Insulin preparations containing carbohydrates

The subject of the invention is an aqueous insulin preparation and the method of its preparation.

Diabetes is a general term used to refer to disorders in people manifested as excessive urine output, as in the case of diabetes mellitus and diabetes insipidus. Diabetes mellitus is a metabolic disorder in which the ability to consume glucose is more or less completely lost. About 2% of all people suffer from diabetes.

Since the introduction of insulin in the 1920s, many steps have been made in improving the treatment of diabetes mellitus. In order to prevent high blood glucose levels, patients with diabetes often receive multiple insulin injections, with which insulin is given with each meal.

188 736

For the treatment of diabetes mellitus, a variety of insulin preparations have been suggested and used, such as normal insulin, Semilente® insulin, isophane insulin, insulin-zinc suspensions, protamine-zinc insulin, and Ultralente® insulin. Because diabetic patients have been treated with insulin preparations for many decades, there is a need for safer and quality-enhancing insulin preparations. Some of the commercially available insulin preparations have been characterized as fast-acting, and others as slow-acting, but with more or less prolonged action. Fast-acting insulin preparations are usually insulin suspensions, whereas delayed insulin preparations can be suspensions containing crystalline and / or amorphous insulin, precipitated by the addition of zinc salts alone or by the addition of protamine, or by a combination of both. In addition, some patients use preparations that have both fast and more prolonged action. Such preparations may be insulin solutions in which protamine insulin crystals are suspended. Some patients prepare their final preparations by mixing insulin solutions and suspension in the ratio required by the patient.

Normally, insulin preparations are administered by subcutaneous injection. The most important for the patient is the action profile of the insulin preparation, which corresponds to the action of insulin on glucose metabolism as a function of time after injection. In this profile, among other things, the time to the start of the activity, the maximum value and the total duration of the activity are important. Patients require a variety of insulin preparations with different action profiles. One patient can use insulin preparations that differ significantly in activity profiles on the same day. The required action profile varies depending on e.g. the time of day and the amount and composition of the meal eaten by the patient.

Equally important to the patient is the physical stability of insulin preparations, especially because of the widespread use of pen injection devices, such as devices containing Penfill cartridges, in which the insulin preparation is stored until the entire cartridge is empty. This may last for at least 1 to 2 weeks for instruments containing 1.5-3.0 ml.

The first stable neutral insulin suspension was developed by Scott and Fischer (J. Pharmacol. Exp. Ther. 58 (1936), 78) who discovered that the presence of excess protamine and zinc salt (2 pg zinc per Ul (International Unit) insulin) can stabilize the protamine insulin preparation described by Hagedom et al.: J. Am. Med. Assn. 106 (1936), 177-180.

The fully crystalline modification of zinc insulin protamine called NPH insulin or Isophane insulin was developed by Krayenbiihl and Rosenberg (see Rep. Steno Mem. Hosp. Nord. Insulinlab. 1 (1946), 60 and Danish Patent No. 64708). They found that insulin and protamine combined in isophane proportions at neutral pH in the presence of a small amount of zinc and phenol, or derivatives of phenol or, preferably m-cresol, will create an amorphous precipitate that, when standing, gradually transforms, but completely into longitudinal tetragonal crystals at the ends through pyramidal walls. Insulin and salmon protamine co-crystallize in a weight ratio corresponding to about 0.09 mg protamine sulfate per mg insulin. Zinc in an amount of at least 0.15 pg per IU and phenol in a concentration higher than 0.1% are necessary to maintain the tetragonal shape of the crystals.

In the early years, this type of crystal was prepared using pig or bovine insulin from natural sources, but since the eighties human insulin has been used for this purpose, produced by means of genetic or semi-synthetic engineering methods.

Human insulin consists of two polypeptide chains, called A and B, containing 21 and 30 amino acids, respectively. The A and B chains are connected to each other by two cysteine ​​disulfide bridges. Insulin from most other species is similarly built, but may not contain the same amino acids at positions that correspond to positions in human insulin chains.

The discovery of genetic engineering has made it possible to easily prepare a large number of various insulin compounds that are analogues of human insulin. In these insulin analogues, one or more amino acids have been substituted with other amino acids that can

188 736 be encoded by the nucleotide sequence. Since human insulin, as explained above, contains 51 amino acids, it is possible to create many insulin analogues, so that many different analogs having interesting properties have been developed. In human insulin solutions containing concentrations suitable for injectables, the insulin molecule is present in associated form as a hexamer (Brange et al., Diabetes Care 13, (1990), 923-954). It is believed that after subcutaneous injection, the degree of absorption by the blood current depends on the size of the molecule and it has been found that insulin analogues with amino acid substitutions that counteract or inhibit the formation of hexamer exhibit extremely fast activity (Brange et al., Supra). This is of great therapeutic importance for patients with diabetes. In crystals of protamine insulin preparations with prolonged action, insulin is also in hexameric form (Balschmidt et al; Acta Chryst. B47, (1991), 975-986).

Pharmaceutical preparations based on human insulin analogues are, for example, known from the following documents:

International publication WO 95/00550 relates to pharmaceutical preparations based on insulin crystals containing Asp^B28insulin and protamine, which, when administered in vivo, are characterized by rapid activity and prolonged activity. The crystals may further contain zinc ions and phenol and / or m-cresol. Glycerin is added to the preparations as an isotonizing agent.

U.S. Patent 5,461,031 discloses various pharmaceutical agents for parenteral administration containing a fast acting monomeric insulin analog, zinc, protamine and a phenolic derivative. The agents also contain glycerin, which plays the role of an isotonic agent.

U.S. Patent 5,474,978 discloses a fast-acting parenteral preparation containing a human insulin analog in the form of a hexameric complex consisting of six monomeric insulin analogs, zinc ions and at least three phenolic derivative molecules. Glycerin is a preferred isotonic agent.

The publication by Vinita Gupta et al. Entitled "Effect of Solvent Additives on the Thermal Stability of Insulin” published in Center for Biotechnology, vol. 70, p. 209212, discloses various preparations of cow insulin containing carbohydrates as additives, such as sorbitol, xylitol, mannitol and trehalose, which were used to improve the thermal stability of these preparations.

U.S. Patent 4,439, 181 relates to a method of preventing the deposition of proteins, such as in hormonal preparations, in drug delivery systems whose good functioning depends on the fluidity of the infusate. To this end, the C-4 to C-18 polyol is mixed with the protein solution before introducing this solution into the drug delivery system. Such polyol is added in an amount of 10 to 90% by weight / volume to prevent protein deposition during long-term storage in the drug delivery system.

Unfortunately, insulin tends to form insoluble and biologically inactive fibers by non-covalent polymerization (cf. e.g. Jens Brange, Galenics of Insulin, Springer-Verlag, 1987 and other references). Increased temperature, e.g. above 30 ° C, and associated movements contribute to fiber formation. The fibrosis process, which is very difficult to avoid, has an upper limit on the time that insulin can be stored and, therefore, in the volume of Penfill® cartridges.

Because fiber formation generally requires insulin monomerization, insulin analogues that are less likely to form di and hexamers make the formulations in which they are used less physically stable due to fibrosis.

It is therefore an object of the invention to provide insulin preparations containing human insulin or an analogue thereof or a derivative thereof, which preparations would have improved physical stability.

The subject of the invention is an aqueous insulin preparation characterized in that it comprises: a) crystals containing a human insulin analogue, in which position B28 is Asp, Lys, Leu, Val or Ala and in position B29 is Lys or Pro; or des (B28-B30), des (B27) or

188 736 des (B30) human insulin and protamine, and b) 100 to 400 mM mannitol, with an insulin analogue concentration of 60 to 3000 nmol / ml.

Preferably, this formulation additionally contains chloride.

The preparation of the invention contains Asp as a human insulin analogue^B28human insulin or LysB2⁸ProB2⁹human insulin.

In the preparation according to the invention, the crystals additionally contain zinc and optionally a phenolic compound.

Preferably, the formulation according to the invention contains both dissolved and crystalline insulin analogues in a weight ratio of 20:80 to 80:20.

The invention also relates to a method of preparing an insulin preparation containing both dissolved and crystalline insulin analogue, characterized in that there are steps in which:

a) an acid solution containing a human insulin analogue, zinc and a subisophane amount of protamine is prepared,

b) preparing a basic solution containing substances acting as a buffer at physiological pH, at least one of the above solutions further comprising a phenolic compound, and in addition at least one of the above solutions further comprising mannitol,

c) the acid solution is mixed with the alkaline solution and the pH is optionally adjusted to a value in the range of 6.5 to 8.0,

d) allowing the suspension obtained to precipitate.

In the method of the invention, the weight ratio of insulin analogue to protamine in the solution in step a) is selected so as to obtain a final product with a weight ratio of dissolved to crystalline insulin analogue in the range 20:80 to 80:20.

The solution of step a) preferably contains 120 to 6000 nmol / ml insulin analog and 0.01 to 5.0 mg / ml protamine.

The solution of step a) preferably additionally contains zinc in an amount corresponding to 10 to 40 [ig Zn / 100 IU insulin.

The solution of step a) and / or the solution of step b) preferably contains chloride in an amount corresponding to 5 to 40 mM in the final product.

In the process according to the invention, the pH of the acid solution from step a) is below 5.

Figure 1 is a micrograph (magnification x 1000) of an agent of the invention comprising AspB28 human protamine insulin crystals and mannitol.

Figure 2 is a micrograph (x1000 magnification) of an agent of the invention containing human protamine insulin crystals and mannitol.

Figure 3 is a graphic representation of the action profile of a formulation according to the invention containing both dissolved and crystalline AspB28 human insulin and a formulation containing both dissolved and crystalline human insulin. Both preparations additionally contain mannitol. The graph shows the blood glucose response following injection of pigs. The graph shows that the highly stable human insulin AspB ^ preparation is responsible for the rapid onset of action.

definitions

The term "human insulin analog" as used herein means human insulin in which one or more amino acids have been deleted and / or substituted with other amino acids, including non-coding amino acids, or human insulin containing additional amino acids, i.e. more than 51 amino acids.

The term "human insulin derivative" as used herein means insulin or an analogue thereof in which at least one organic substituent is attached to one or more amino acids.

The term "water-soluble" as used herein corresponds to a water solubility of at least 10 mmol / 1, preferably at least 50 mmol / 1, at 20 ° C.

The term carbohydrate, reduced carbohydrate, monosaccharide, disaccharide, ester and ether derivatives of these compounds as used herein was used in accordance with the terminology used in KA, Jensen "Grundrids af den organiske kemi, Almen Kemi III", 1st edition, Jul. Gjellerups forlag, 1969, pages 299-316.

188 736

The term "non-reducing carbohydrate," as used herein, means a carbohydrate that is essentially unable to react with the amino groups of insulin in the formulations of the invention and form glycated insulin. This definition also applies to carbohydrates in which they have been inactivated or blocked by a carbonyl group (s), e.g. by the formation of anhydrides or derivatives.

In a first embodiment, the present invention relates to aqueous insulin preparations containing:

dissolved or precipitated human insulin, an analogue and / or a derivative thereof, and

100 up to 400 mM, preferably 150 to 250 mM, more preferably 180 to 230 mM, water-soluble, reduced or non-reducing carbohydrate, containing at least 4 carbon atoms in the main carbohydrate structure or water-soluble, non-reducing ester and / or ether derivative of the carbohydrate or reduced carbohydrate containing at least 4 carbon atoms in the main carbohydrate structure, or mixtures thereof.

In another embodiment, the invention relates to aqueous insulin preparations containing: dissolved or precipitated human insulin, its analogue and / or its derivative, and water-soluble, reduced or non-reducing carbohydrate, containing at least 4 carbon atoms in the main carbohydrate structure or water-soluble, non-reducing ester and / or ether derivative of carbohydrate or reduced carbohydrate at least 4 carbon atoms in the main carbohydrate structure, or a mixture thereof.

In yet another embodiment, the invention relates to aqueous preparations of insulin analogues comprising:

dissolved or precipitated human insulin analogue, and a water-soluble, reduced or non-reducing carbohydrate, containing at least 4 carbon atoms in the main carbohydrate structure or a water-soluble, non-reducing ester and / or ether derivative of a carbohydrate or reduced carbohydrate containing at least 4 carbon atoms in the main structure carbohydrate, or a mixture thereof.

The carbohydrate or carbohydrate derivative used in the insulin preparation of the invention preferably contains from 5 to 18 carbon atoms in the main carbohydrate structure and is preferably selected from the following compounds:

i) monosaccharides selected from the group of non-reducing aldoses and ketoses, preferably non-reducing aldotetroses, ketotetroses, aldopentoses, ketopentoses, aldohexoses and ketohexoses, more preferably non-reducing aldopentoses, ketopentoses, aldohexoses and ketohexoses;

ii) reduced monosaccharides, i.e. polyhydric alcohols such as alditols, preferably selected from the group consisting of reduced forms of aldotetroses, ketotetroses, aldopentoses, ketopentoses, aldohexoses and ketohexoses, more preferably reduced forms of aldopentoses, ketopentoses and ketohexosolees. ).

iii) non-reducing disacchrides, preferably selected from non-reducing dihexoses.

Particular examples of suitable reduced or non-reducing carbohydrates are mannitol, sorbitol, xylitol, inositol, sucrose and trehalose.

The preferred compounds i) to iii) are mannitol, sorbitol, and most preferred is mannitol.

Preferred ester and ether derivatives are C1-C4 fatty acid ester derivatives and C1-C4 alkyl ether derivatives, respectively.

In a preferred embodiment of the invention, the insulin preparation further comprises a halide, preferably chloride, more preferably sodium chloride. The presence of a halide has been shown to provide better physical stability to the formulation.

Since insulin preparations containing fast-acting human insulin analogues generally exhibit rather low physical stability, the present invention is particularly useful in combination with preparations containing such analogs. Thus, the insulin preparations in accordance with the invention preferably contain one or more fast-acting human insulin analogues, in particular analogues in which Asp, Lys, Leu, Val or Ala is in position B28 and Lys or Pro in position B29; or des (B28-B30), des (B27) or des (B30) human

188 736 insulin. The insulin analogue is preferably selected from human insulin analogues in which the position B28 is Asp or Lys and in Dosage B29 is Lys or Pro. The most preferred analogs are Asp^B28human insulin and LysB²⁸Pro^B29human insulin.

In another embodiment, the formulation of the invention comprises an insulin derivative having a prolonged action profile, such as insulins having one or more lipophilic substituents. Preferred lipophilic insulins are acylated insulins, including those described in WO 95/07931 (Novo Nordisk A / S), B29, e.g. human insulin derivatives in which the Lys εamino group has an acyl substituent consisting of at least 6 carbon atoms.

Preferred insulin derivatives are:

B29-N^E-mirstoyl-des (B30) human insulin, B29-N^E-mirstoyl human insulin, B29N^E-palmitoilo human insulin, B28-N®-myristoyl Lys^B2⁸Pro2⁹ human insulin, B28-N¹'palmitoilo Lys^B2⁸Pro2⁹ human insulin, B30-N^e-mirystoilo-Thr^B2⁹lys³⁰ human insulin, B30N^Epalmitoyl-Thr^B2⁹lys³⁰ human insulin, B29-N^E- (N-palmitoyl-γ-glutamyl) -des (B30) human insulin, B29-N^E- (N-lithocholyl-γ-glutamyl) -des (B30) human insulin, B29-N^Ecarboxyheptadecanoyl) -des (B30) human insulin; the most preferred is B29-NE-myristoylodes (B30) human insulin.

In a preferred embodiment, the insulin preparation comprises both soluble and precipitated, preferably crystalline insulin, an analogue or derivative thereof in a weight ratio of 1:99 to 99: 1, preferably 20:80 to 80:20, more preferably 30:70 to 70:30.

In this embodiment of the invention, the insulin preparation preferably contains crystals comprising: insulin or an analogue or derivative thereof and protamine, in addition zinc and / or a phenolic compound such as phenol, m-cresol or a mixture thereof. The amount of protamine in the crystals preferably corresponds to 0.20 to 0.40 mg protamine base / 100 IU insulin or insulin analogue. The ratio of protamine to insulin in the crystals more preferably corresponds to the isophane ratio. Zinc is preferably present in an amount of 10 to 40 pg Zn / 100 IU insulin, preferably 15 to 35 pg Zn / 100 U insulin. The phenol and m-cresol are suitably preferably present in an amount corresponding to 0 to 4 mg / ml. However, a mixture of 1.4 to 2.0 mg / ml m-cresol and 0.6 to 2.0 mg / ml phenol is most preferred.

In a preferred embodiment of the invention, the insulin preparation comprises:

a) 60 to 3000 nmol / ml, preferably 240 to 1200 nmol / ml human insulin or analogue and / or insulin derivative,

b) reduced or non-reducing carbohydrate, preferably mannitol, in a concentration of 100 to 400 mM, preferably 150 to 250 mM, more preferably 180 to 230 mM.

c) chloride, preferably sodium chloride, in a concentration of 0 to 100 mM, preferably 5 to 40 mM, more preferably 5 to 20 mM; and

d) a physiologically tolerable buffer, preferably a phosphate buffer such as disodium phosphate dihydrate in an amount of 1 to 4 mg / ml.

The formulation of the invention may further contain one or more compounds commonly used as isotonizing agents, such as glycerin.

The pH of the insulin preparations is preferably from 7.0 to 7.8.

In addition, the present invention relates to a method of preparing an insulin preparation containing both dissolved and precipitated insulin analogues, which comprises the following steps:

a) providing an acidic solution containing a human insulin analogue, zinc and a subisophane amount of protamine;

b) providing a basic solution containing substances that act as a buffer at physiological pH;

at least one of the above solutions further comprising a phenolic compound;

c) mixing the acid and base solution and, in addition, adjusting the pH to a value in the range 6.5 to 8.0, preferably 7.0 to 7.8; and

d) allowing the resulting suspension to precipitate.

In this way, it is very easy to obtain an insulin preparation containing both dissolved and precipitated insulin analogues. In addition, the suspension loss contains rod-shaped crystals that are beneficial in so-called PreMix insulin preparations.

188 736

The weight ratio of insulin analogue to protamine in the solution of step a) is preferably selected so as to achieve the final product in a weight ratio of dissolved to precipitated insulin analogue in the range 1:99 to 99: 1, preferably 20:80 to 80:20, more preferably 30: 70 to 70:30. In particular, the solution of step a) preferably contains 120 to 6000 nmol / ml insulin analog and 0.01 to 5.0 mg / ml protamine.

The solution of step a) further contains zinc, preferably in an amount corresponding to 10 to 40 (ig Zn / 100 IU insulin, preferably 15 to 35 pg Zn / 100 U insulin).

In a preferred embodiment solution a) and / or solution b) contain chloride, preferably sodium chloride, in an amount corresponding to 0 to 100 mM, preferably 5 to 40 mM, more preferably 5 to 20 mM of final product.

The pH of the acid solution in step a) is preferably below 5, more preferably it ranges from 2 to 3.5.

The insulin analogue is preferably insulin having an Asp, Lys, Leu, Val or Ala position B28 and a Lys or Pro position b29; or des (B28-B30), des (B27) or des (B30) human insulin, more preferably AspB2⁸human insulin or LysB2⁸ProB ^ human insulin, most preferably Asp human insulin.

The phenolic compound used in the solutions of step a) and / or step b) is preferably phenol, m-cresol or a mixture thereof.

Preferably, solution a) and / or b) further comprises a water-soluble, reduced or non-reducing carbohydrate containing at least 4 carbon atoms in the main carbohydrate structure or water-soluble, non-reducing ester and / or ether derivative of a carbohydrate or reduced carbohydrate containing at least 4 atoms carbon in the main carbohydrate structure, or a mixture thereof.

Said carbohydrate or carbohydrate derivative preferably contains 5 to 18 carbon atoms in the main carbohydrate structure.

In a particularly preferred embodiment, the solution of step a) and / or step b) comprises mannitol, sorbitol, xylitol, inositol, trehalose, sucrose or any mixture thereof, preferably mannitol and / or sorbitol, and most preferably mannitol.

The buffering compound used in the alkaline solution of step b) is preferably a physiologically tolerable buffer, more preferably a phosphate buffer, most preferably disodium phosphate dihydrate.

The precipitated insulin analogue is preferably in the form of crystals containing insulin analogue and protamine.

The suspension obtained in step a) is allowed to precipitate preferably at a temperature in the range of 5 ° C to 40 ° C, more preferably 20 ° C to 36 ° C, most preferably 30 ° C to 34 ° C.

The invention will then be illustrated by the following examples, which do not limit its scope.

Example 1

Preparation 1

An insulin preparation containing both dissolved and crystalline AspB2 was prepared⁸human insulin as follows:

Solution A:

A solution of Asp human insulin at a concentration of 200 IU / ml was prepared by dissolving 76.5 mg of AspB28 human insulin in water, by adding 326 µl of 0.2N hydrochloric acid and 163 µl of zinc chloride solution (0.4 mg / ml). Then 6.35 mg protamine sulfate in solution and a mixture consisting of 17.2 mg mcresol, 15 mg phenol and 455 mg mannitol were added with stirring. The pH of the clear solution was measured to 2.6-2.9. Then water was added to 10 ml. The solution was equilibrated at 28-32 ° C.

Solution B:

mg dvmodium phosphate was added in the injection water. 17.2 mg m-cresol, 15 mg phenol and 455 mg mannitol were added with stirring. The pH of the clear solution was measured to 9. Water was added to 10 ml. The solution was equilibrated at 28-32 ° C.

Mixing of solutions A and B

Solution B was added to solution A, the pH was adjusted to 7.30. The resulting suspension was allowed to crystallize at 30 ° C for 6 days.

188 736

In the resulting formulation, the weight ratio of precipitated to dissolved insulin was 70:30.

The formulation was used to fill 1.5 ml Penfill® cartridges.

Example II

Preparation 2

An insulin preparation was prepared containing both dissolved and crystalline Asp^B28human insulin as follows:

i) crystalline fraction.

Solution A:

A solution of Asp human insulin at a concentration of 200 IU / ml was prepared by dissolving 190.3 mg B ^ Asp human insulin in water by adding 813 μΐ 0.2N hydrochloric acid and 410 μl zinc chloride solution (0.4 mg / ml). Then, 16.1 mg protamine sulfate in solution and a mixture consisting of 43.0 mg m-cresol, 37.5 mg phenol and 909 mg mannitol and 14.6 mg sodium chloride were added with stirring. The pH of the clear solution was measured to 2.6-2.9. Then water was added to 22 ml. The solution was equilibrated at 32 ° C.

Solution B:

62.4 mg of disodium phosphate dihydrate were dissolved in water. 43.0 mg m-cresol, 37.5 mg phenol and 909 mg maooitrile and 14.6 mg sodium chloride were added with stirring. The pH of the clear solution was measured to 9. Water was added to 22 ml. The solution was equilibrated at 32 ° C.

Mixing of solutions A and B

Solution B was added to solution A, the pH was adjusted to 7.30. Water was added to 50 ml. The resulting suspension was allowed to crystallize at 32 ° C for 4 days.

In the resulting formulation, the weight ratio of precipitated to dissolved insulin was 70:30.

The preparation was introduced into 1.5 ml Penfill® cartridges.

Examples III-VI

Preparations 3 to 6

Insulin preparations containing both dissolved and crystalline AspB2 were prepared⁸human insulin as described in Example II, except that the amount of mannitol used in Solution A and B was 818 mg, 1005 mg, 1047 mg and 1137 mg, respectively.

The preparations were introduced into 1.5 ml Penfill® cartridges.

Example VII

Preparation 7

An insulin preparation containing both dissolved and crystalline LysB2 was prepared⁸ProB²⁹human insulin as follows:

i) crystalline fraction.

Solution A:

Lys Pro solution for human insulin at a concentration of 200 IU / ml was prepared by suspending 69.7 mg LysB2⁸PrnB2⁹human insulin in water. Then a mixture consisting of 16.0 mg m-cresol, 6.5 mg phenol and 364 mg mannitol and 25.1 mg disodium phosphate dihydrate was added with stirring. Then 50 μΐ zinc chloride solution (10 mg / ml) was added. The pH of the clear solution was measured to 7.40. Then water was added to 10 ml. The solution was equilibrated at 15 ° C.

Solution B:

A solution of protamine sulfate was prepared by dissolving 7.61 mg of protamine sulfate and 25.1 mg of disodium phosphate dihydrate in water. Mixtures of 16.0 mg m-cresol, 6.5 mg phenol and 364 mg mannitol and 14.6 mg sodium chloride were added with stirring. The pH of the clear solution was measured to 7.40. Water was added to 10 ml. The solution was equilibrated at 15 ° C.

Mixing of solutions A and B

Solution B was added to solution A, the pH was adjusted to 7.30. The resulting suspension was allowed to crystallize at 15 ° C for 3 days.

188 736 ii) Protein fraction ^ A Lys solution was prepared^B28Pro^B29human insulin by dissolving 34.9 mg of Lys ^ ProB ^ human insulin in water by adding 33 p and 1N hydrochloric acid and 25 pl of zinc chloride solution (10 mg / ml). Then a mixture consisting of 26.1 mg disodium phosphate dihydrate, 6.5 mg phenol, 16.0 mg m-cresol and 364 mg mannitol was added with stirring. The pH of the clear solution was measured to 7.3. Then water was added to 10 ml.

ml of the dissolved fraction was added to 14 ml of the crystalline fraction, the pH was adjusted to 7.30.

In the resulting formulation, the weight ratio of precipitated to dissolved insulin was 70:30.

The preparation was introduced into 1.5 ml Penfill® cartridges.

Example VIII Preparation 8

An insulin preparation containing both dissolved and crystalline human insulin was prepared as follows:

i) crystalline fraction.

Solution A:

A solution of human insulin was prepared by dissolving 69.7 mg of human insulin in water by adding 65 µL of 1N hydrochloric acid and 26 µL zinc chloride chloride root (10 mg / ml). Next, 6.0 mg of sulfur, again potassium in solution, and the mixture were added. consisting of 15 mg phenol, 17.2 mg m-cresol. The pH of the clear solution was 2.7-3.2 Then water was added to 10 ml.

Solution B:

24.9 mg of sodium phosphate dihydrate were dissolved in water. 15 mg phenol, 17.2 mg m-cresol and 728 mg mnkkitol and 11.7 mg sodium chloride were added with stirring. The pH of the clear solution was measured to 9. Water was added to 10 ml.

Solution B added to solution A, the pH was adjusted to 7.30. The resulting suspension remained at 22-24 ° C until the next day.

ii) Dissolved fraction

A solution of human insulin was prepared by dissolving 34.9 mg of human insulin in water by adding 33 µL of 1N hydrochloric acid and 13 µL of zinc chloride solution (10 mg / ml). Then by mixing the mixture consisting of 12.5 mg disodium phosphate dihydrate, 15 mg phenol, 17.2 mg m-cresol, 364 mg maocitol and 5.8 mg sodium chloride. The measured pH of the Ularovoyg solution was 7.3. Then water was added to 10 ml.

ml of the dissolved fraction was added to 14 ml of the Urystniizzka fraction, pH adjusted to 7.30.

In the resulting formulation, the weight ratio of precipitated to dissolved insulin was 70:30.

The preparation was introduced into 1.5 ml Penfill® cartridges.

Example IX

Preparation 9

An insulin preparation containing both dissolved and Ucnstalizzone AspB2 was prepared⁸Loose insulin as follows:

Solution A:

nio

A solution of Asp human insulin at a concentration of 200 IU / ml was prepared by dissolving 189.9 mg of AspB2⁸human insulin in water with dddnniy 163 pl of 1N hydrochloric acid and 163.5 pl of zinc chloride solution (10 mg / ml). Then, 11.5 mg protamine sulfate in solution and a mixture consisting of 44.3 mg m-cresol, 38.6 mg phenol, 1048 mg maonitol and 7.3 mg sodium chloride were added with stirring. The pH of the calcified solution was measured to 2.6-2.9. Then water was added to 25 ml. The solution was equilibrated at 22-24 ° C.

Solution B:

62.3 mg disodium phosphate dihydrate was dissolved in water. By mixing 0odaoo 44.3 mg m-cresol, 38.6 mg phenol, 1048 mg manoitol and 7.3 mg sodium chloride. The measured pH of the clear solution was 9. Water was added to 25 ml. The solution was equilibrated at 22-24 ° C.

Mixing of solutions A and B

Solution B was added to solution A, the pH was adjusted to 7.30. The resulting suspension was allowed to crystallize at 32 ° C for 2 days.

In the resulting formulation, the weight ratio of precipitated to dissolved insulin was 50:50.

The formulation was used to fill 1.5 ml Penfill® cartridges.

Example X (Comparing)

An insulin preparation containing both dissolved and crystalline Asp828 human insulin was prepared as follows.

Solution A:

dao

A solution of Asp human insulin at a concentration of 200 IU / ml was prepared by dissolving 76.5 mg of AspB28 human insulin in water by adding 326 µl of 0.2N hydrochloric acid and 163 µl of zinc chloride solution (0.4 mg / ml). Then 6.35 mg protamine sulfate in solution was added while mixing, and a mixture consisting of 17.2 mg m-cresol, 15 mg phenol and 160 mg glycerin was added. The pH of the clear solution was measured to 2.6-2.9. Then water was added to 10 ml. The solution was equilibrated at 28-32 ° C.

Solution B:

mg diode phosphate dihydrate was dissolved in Water for Injections. 17.2 mg m-cresol, 15 mg phenol and 160 mg glycerin were added with stirring. The pH of the clear solution was measured to 9. Water was added to 10 ml. The solution was equilibrated at 28-32 ° C.

Solution B was added to solution A, the pH was adjusted to 7.30. The resulting suspension was allowed to crystallize at 28-32 ° C for 2 days.

In the resulting formulation, the weight ratio of precipitated to dissolved insulin was 70:30.

The formulation was used to fill 1.5 ml Penfill® cartridges.

Example XI

Physical stress tests on samples of each insulin preparation were introduced into Penfill® cartridges and subjected to the following physical stress tests:

Penfill® cartridges were placed in a rotator which was placed in an incubator and rotated 360 ° for four hours at 30 rpm and at a constant temperature of 37 ° C ± 2 ° C. While not rotating, Penfill® cartridges were stored at 37 ° C ± 2 ° C.

Penfill 'cartridges were examined macroscopically 5 times a week and changes in the appearance of the preparations were noted according to the following principles:

i) Cartridges containing a suspension resuspending after shaking and free of lumps and granules were classified as "non-fibrotic".

ii) Cartridges containing a slurry with lumps and / or granules that do not re-suspend after shaking and / or deposited on the walls of the cartridge are classified as "fibrous". This was confirmed by the addition of 6 μ1 6N HCl to the cartridge: the fibrotic cartridges did not become clear when the acid was added.

Cartridge rotation continued until all samples were fibrosis

The results are summarized in Table 1.

188 736

Table 1

<td>Preparation No.</td><td>Composition</td><td>Average number of days to fibrosis</td><td>First day of fibrosis</td>

<td> 1</td><td>AspB2⁸insulin 30/70 7 mM phosphate 250 mM mannitol</td><td> 20</td><td> 20</td>

<td> 2</td><td>AspB2⁸ insulin 30/70 7 mM phosphate 200 mM mannitol 10 mM sodium chloride</td><td> 23</td><td> 23</td>

<td> 3</td><td>AspB2⁸insulin 30/70 7 mM phosphate 180 mM mannitol 10 mM sodium chloride</td><td> 21</td><td> 17</td>

<td> 4</td><td>AspB2⁸jnsulin 30/70 7 mM phosphate 220 mM mannitol 10 mM sodium chloride</td><td> 19</td><td> 17</td>

<td> 5</td><td>AspB2⁸insulin 30/70 7 mM phosphate 230 mM mannitol 10 mM sodium chloride</td><td> 21</td><td> 18</td>

<td> 6</td><td>AspB2⁸insulin 30/70 7 mM phosphate 250 mM mannitol 10 mM sodium chloride</td><td> 22</td><td> 22</td>

<td> 9</td><td>AspB28jnsulin 50/50 7 mM phosphate 230 mM mannitol 10 mM sodium chloride</td><td> &gt;30</td><td> &gt;30</td>

<td>10 (Comparing)</td><td>AspB28 insulin 30/70 7 mM phosphate 174 mM glycerin</td><td> 11</td><td> 9</td>

UP Department of Publications. Circulation of 50 copies Price PLN 4.00