The drug is insulin, containing nacl, parenteral ready form, the method of increasing the chemical stability of the drug insulin

 

(57) Abstract:

In the invention disclosed insulin preparations with high chemical stability, including human insulin or an analogue or derivative, glycerine and/or mannitol, and 5 to 100 mm halide, and presents parenteral ready form and method of increasing the chemical stability of the drug insulin. The invention provides improved chemical stability of insulin at low concentrations of sodium chloride. 3 C. and 15 C.p. f-crystals, 8 PL.

This invention relates to aqueous insulin preparations containing human insulin or an analogue or derivative, and these drugs have excellent chemical stability. In addition, this invention relates to parenteral drugs, including insulin, and to a method of increasing the chemical stability of insulin preparations.

Diabetes is a General term for disorders a person with excessive excretion as in diabetes mellitus and diabetes insipidus. Diabetes mellitus is a metabolic disorder in which the ability to dispose of glucose to a greater or lesser extent Dit insulin, ongoing research to improve the treatment of diabetes. In order to help avoid extreme blood glucose levels, patients with diabetes often in practice, the use of multiple injection therapy, in which insulin is administered with each meal.

For diabetes treatment proposed and used a large variety of insulin preparations, such as regular insulin, SemilenteR(SemilenteR) insulin, isophane insulin, insulin zinc suspension, Protamine zinc insulin and UltralenteR(UltralenteR) insulin. Because patients with diabetes treated with insulin for several decades, for the safety and improve quality of life (patients) there is a continuing need to improve insulin preparations. Some of the commercially available insulin preparations are characterized by a rapid onset of action, and other drugs have a relatively slow start (), but show a more or less prolonged action. Fast-acting insulin preparations are usually solutions of insulin, whereas insulin delayed action can be suspensions, soderzalisj Protamine, or a combination of both. In addition, some patients using drugs with a rapid onset of action and a more prolonged action. Such a drug may be insulin solution in which suspended the Protamine-insulin crystals. Some patients receive the drug, mixing insulin solution with a suspension of the drug in relation to the required patient.

Human insulin consists of two polypeptide chains, the so-called a and b, which contain 21 and 30 amino acids, respectively. A and b chains connected to each other by two cystine disulfide bridges. Insulin most varieties have a similar structure, but may not contain the same amino acids in the corresponding positions in the chains, as in human insulin.

The development of the method, known as genetic engineering have made it possible to easily obtain a large number of insulin compounds similar to human insulin. In these insulin analogs, one or more of amino acids are substituted by other amino acids that can be encoded in the nucleotide sequences. Because the human insulin as described above, contains 51 amino acid residue, it is obvious that wozmovny. In solutions of human insulin with a concentration of interest for injection of the drug, the insulin molecule is present in the associated form hexamer (Brange et al. Diabetes Care 13, (1990), 923-954). After subcutaneous injection, it is believed that the absorption rate of the flow depends on the size of the molecule, and it was found that insulin analogs with amino acid substitutions that counteract or inhibit the formation of this hexamer, have unusually rapid onset of action (Brange et al.: Ibid). It is of great therapeutic value for the diabetic patient.

Pharmaceuticals, which is based on analogues of human insulin, represented, for example, Heinemann et al., Lutterman et al Wiefels and et al. at the "Frontiers in Insulin Pharmacology International Symposium in Hamburg, 1992.

In addition, US 5474978 reveals fast-acting parenteral preparation, including review of the complex analogue of human insulin, consisting of six Monomeric insulin analogs, zinc ions and at least three molecules of phenolic derivative.

Usually insulin is administered via subcutaneous injection. Patient-important action profiles of insulin drug profile is important in addition, the start time (steps), maximum value, and the total duration. There is a need and demand for different insulin preparations with different profiles of action. One patient for one day uses insulin preparations with different profiles of action. The desired mode of action depends on the time of day and the number and composition of the food taken by the patient.

Equally important for the patient and the chemical stability of insulin preparations especially due to the abundance of devices used for injection-type handles, such as devices that contain PenfillRcartridges, in which insulin is stored up until the entire cartridge is not empty. This can continue for at least 1 to 2 weeks for devices containing cartridges 1,5-3,0 ml During storage occur covalent chemical changes in the structure of insulin. This can lead to the formation of molecules, which are less active and potentially immunogenic, such as products deliciouse and transformation products with higher molecular weight (dimers, polymers, and so on). A comprehensive study on Himich, pp.149-158 described insulin, in which the concentration of sodium chloride ranges from 0 to 250 mm. However, the main part of the preparations, including all drugs, which include glycerin, contains a fairly high amount of sodium chloride, i.e. 0.7 per cent, corresponding to approximately a concentration of 120 mm. This document indicates that while the sodium chloride generally has a stabilizing effect on insulin, glycerol and glucose leads to chemical deterioration.

Unexpectedly, however, it was discovered that you can get insulin with excellent chemical stability in the presence of glycerol and/or mannitol, and very low concentrations of halide.

Used herein, the term "analogue of human insulin" refers to human insulin in which one or more amino acids deleted and/or replaced by other amino acids, including non-coding amino acids, or human insulin, including additional amino acids, i.e. more than 51 amino acid.

Used herein, the term "derivative of human insulin" refers to human insulin or an analogue, in which at least the e unit "U" corresponds to 6 nmol.

This invention relates to aqueous insulin preparation, including:

human insulin, an analogue and/or derivative thereof;

glycerine and/or mannitol;

from 5 to 100 mm halide.

The above-mentioned insulin drug has a high chemical stability, which, for example, is reflected in the reduction in the formation of dimers and polymers, desamino-insulin after storage. In addition, the physical stability does not deteriorate due to the presence of very low amounts of halide, and not insulin precipitates during long-term storage of insulin preparations.

The halide is preferably a halide of the alkali or alkaline earth metal, more preferably a chloride such as sodium chloride.

Glycerine and/or mannitol is preferably present in an amount corresponding to the concentration from 100 to 250 mm, more preferably from 140 to 250 mm, even more preferably from 160 to 200 mm.

This invention particularly useful in connection with drugs, including analogues and/or derivatives of human insulin. Thus, the insulin preparation according to this invention preferably includes one or more fast de the Ala and position B is Lys or Pro; or des(B28-B30), des(B27) or des(B30) human insulin. The insulin analogue is preferably selected from a human insulin analogue, where the position B28 is Asp or Lys, and position B is Lys or Pro. The most preferred analogs are AspB28human insulin or LysB28ProWhuman insulin.

In this variant embodiment of the invention the insulin preparation preferably comprises from 5 to 60 mm, more preferably from 5 to 40 mm halide.

In another variant embodiment of the insulin preparation according to this invention includes a derived insulin with prolonged profile of action, such as insulin, having one or more lipophilic substituents. Preferred lipophilic insulin is acylated insulins, including those described in WO 95/07931 (Novo Nordisk A/S), for example derivatives of human insulin where-amino group of LysWcontains acyl Deputy, which includes at least 6 carbon atoms.

Preferred insulin derivative are as follows: B29-N-myristoyl-des(B30) human insulin, B29-N-Palmitoyl-des(B30) human is insulin, B28-N-myristoyl LysB28ProB29human insulin, B28-N-Palmitoyl LysB28ProB29human insulin, B30-N-myristoyl-ThrB29LysB30human insulin, B30-N-Palmitoyl ThrB29LysB30human insulin, B29-N(N-Palmitoyl-glutamyl)-des(B30) human

insulin, B29-N-(N-lithocholyl--glutamyl)-des(B30) human insulin, B29-N-(-carboxypropanoyl)-des (B30) human insulin, B29-N-(-carboxypropanoyl) human insulin; and most preferred is B29-N-myristoyl-des(B30) human insulin.

In this variant embodiment the insulin preparation preferably includes from 10 to 100 mm, more preferably from 10 to 70 mm halide.

In a particular variant embodiment of an insulin drug of this invention includes an insulin analog, and a derivative of insulin.

In a preferred variant embodiment of the present invention the insulin preparation includes:

from 60 to 3000 nmol/ml, preferably from 240 to 1200 nmol/ml human insulin or an insulin analogue or derivative,

Mainly used as phenolic compounds from 0.5 to 4.0 mg/ml, preferably from 0.6 to 4.0 mg/ml m-cresol, or from 0.5 to 4.0 mg/CL, preferably from 1.4 to 4.0 mg/ml phenol, or a mixture of them.

Insulin drug of this invention can also contain other ingredients characteristic of insulin preparations, such as tools, components zinc, such as citrate, and phosphate buffers.

This invention also relates to a parenteral pharmaceutical preparation comprising an insulin drug of this invention.

In addition, this invention relates to a method of improving the chemical stability of insulin preparation comprising human insulin or an analogue or derivative, and this method includes the addition of glycerol and/or mannitol and 5 to 100 mm halide to the specified drug.

The invention is additionally illustrated by the following examples, which, however, should not be considered as limiting it.

Example I

Get solutions containing 100 u/ml AspB28human insulin, 2.6 mg/ml phenol, 16 mg/ml glycerin and variouscassini) are presented in table. 1.

Example II

Insulin preparations containing dissolved AspB28human insulin with varying concentrations of sodium chloride, was prepared as follows/

370,4 mg AspB28human insulin was dissolved in water by adding 1.6 ml of 0.2 N HC1 and 49 μl of a solution of zinc chloride (40 mg Zn/ml). 40 g of a solution containing 40 mg/ml glycerol, 3.75 mg/g of phenol and 4,30 mg/g of m-cresol, is added to the insulin solution while stirring. 20 g of a solution containing: (a) to 12.0 mg/g of disodium phosphate dihydrate + 5 µl/g 2 N sodium hydroxide, (b) to 12.0 mg/g of disodium phosphate dihydrate + 5 µl/g 2 N sodium hydroxide + 5 mg/g sodium chloride or (C) of 12.0 mg/g of disodium phosphate dihydrate + 5 µl/g 2 N sodium hydroxide + 10 mg/g of sodium chloride, added under stirring. the pH is brought to 7,400,05 and add water to 100 ml Preparations AspB28human insulin injected into PenfillRcartridges and subjected to stability testing at 25 and 37oC. stability Data obtained at two different temperatures and at a phosphate concentration of 13.5 mm and 19.6 μg Zn/100 E insulin and pH=7,4 presented in table. 2.

Example III

Insulin preparations containing dissolved AspB28human insulin, with different concentrated in water, adding 1.6 ml of 0.2 N HC1 and 49 μl of a solution of zinc chloride (40 mg Zn/ml). To the solution was added with stirring 40 g of a solution containing 40 mg/g of glycerin, 3.75 mg/g of phenol and 4,30 mg/g of m-cresol. 10 g of a solution containing 24,0 mg/g of disodium phosphate dihydrate and 11 µl/g 2 N sodium hydroxide, add with stirring. Finally, different amounts (from 0 up to 4.38 g) of a solution containing 40 mg/g of sodium chloride, is added under stirring until the concentration of sodium chloride indicated in the table. 4. the pH is brought to 7,400,05 and add water to 100 ml Preparations AspB28human insulin injected into PenfillRcartridges and subjected to stability testing at 25 and 37oC. stability Data obtained at two different temperatures and at a phosphate concentration of 13.5 mm, summarized in table. 3.

Example IV

Insulin preparations containing dissolved AspB28human insulin with varying concentrations of phosphate and sodium chloride was prepared as follows

375,7 mg AspB28human insulin was dissolved in water by adding 1.6 ml of 0.2 N Hcl and 49 μl of a solution of zinc chloride (40 mg Zn/ml). 20 g of a solution containing 80 mg/g of glycerin, 7,50 mg/g of phenol and at 8.60 mg/g of m-cresol, are added to a solution when peremeshivajutsa sodium, add with stirring, finally, different amounts (from 0 to the 3.65 g) of a solution containing 40 mg/g of sodium chloride, added under stirring so as to obtain a concentration of sodium chloride indicated in the table. 6. the pH is brought to 7,400,05 and add water to 100 ml Preparations AspB28human insulin injected into PenfillRcartridges and subjected to stability testing at 25 and 37oC. stability Data at two different temperatures and three different concentrations of phosphate and a 19.6 μg Zn/100 u insulin and pH=7,4, summarized in table. 4 - 6.

Example V

Get solutions containing 0.6 mm B29-N-myristoyl-des(B30) human insulin, 1.5 or 4.0 mg/ml phenol, 5 mm of sodium phosphate, of 13.1 ág/ml Zn, and different amounts of sodium chloride and mannitol. the pH was adjusted to 7.4. The stability data (formation of dimers and polymers) after storage at 25oWith over 13 weeks or at 37oWith over 8 weeks are shown in table. 7.

Example VI

Get solutions containing 0.6 mm B29-N-myristoyl(B30) human insulin, 1.5 mg/ml phenol and 1,72 mg/ml m-cresol, 16 mg/ml glycerin or 36 mg/ml mannitol, 13,1 μg/ml Zn, 7 mm of sodium phosphate and various amounts of chlorineWith over 13 weeks or at 37oWith over 8 weeks are shown in table. 8.

1. Aqueous preparation of insulin, including human insulin, an analogue and/or derivative, glycerol or mannitol, and 5 to 100 mm chloride.

2. The drug is insulin p. 1, wherein the chloride is the chloride of the alkali or alkaline earth metal, preferably sodium chloride.

3. The drug is insulin under item 1 or 2, characterized in that it comprises from 100 to 250 mm, preferably from 140 to 250 mm, more preferably from 160 to 200 mm glycerol or mannitol.

4. The drug is insulin according to any one of the preceding paragraphs, characterized in that it includes analogue of human insulin, where the position B28 is sp, Lys, Leu, Val or A1A and position 29 is Lys or Pro, or des (B28-B30), des (B27) or des (B30) human insulin.

5. The drug is insulin p. 4, characterized in that it includes analogue of human insulin, where the position 28 is sp or Lys, and position B is Lys or Pro, preferably spB28human insulin or LysB28ProB29human insulin.

6. The drug is insulin p. 4, characterized in that it includes (B30) chaloon includes from 5 to 60 mm, preferably from 5 to 40 mm, chloride.

8. The drug is insulin according to any one of paragraphs. 1-3, characterized in that it includes a derivative of human insulin having one or more lipophilic substituents, preferably acylated insulin.

9. The drug is insulin p. 8, characterized in that a derivative of insulin selected from the group consisting of B29-N-myristoyl-des(B30) human insulin, B29-N-Palmitoyl-des(B30) human insulin, B29-N-myristoyl human insulin, B29-N-Palmitoyl human insulin, B28-N-myristoyl LysB28ProB29human insulin, B28-N-Palmitoyl LysB28ProB29human insulin, B30-N-myristoyl-ThrB29ProB30human insulin, B30-N-Palmitoyl ThrB29LysB30human insulin, B29-N(N-Palmitoyl-glutamyl-(B30) human insulin, B29-N-(N-lithocholyl--glutamyl-(B30) human insulin, B29-N--carboxypropanoyl)-des(B30) human insulin, B29-N-(-carboxypropanoyl) human insulin.

10. The drug insuli insulin.

11. The drug is insulin according to any one of paragraphs. 8-10, characterized in that it comprises from 10 to 100 mm, preferably from 10 to 70 mm, chloride.

12. The drug is insulin according to any one of the preceding paragraphs, characterized in that it includes an insulin analog and derivative of insulin.

13. The drug is insulin according to any one of the preceding paragraphs, characterized in that it comprises from 60 to 3000 nmol/ml, preferably from 240 to 1200 nmol/ml human insulin, insulin analogue or derivative of insulin.

14. The drug is insulin according to any one of the preceding paragraphs, characterized in that it comprises from 10 to 40 μg Zn/100 u insulin, preferably from 10 to 26 μg Zn/100 u insulin.

15. The drug is insulin according to any one of the preceding paragraphs, characterized in that it comprises from 0 to 5 mg/ml, preferably from 0 to 4 mg /ml, phenol compounds.

16. The drug is insulin p. 15, characterized in that it comprises from 0.5 to 4.0 mg/ml, preferably from 0.6 to 4.0 mg/ml m-cresol, or from 0.5 to 4.0 mg/ml, preferably from 1.4 to 4.0 mg/ml, phenol, or a mixture of them.

17. Parenteral pharmaceutical finished form, including the preparation of insulin according to any one of the preceding paragraphs.

18. The method above is the initial, including the addition of glycerol or mannitol, and 5 to 100 mm chloride to the specified drug.

 

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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

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