WO2006096079A2

WO2006096079A2 - Pharmaceutical composition comprising a biosynthetic analog of human insulin, and its use in the treatment of diabetes mellitus.

Info

Publication number: WO2006096079A2
Application number: PCT/PL2006/000016
Authority: WO
Inventors: Piotr Borowicz; Jerzy Mikolajczyk; Monika Bogiel; Henryk Klukowski; Anna Krejer
Original assignee: Instytut Biotechnologii i Antybiotykow
Current assignee: Instytut Biotechnologii i Antybiotykow
Priority date: 2005-03-10
Filing date: 2006-03-09
Publication date: 2006-09-14
Anticipated expiration: 2007-09-10
Also published as: PL373543A1; WO2006096079A3

Abstract

The invention relates to a pharmaceutical composition comprising a recombinant human insulin derivative, and consisting in that it includes an effectively acting amount of the recombinant human insulin derivative of formula (I) or its physiologically acceptable salt, and excipients. The invention relates also to a method of preparation of the composition and its use in the treatment of diabetes mellitus.

Description

Pharmaceutical composition comprising a biosynthetic analog of human insulin, and its use in the treatment of diabetes mellitus.

An object of the invention is a pharmaceutical composition comprising a biosynthetic analog of human insulin, and its use in the treatment of diabetes mellitus.

Insulin and its various derivatives are used in large amounts in the treatment of diabetes mellitus and are often manufactured on large industrial scale. Although there is a number of various modified derivatives of insulin and pharmaceutical formulations exhibiting various activity profiles, a search is being continued to find a drug that would maintain a constant glucose level in human body for a long time.

To provide an effect of delayed and/or extended action, some formulations of typical human insulin include specific additives, e.g. various amounts of protamine, a protein forming with insulin an insoluble complex that deposits in the subcutaneous tissue, from which insulin is gradually released. There are known various derivatives of human insulin used in the therapy of diabetes mellitus that include additional amino acids or an altered sequence of some amino acids. Alterations in the primary insulin structure affect its secondary and tertiary structure, which in turn induces changes in chemical and biological properties, and - as a consequence - results in pharmacokinetic and pharmacodynamic effects. These changes are of various nature. They lead to acceleration or delay and extension of action of the administered modified insulin. An active form of insulin is its monomeric form, which readily penetrates into the blood upon subcutaneous injection. As it is known, the normal human insulin in the solution is in a form of hexamers, which upon administration dissociate into dimers, and, then, into monomers before penetrating the blood. Insulin derivatives characterized by an accelerated action include lispro-lnsulin (Humalog®), where the sequence proline(28)-lysine(29) in B chain has been reversed. In such a case, formation of insulin dimers in the solution is difficult, because of steric effects. In another insulin derivative of accelerated action, proline in position 28 in B chain was replaced with aspartic acid. The negative charge introduced in this way hinders self-association of insulin monomers. Both these insulin derivatives are faster absorbed thanks to their structure. In another known biosynthetic analog of human insulin, asparagine (21) in A chain was replaced with glycine (21) and two arginine residues were added to C- terminus of B chain. It is referred to as glargine derivative of insulin and manufactured under the name Lantus^® (U.S. Patent No. 5,656,722).

We have found that chemical and biological properties similar to that of glargine derivative are exhibited by the human insulin derivative - referred to as Lys-Arg insulin - of formula 1 , in which lysine (B31 Lys) and arginine (B32Arg) residues have been added to C-terminus of B chain.

Formula 1

The above-mentioned compound can be prepared by a known method from a hybrid polypeptide of formula 2 - disclosed in Polish Patent No. 180 818 and obtained by expression in a bacteria cell - by digesting it with trypsin and, then, purifying it by known methods, e.g. such as chromatography and crystallization.

I — leader protein

Formula 2

We have found that these modifications have resulted in a shift of isoelectric point to pH 5-8 and, thus, in a solubility decrease of the new insulin derivative at the physiological pH of an injection site. This causes precipitation of microdepot of insulin derivative at the subcutaneous tissue, and, then, its gradual, slow release to the blood, thanks to which a therapeutic level is longer maintained. It is also important that the derivative - Lys-Arg insulin, used in the present invention, starts its action almost immediately. This means that the compound surprisingly exhibits features of biological activity of known insulin analogs of the accelerated time of action as well as those of the extended time of action.

The main physicochemical property of the insulin derivative B31Lys-32Arg, distinguishing it from human insulin, is its isoelectric point value within the range from about 5 to about 8. This determines good solubility of the compound in solutions at acidic-to-slightly-acidic pH as well as at alkaline pH. Thanks to that property, it was possible to prepare B31 Lys-32Arg composition-solutions at acidic and alkaline pH, then subjected to biological activity testing.

The main aspect of the invention is a pharmaceutical composition comprising an effectively acting amount of biosynthetic analog of human insulin of formula 1 or its physiologically acceptable salt, and excipients.

Formula 1

The salt of biosynthetic analog of human insulin derivative of formula 1 should be for example metal alkaline or ammonium salt.

The composition is designed for administration in the solution or suspension form. It includes an effectively acting amount of the biosynthetic analog of human insulin of formula 1 , referred herein to as LysArg-insulin, or its physiologically acceptable salt, and excipients, such as isotonizing agents, preservatives, stabilizing agents and, optionally, a buffer substance.

The amount of active substance used in the composition is about 1-1600

IU/mL, preferably 10-1200 IU/mL, more preferably 10-500 IU/mL. pH value of the solution of pharmaceutical composition according to the invention is from about

3,5 to about 8.5.

In general, the same substances are used as excipients in the compositions of the invention, which are used in formulations comprising the normal recombinant human insulin. A preferable isotonizing agent in the composition of the invention can be any substance, which makes it possible to prepare a solution isotonic with the human blood plasma. Some typical isotonizing agents used in pharmacy include such agents as sodium chloride, mannitol, glycine and glycerol. A use of glycerol is preferable. Useful preservatives to be used in the compositions of the invention are compounds selected from the group consisting of m-cresol, phenol, methylparaben, propylparaben and their mixtures. A choice of a preservative and/or an isotonizing agent can influence solubility of the insulin derivative. It has been found that the best preservatives for a formulation at pH 7.8 are 4- hydroxybenzoic acid derivatives (parabens).

Various additional excipients usually used in insulin preparations may also be components of pharmaceutical composition according to the invention. Among others there are buffer substance such as for example phosphate buffer, glycine buffer, TRIS, citrate buffer, whereas, the last one may take a function of buffer and/or zink ions complexing agent.

The Lys-Arg insulin, like the normal recombinant human insulin, is stabilized by addition of zinc ions, introduced to the solution in a form of zinc chloride or oxide. The zinc content can be from about 5 to about 150 mcg/mL

Useful stabilizing agents to be used are nonionic surface-active agents such as for example polyoxyethylene and polyoxypropylene copolymers or various polysorbates. Obtional, additional polysorbate content in composition according to the invention is 0,001 - 0,2 mg/mL. The following content of the composition of the invention is proposed: 10-

500 IU/mL of the biosynthetic analog of human insulin of formula 1 , or its physiologically acceptable salt, 16 mg/mL of glycerol, 3 mg/mL of m-cresol, 10-50 mcg/mL of zinc and water for injections up to 1 mL.

Another following content of the composition of the invention is proposed: 10-500 IU/mL of the biosynthetic analog of human insulin of formula 1 , or its physiologically acceptable salt, 16 mg/mL of glycerol, 1 mg/mL of methylparaben- propylparaben mixture (7:3), 10-50 mcg/mL of zinc and water for injections up to 1 mL. The composition includes phosphate buffer - pH 7.8-8.0.

A method of preparation of the pharmaceutical composition consisting in that (a) the biosynthetic analog of human insulin of formula 1 is dissolved while adjusting solution pH to about 3,5 -8,5 and the suitable amount of water is added to the volume; (b) a preservative, an isotonizing agent, a stabilizing agent and, optionally, a buffer substance are dissolved in an appropriate amount of water; and (c) the solutions (a) and (b) are mixed and, when necessary, the suitable amount of water is added to the volume.

A method of preparation of the basic pharmaceutical composition of the invention consists in that the biosynthetic analog of human insulin of formula 1 is dissolved in water with a use of acidifying agent, preferably diluted hydrochloric acid, and, then, pH of the solution is adjusted to about 3,5 - 5 with an alkalizing agent, preferably diluted sodium hydroxide solution, or it is dissolved in water with a use of an alkalizing agent, preferably phosphate buffer with pH 11 at a temperature below the room temperature, and, then, pH of the solution is adjusted to about 7.8 with phosphate buffer pH 5.5. The further aspect of the invention is the medical use of the biosynthetic analog of human insulin derivative of formula 1 or its physiologically acceptable salt, of isoelectric point between 5 and 8, for preparation of a drug for the treatment of patients suffering from diabetes mellitus, and also a use of the pharmaceutical composition of the invention for preparation of the drug for the treatment of patients suffering from diabetes mellitus.

An aspect of the invention is also a method of treatment of patients suffering from diabetes mellitus, consisting in that to patients in a need of thereof is administered an effective amount of pharmaceutical composition of the invention. An influence of B31Lys-32Arg biosynthetic analog of human insulin in the formulation of pH 5.0, prepared according to Example 1 (LAI-5.0) and of B31 Lys- 32Arg biosynthetic analog in the alkaline solution, prepared according to Example 8 (LAI -7.8) on glucose level in serum normoglycemic rats upon subcutaneous administration of a dose corresponding to 1 , 2 and 5 I. U. of insulin per kg body mass was studied.

Results of the study are shown in Table 1.

It was assumed that 1 mg of Lys-Arg insulin corresponds to 28.82 I. U. (as in case of a normal human insulin).

For further analogous study of the same formulations with a use of h perglycemic rats (Nakhoda A., Wong H. A., Experientia 1979, 35, 1679-1680), a dose corresponding to 2 I. U. /kg body mass was selected and the study was conducted in comparison with the reference formulations of the fast-acting human insulin - Gensulin R (RHI-40) and the long-acting human insulin - Gensulin N (NHI-40). Results of the study are shown in Table 2 and presented graphically in Fig. 1 as an effect of insulin formulations on a glucose level in the blood serum of hyperglycemic rats.

The study has shown that B31 Lys-32Arg of biosynthetic analog exhibits hypoglycemic activity in both indicated pharmaceutical compositions. Table 1. Effect of LAI-5.0, LA1-7.8 formulations and Gensulin R, Gensulin N (the reference formulations) on a glucose level in the blood serum of normoglycemic rats.

Table 2. Effect of LAI-5.0, LAI-7.8 formulations and Gensulin R, Gensulin N (the reference formulations) on a glucose level in the blood serum of hyperglycemic rats.

The experiments with hypoglycemic rats have proved that the formulations significantly decreased sugar levels in the blood and the start of action occurred already after 0.16 h., as in the case of the human insulin solution RHI-40 formulation, whereas in the case of isophan-insulin NHI-40 formulation, the activity was observed after 0.5 h.

The maximum activity of the formulation LAI-7.8i occurs after about 2 h, like in the case of the human insulin solution RHI-40 formulation and a decrease in glucose level has more mild nature and lasts much longer, about 1O h. Comparison of activity of the analogue of human insulin LAI-5.0 with that of isophan-insulin (Gensulin N) NHI-40 has proved that LAI - 5.0 lowers a glucose level in blood serum of hyperglycemic rats more rapidly; that activity was observed in 10 minutes upon the administration and reached its maximum in 1 hour; long- term activity of both formulations is maintained for about 24 h. A significant difference in activity of both formulations consists in an unexpected different nature of the biological action - the formulation LAI-5.0 reduces a glucose level immediately upon the administration and exhibits a weak maximum, indicating for permanent release of the active compound from a microdepot. The stability examinations of preparations containing composition according to the invention show, the addition of stabilizer, especially polysorbate takes an advantage for the effect of stability during the storage. The composition according to the invention stored during 6 months period was stable without any signs of changes.

The below examples are going to illustrate but not to limitate solution according to the invention.

Example I. 362.3 mg of Lys-Arg insulin including 3.5% Zn was dissolved in 40 mL of 0.01 M hydrochloric acid. Then, to the resulting solution 0.01 M sodium hydroxide solution was added very slowly under stirring to reach pH 5.0. After that, water was added to the solution to the volume of 50 mL (Solution A). Separately, 0.3 g of m-cresol, 1.6 g of anhydrous glycerol and zinc chloride (the amount necessary to reach the final concentration of Zn ions of 30 mcg/mL) were dissolved in 45 mL of water. After that, water was added to the solution to the volume of 50 mL (Solution B). To Solution A, Solution B was added slowly under stirring, and the resulting mixture was filtered through a sterilizing filter. The resulting solution, including 100 IU/mL of Lys-Arg insulin, was distributed under aseptic conditions into vials (10 mL each).

Example II. 362.3 mg of Lys-Arg insulin including 3.5% Zn was dissolved in 40 mL of 0.01 M hydrochloric acid. Then, to the resulting solution 0.01 M sodium hydroxide solution was added very slowly under stirring to reach pH 4.5. After that, water was added to the solution to the volume of 50 mL (Solution A). Separately, 150 mg of m-cresol, 65 mg phenol, 1.6 g of anhydrous glycerol and zinc chloride (the amount necessary to reach the final concentration of Zn ions of 30 mcg/mL) were dissolved in 45 mL of water. After that, water was added to the solution to the volume of 50 mL (Solution B). To Solution A, Solution B was added slowly under stirring, and the resulting mixture was filtered through a sterilizing filter. The resulting solution, including 100 IU/mL of Lys-Arg insulin, was distributed under aseptic conditions into vials (1O mL each).

Example III. 362.3 mg of Lys-Arg insulin including 3.5% Zn was dissolved in 40 mL of 0.01 M hydrochloric acid. Then, to the resulting solution 0.01 M sodium hydroxide solution was added very slowly under stirring to reach pH 4.5. After that, water was added to the solution to the volume of 50 mL (Solution A). Separately, 0.3 g of m-cresol, 3.5 g of mannitol and zinc chloride (the amount necessary to reach the final concentration of Zn ions of 30 mcg/mL) were dissolved in 45 mL of water. After that, water was added to the solution to the volume of 50 mL (Solution B)

To Solution A, Solution B was added slowly under stirring, and the resulting mixture was filtered through an sterilizing filter. The resulting solution, including 100 IU/mL of Lys-Arg insulin, was distributed under aseptic conditions into vials (1O mL each).

Example IV. 362.3 mg of Lys-Arg insulin including 3.5% Zn was dissolved in 40 mL of 0.01 M hydrochloric acid. Then, to the resulting solution 0.01 M sodium hydroxide solution was added very slowly under stirring to reach pH 4.5. After that, water was added to the solution to the volume of 50 mL (Solution A). Separately, 0.3 g of m-cresol, 1.6 g of anhydrous glycerol, 1 g polysorbate 80 and zinc chloride (the amount necessary to reach the final concentration of Zn ions of 30 mcg/mL) were dissolved in 45 mL of water. After that, water was added to the solution to the volume of 50 mL (Solution B).

To Solution A, Solution B was added slowly under stirring, and the resulting mixture was filtered through a sterilizing filter. The resulting solution, including 100 IU/mL of Lys-Arg insulin, was distributed under aseptic conditions into vials (10 mL each).

Example V. 22 mg of zinc oxide was dissolved in 400 mL of 0.01 M hydrochloric acid, than 3,865 g of lizargτinsuline was added. Then, in the resulting solution 0.2

N sodium hydroxide solution was added very slowly under stirring to reach pH 4,5±0,2. Then, water was added to the solution up to the volume of 500 mL

(Solution A).

Separately, the solution containing 2,7 g of m-cresol, 16 g of anhydrous glycerol, in 500 mL of water was prepared. (Solution B).

To Solution A, Solution B was added under stirring during 15 minutes, and after pH correction to the value 4,5±0,2, the resulting mixture was filtered through a sterilizing filter. The resulting solution, was distributed under aseptic conditions to injection pen cartridges (3,3 mL each).

Example Vl. 724.6 mg of Lys-Arg insulin including 3.5% Zn was dissolved in 40 mL of 0.01 M hydrochloric acid and, then, 0.01 M sodium hydroxide solution was added very slowly under stirring to reach pH 4.1. Then, water was added to the solution up to the volume of 50 mL (Solution A).

Separately, 0.3 g of m-cresol, 1.6 g of anhydrous glycerol and zinc chloride (the amount necessary to reach the final concentration of Zn ions of 30 mcg/mL) was dissolved in 45 mL of water. Then, water was added to the solution up to the volume of 50 mL (Solution B).

To Solution A, Solution B was slowly added under stirring, and the resulting mixture was filtered through a sterilizing filter. The resulting solution, including 200 IU/mL of Lys-Arg insulin, was distributed into vials (10 ml_ each) under aseptic conditions.

Example VII. 1086.9 mg of Lys-Arg insulin including 3.5% Zn was dissolved in 40 mL of 0.01 M hydrochloric acid and, then, 0.01 M sodium hydroxide solution was very slowly added under stirring to reach pH 5.0. Then, water was added up to the volume of 50 mL (Solution A).

Separately, 0.3 g of m-cresol, 1.6 g of anhydrous glycerol and zinc chloride (the amount necessary to reach the final concentration of Zn ions of 40 mcg/mL) were dissolved in 45 mL of water. Then, water was added up to the volume of 50 mL (Solution B)

To Solution A, Solution B was added slowly under stirring, and the resulting mixture was filtered through a sterilizing filter. The resulting solution, including 300 IU/mL of Lys-Arg insulin, was distributed under aseptic conditions to injection pen cartridges (3 mL each).

Example VIII. 80 mg of Lys-Arg insulin including 3.5% Zn was dissolved in 40 mL of cooled (to a temperature below the room temperature) 0.25 M phosphate buffer solution (pH 11), and, then, under stirring pH was rapidly adjusted to 7.8 with 0.25 M phosphate buffer solution (pH 5.5). A precipitate appeared, which dissolved upon stirring within about 30 minutes.

Separately, 70 mg of methylparaben, 30 mg of propylparaben, 1.6 g of anhydrous glycerol and zinc chloride (the amount necessary to reach the final concentration of Zn ions of 30 mcg/mL) were dissolved in 25 mL of water. Then, water was added up to the volume of 50 mL (Solution B).

To Solution A, Solution B was added slowly under stirring. Then, water was added to reach the volume of 100 mL and the mixture was filtered through a sterilizing filter. The resulting solution including 20 IU/mL Lys-Arg insulin was distributed under aseptic conditions into vials (10 mL each).

Claims

Patent claims

1. A pharmaceutical composition comprising a biosynthetic analog of human insulin, wherein an effectively acting amount of the biosynthetic analog of human insulin of formula 1 or its physiologically acceptable salt, and excipients, are included.

Formula 1

2. A pharmaceutical composition according to Claim 1 , wherein: a. an effectively acting amount of the biosynthetic analog of human insulin of formula 1 or its physiologically acceptable salt and: b. an isotonizing agent; c. a preservative; d. a stabilizer; e. optionally, a buffer substance, are included.

3. A pharmaceutical composition according to Claim 1 - 2, wherein pH of the solution is from about 3,5 to about 8.5

4. A pharmaceutical composition according to Claim 1 - 3, wherein the isotonizing agent includes sodium chloride, mannitol or glycerol.

5. A pharmaceutical composition according to Claim 1 - 3, wherein the stabilizing agent includes zinc oxide or chloride or optionally surface-active agent such as polysorbate.

6. A pharmaceutical composition according to Claim 1 - 3, wherein a preservative selected from the group consisting of m-cresol, phenol, methylparaben, propylparaben and a mixture of these compounds, is included.

7. A pharmaceutical composition according to Claim 1 - 3, wherein 10-500 IU/mL of the biosynthetic analog of human insulin of formula 1 or its physiologically acceptable salt 16 mg/mL of glycerol, 3 mg/mL of m-cresol, 10-50 mcg/mL of zinc and water for injections up to 1 mL , are included.

8. A pharmaceutical composition according to Claim 5, wherein optionally 0,001- 0,2 mg/mL polysorbate is additionally included.

9. A pharmaceutical composition according to Claim 1-3 wherein 10-500 IU/mL of the biosynthetic analog of human insulin of formula 1 , or its physiologically acceptable salt 16 mg/mL of glycerol, 1 mg/mL of methylparaben- propylparaben mixture (7:3), 10-50 mcg/mL of zinc and water for injections up to 1 mL, are included.

10. A pharmaceutical composition according to Claim 2 or 9 wherein a phosphate buffer of pH 7.8-8.0 is included.

11. A use of the biosynthetic analog of human insulin of the isoelectric point between 3,5 and 8,5 of formula 1 or its physiologically acceptable salt, for preparation of a drug for the treatment of patients suffering from diabetes mellitus.

12. A use of the pharmaceutical composition according to Claim 1 - 2 for preparation of a drug for the treatment of patients suffering from diabetes mellitus.

13. A method of treatment of patients suffering from diabetes mellitus, wherein the effectively active amount of pharmaceutical composition according to Claim 1 - 3 is administered to them.