at a temperature of 15-30 ^o With four hours of stirring 24 hours after the suspension is bottled.

 The developed method allows to prepare a suspension of domestic human protamine insulin for vials and cartridges (inserts), which retains its physicochemical and biological properties for a long time (see tables 1 and 2). A draft Pharmacopoeia article of the enterprise (FSP) was developed for the drug.

 The inventive preparation allows providing healthcare with a new effective and inexpensive domestic antidiabetic agent and reducing the penetration of expensive imported insulin preparations into the Russian market.

 The claimed method is illustrated by the following examples.

 Example 1

 Preparation of the first solution. 2.4 g of sodium phosphate disubstituted, 0.68 g of m-cresol and 0.3 g of phenol are dissolved in 200 ml of injection water. After complete dissolution of the introduced components, 1 M hydrochloric acid is added to the solution (to pH 7.4), and then the volume of the solution is adjusted with injection water to 500 ml.

 Preparation of the second solution. 0.68 g of m-cresol and 0.3 g of phenol are dissolved in 150 ml of injection water. After complete dissolution of the introduced components, the volume of the solution was adjusted with injection water to 200 ml.

Preparation of the third solution. In 5 ml of injection water, heated to a temperature of 45 ^o With, dissolve 0.27 g of protamine sulfate. After dissolving the introduced component, 5 ml of injection water cooled to a temperature of 4 ^{° C.} is added to the solution, then 8.0 g of glycerol is added. Mixing the mixture is carried out until glycerol is completely dissolved, after which the volume of the solution is adjusted with injection water to 30 ml.

Preparation of the fourth solution. 3.85 g of domestic genetically engineered human insulin with an activity of 26 U / mg and a zinc content of 0.12% is poured into 25 ml of injection water cooled to a temperature of 4 ^o C and stirred, then a 0.2 M hydrochloric acid solution is added with stirring to pH 3.0-3.4. The suspension is stirred until complete dissolution of the insulin, after which 1.21 ml of a 1% solution of zinc chloride containing 4.8 mg of zinc in 1 ml is added to the solution while stirring. Stirring after adding zinc chloride is carried out for 5 minutes, and then, while stirring, 8.0 g of glycerol are added to the solution. After complete dissolution of glycerol, the volume of the solution is injected with injection water to 100 ml.

 Preparation of the fifth solution. 30 ml of the third and 100 ml of the fourth solutions are added to the second solution with constant stirring. The volume of the resulting solution was adjusted with injection water to 450 ml.

 Preparation of a suspension of insulin with protamine sulfate. Through a sterilizing filter with a pore diameter of 0.22 μm, 500 ml of the first solution and 25 ml of injection water are filtered into a sterile container, and then 450 ml of the fifth solution and 25 ml of injection water are added to it through a sterilizing filter with constant stirring.

 Pouring suspensions. The suspension formed by mixing the first and fifth solutions is poured into cartridges of 1.5 or 3 ml or into bottles of 5 or 10 ml. Cartridges or vials are corked and caps are rolled on them.

Crystallization. Run-in cartridges or vials are placed in cassettes and kept at a temperature of 15 ^° C for 24 hours, then the cassettes are placed on a shaker and mixing is carried out for 4 hours, after which the cassettes are removed and the suspension is continued to withstand for 20 hours at a temperature of 15 ^° C.

 Example 2

 Preparation of the first solution. 2.4 g of sodium phosphate disubstituted, 0.83 g of m-cresol and 0.36 g of phenol are dissolved in 200 ml of injection water. After complete dissolution of the introduced components, 1 M hydrochloric acid is added to the solution (to pH 7.5), and then the volume of the solution is adjusted with injection water to 500 ml.

 Preparation of the second solution. 0.83 g of m-cresol and 0.36 g of phenol are dissolved in 150 ml of injection water. After complete dissolution of the introduced components, the volume of the solution was adjusted with injection water to 200 ml.

Preparation of the third solution. 0.40 g of protamine sulfate is dissolved in 5 ml of injection water, heated to a temperature of 55 ^{o C.} After dissolving the introduced component, 5 ml of injection water cooled to a temperature of 4 ^{° C.} is added to the solution, then 8.0 g of glycerol is added. Mixing the mixture is carried out until glycerol is completely dissolved, after which the volume of the solution is adjusted with injection water to 30 ml.

Preparation of the fourth solution. 3.85 g of genetically engineered human insulin with an activity of 26 PIECES / mg and a zinc content of 0.12% is poured into 25 ml of injection water cooled to a temperature of 4 ^° C and stirred, then a 0.2 M hydrochloric acid solution is added with stirring to pH 3 , 0-3.4. The suspension is stirred until complete dissolution of the insulin, after which 7.6 ml of a 1% solution of zinc chloride containing 4.8 mg of zinc in 1 ml is added to the solution while stirring. Stirring after adding zinc chloride is carried out for 15 minutes, and then, while stirring, 8.0 g of glycerol are added to the solution. After complete dissolution of glycerol, the volume of the solution is injected with injection water to 100 ml.

 Preparation of the fifth solution. 30 ml of the third and 100 ml of the fourth solution are added to the second solution with constant stirring. The volume of the resulting solution was adjusted with injection water to 450 ml.

 Preparation of a suspension of insulin with protamine sulfate. Through a sterilizing filter with a pore diameter of 0.22 μm, 500 ml of the first solution and 25 ml of injection water are filtered into a sterile container, and then 450 ml of the fifth solution and 25 ml of injection water are added to it through a sterilizing filter with constant stirring.

 Pouring suspensions. The suspension formed by mixing the first and fifth solutions is poured into cartridges of 1.5 or 3 ml or into bottles of 5 or 10 ml. Cartridges or vials are corked and caps are rolled on them.

Crystallization. Run-in cartridges or vials are placed in cassettes and kept at a temperature of 30 ^o C for 24 hours, then the cassettes are placed on a shaker and mixing is carried out for 4 hours, after which the cassettes are removed and the suspension is continued to withstand for 20 hours at a temperature of 30 ^o C.

 Data on monitoring the preservation of the drug at different shelf life in cartridges and vials are given in table. 1 and 2.

Sources of information
1. US patent 386358, CL. NKI 260 / 112.7, cl. MKI C 07 C 103/52, 02.25.1975 (Eli Lily and Company, Indiapolis).

 2. Register of medicines of Russia (Encyclopedia of medicines). - M .: Publishing house "RLS-2000", 1999, p. 948.

 3. RF patent 2157698, cl. MKI A 61 K 38/28, 20.10.2000 (State Institute of Blood Substitutes and Medications).

Claims (3)

1. A method of preparing a suspension of an insulin-based pharmaceutical composition for cartridges and / or vials, comprising preparing an insulin preparation in the form of an aqueous suspension with a concentration of 100 IU / ml containing protamine sulfate, glycerin, a preservative, characterized in that domestic preparation is used substance of genetically engineered (recombinant) human insulin, and zinc chloride is additionally added to the substance solution in the amount of 5.0-40.0 μg per 100 IU of insulin, while solutions containing per 1 00 IU of insulin: 2.4 mg of sodium phosphate disubstituted, 0.7-0.8 mg of m-cresol and 0.3-0.4 mg of phenol (first solution); 0.7-0.8 mg of m-cresol and 0.3-0.4 mg of phenol (second solution); 0.3-0.4 mg of protamine sulfate and 7.5-8.5 mg of glycerol (third solution); insulin, zinc chloride and 7.5-8.5 mg of glycerol (fourth solution); then the second solution is mixed with the third and fourth (fifth solution), and then the first solution is sterilely filtered in the first solution and the fifth, the resulting suspension is poured into cartridges and / or vials, sealed, caps are rolled on them and crystallized for 48 hours at a temperature of 15-30 ^o C.  2. The method according to p. 1, characterized in that the filling and subsequent crystallization is carried out in cartridges having a volume of 1.5 or 3.0 ml.  3. The method according to p. 1, characterized in that the bottling and subsequent crystallization is carried out in bottles of 5.0 or 10.0 ml.

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