RU2204411C2 - Carbohydrate-containing insulin preparations - Google Patents

Abstract

FIELD: medicine, endocrinology, hormones. SUBSTANCE: invention relates to insulin preparations with the excellent physical stability comprising dissolved and/or precipitated human insulin or its analogue, or its derivative and water-soluble reduced or non-reduced carbohydrate containing at least 4 carbon atoms in the basic carbohydrate structure, or water-soluble non-reducing ester and/or ether derivative of carbohydrate or reduced carbohydrate containing at least 4 carbon atoms in basic carbohydrate structure, or their mixture. Invention provides preparing the insulin preparation with enhanced physical stability. EFFECT: improved preparing method. 58 cl, 3 dwg, 1 tbl

Description

 This invention relates to aqueous insulin preparations containing human insulin or an analogue or derivative thereof, and these preparations have excellent physical stability. In addition, this invention relates to parenteral formulations comprising such insulin preparations, and to a method for increasing the physical stability of insulin preparations.

State of the art
Diabetes is a general term for disorders in a person who have excessive urination in both diabetes and diabetes mellitus. Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is more or less lost. About 2% of the total population suffers from diabetes.

 Since insulin was introduced in the 1920s, continuous research has been ongoing to improve the treatment of diabetes. In order to help avoid extreme glycemic levels, patients with diabetes often practice multiple injections with which they can administer insulin with each meal.

A wide variety of insulin preparations have been proposed and used for the treatment of diabetes, such as regular insulin, Semilente ^® insulin, isofan insulin, insulin zinc suspension, protamine zinc insulin and Ultralente ^® insulin. Since patients with diabetes have been treated with insulin for several decades, there is a need to improve insulin preparations to improve safety and improve the quality of life of patients. Some of the commercially available insulin preparations are characterized by a rapid onset of action, and other drugs have a relatively delayed (delayed) onset (s), but show a more or less prolonged effect. Fast acting insulin preparations (short-acting drugs) are usually insulin solutions, while delayed-acting insulin preparations can be suspensions containing insulin in crystalline and / or amorphous form, precipitated by adding only zinc salts or by adding protamine, or a combination of both of another. In addition, some patients use drugs that have both a fast onset of action and a more prolonged action. Such a preparation may be an insulin solution in which crystals of protamine-insulin are suspended. Some patients themselves receive the final drug by mixing a solution of insulin with the drug in the form of a suspension in the ratio required by the patient in question.

 Typically, insulin preparations are administered by subcutaneous injection. For the patient, the profile of the action of the insulin preparation is important, which is the effect of insulin on glucose metabolism as a function of time from the (moment) of administration. In this profile, among other things, the start time (action), the maximum value and the total (total) duration of the action are important. For patients, there is a need and demand for a range of insulin preparations with different profiles of action. One patient can use insulin preparations with very different action profiles for one day. The desired profile of action depends, for example, on the time of day and the amount and composition of food taken by the patient.

The physical stability of insulin preparations is also important due to the abundance of pen-type injection devices used, such as devices that contain Penfill ^® cartridges, in which the insulin preparations are stored until the entire cartridge is empty. This can last for at least 1 to 2 weeks for devices containing 1.5-3.0 ml cartridges.

Prior level
The first stable neutral suspension of insulin was developed by Scott and Fischer (J. Pharmacol. Exp. Ther. 58 (1936), 78), which found that the presence of excess protamine and zinc salt (2 μg zinc per ME (IU) (international Unit ) insulin) can stabilize the protamine-insulin preparation described by Hagedorn et al .: J. Am. Med. Assn. 106 (1936), 177-180.

 Protamine-zinc insulin, made according to the pharmacopoeias of the United States and Europe (United States or European Pharmacopoeias), contains amorphous protamine-zinc insulin, as well as crystalline protamine-zinc insulin. Freshly prepared protamine-zinc insulin contains mainly an amorphous precipitate, which usually partially transforms into crystalline particles during storage, leading to a more delayed action.

и Rosenberg (см. Rep. Steno Mem. Hosp. Nord. Insulinlab. 1 (1946), 60; и Danish patent 64 708). Они обнаружили, что инсулин и протамин, вносимые вместе в пропорциях изофана при нейтральном значении рН в присутствии небольшого количества цинка и фенола или производных фенола, или предпочтительно м-крезола, будут образовывать аморфный осадок, который при стоянии постепенно, но полностью трансформируется в удлиненные тетрагональные кристаллы, ограниченные по концам пирамидальными гранями. Инсулин и оранжево-розовый протамин совместно кристаллизуются в массовом отношении, отвечающем приблизительно 0,09 мг протамин-сульфата на мг инсулина. Цинк в количестве, по крайней мере, 0,15 мкг на ME, и фенольного соединения в концентрации выше, чем 0,1%, является необходимым для получения тетрагональных кристаллов.A modification of fully crystalline insulin protamine-zinc, designated NPH insulin or isophan insulin, has been developed

and Rosenberg (see Rep. Steno Mem. Hosp. Nord. Insulinlab. 1 (1946), 60; and Danish patent 64 708). They found that insulin and protamine, taken together in isophan proportions at a neutral pH in the presence of a small amount of zinc and phenol or phenol derivatives, or preferably m-cresol, will form an amorphous precipitate, which, when standing, gradually but completely transforms into elongated tetragonal crystals bounded at the ends by pyramidal faces. Insulin and orange-pink protamine co-crystallize in a mass ratio corresponding to approximately 0.09 mg of protamine sulfate per mg of insulin. Zinc in an amount of at least 0.15 μg per ME, and a phenolic compound in a concentration higher than 0.1%, is necessary to obtain tetragonal crystals.

 Previously, this type of crystals was obtained using pork and bovine insulin from natural sources, but since the 80s, human insulin has been used, obtained by genetic engineering or in a semi-synthetic way.

 Human insulin consists of two polypeptide chains, the so-called chains A and B, which contain 21 and 30 amino acids, respectively. Chains A and B are connected to each other by two cystine disulfide bridges. Insulin from most other varieties (sources) has 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 a method known as genetic engineering has made it possible to easily obtain a large number of insulin compounds like human insulin. In these insulin analogs, one or more of the amino acids is replaced by other amino acids that can be encoded by nucleotide sequences. Since human insulin, as indicated above, contains 51 amino acid residues, it is obvious that a large number of insulin analogues is possible, and in fact a large number of analogues with interesting properties have been obtained. In solutions of human insulin with a concentration of interest for the injectable preparation, the insulin molecule is present in an associated form as a hexamer (Brange et al. Diabetes Care 13, (1990), 923-954). After subcutaneous injection, it is believed that the rate of absorption by blood flow 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 this hexamer have an unusually fast onset of action (Brange et al .: Ibid). This is of great therapeutic value to a patient with diabetes. In sustained release protamine insulin crystals, insulin has also been found to be in hexameric form (Balschmidt et al .: Acta Chryst. B47, (1991), 975-986).

Pharmaceutical preparations that are based on analogues of human insulin, for example, are known from the following documents:
WO 95/00550 describes pharmaceutical preparations based on insulin crystals, including Asp ^B28 insulin and protamine, which exhibit rapid onset of action and prolonged activity when administered in vivo. In addition, the crystals may contain zinc ions and phenol and / or m-cresol. As a means of isotonicity, glycerin is added to the preparations.

 US 5,461,031 discloses various parenteral pharmaceutical formulations that include a fast-acting analogue of insulin monomer, zinc, protamine, and a phenolic derivative. Finished forms, in addition, contain glycerin, which acts as a means of isotonicity.

 US 5,474,978 discloses a fast-acting parenteral dosage form comprising a complex of the hexameric analogue of human insulin, consisting of six analogs of monomeric insulin, zinc ions and at least three phenolic derivative molecules. Glycerin is the preferred isotonicity agent.

Unfortunately, insulin tends to form insoluble and biologically inactive fibrils by non-covalent polymerization (cf., for example, Jens Brange, Galenics of Insulin, Springer-Verlag, 1987 and references therein). The formation of fibrils is accelerated at elevated temperatures, for example above 30 ^o With, and related effects. This process of fibrillation, which is very difficult to avoid, raises the question of the upper limit of the period of time during which the insulin preparation can be stored, and therefore the volume of the Penfills ^® cartridge.

 Since the formation of fibrils usually requires insulin monomerization, insulin analogues that are more or less susceptible to the formation of di- and hexamers lead to drugs with less physical stability due to fibrillation.

 Thus, it is an object of the present invention to provide an insulin preparation comprising human insulin or an analogue thereof or a derivative with enhanced physical stability.

 According to the invention, this goal is achieved by creating an aqueous insulin preparation comprising dissolved and / or precipitated human insulin or an analogue or derivative thereof, and a water-soluble reduced or non-reducing carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate, or water-soluble non-reducing ester and / or ester derivative of a carbohydrate or reduced carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate , or mixtures thereof.

A brief description of figures 1-3:
- FIG. 1 is a photomicrograph (x1000 magnification) of the finished form of the present invention, including Asp ^B28 human insulin-protamine crystals and mannitol;
- FIG. 2 is a micrograph (x1000 magnification) of the finished form of the present invention, including human insulin-protamine crystals and mannitol;
- figure 3 is a graphical representation of the profile of the action of the drug of the present invention, containing both dissolved and crystalline Asp ^B28 human insulin, and a preparation containing both dissolved and crystalline human insulin. Both drugs also contain mannitol. The graph represents the blood glucose response after injection in pigs. In FIG. Figure 3 shows that a fast onset of action is maintained for the highly stable preparation of Asp ^B28 human insulin.

SUMMARY OF THE INVENTION
Definitions
As used herein, the term “human insulin analogue” means human insulin in which one or more amino acids are removed and / or replaced by other amino acids, including non-encoded amino acids, or human insulin, including additional amino acids, i.e., more than 51 amino acids.

 As used herein, the term “human insulin derivative” means human insulin or an analogue thereof, in which at least one organic substituent is bound to one or more amino acids.

In this context, the term "water soluble" corresponds to a solubility in water of at least about 10 mmol / L, preferably at least 50 mmol / L at a temperature of 20 ^o C.

 The terms “carbohydrate”, “reduced carbohydrate”, “monosaccharide”, “disaccharide”, and ester and ester derivatives of such compounds are used as directed by K. A. Jensen in “Grundrids af den organiske kemi”, Almen Kemi III, 1. Ed ., Jul. Gjellrups forlag, 1969, pp. 299-316.

 As used herein, the term “non-reducing carbohydrate” means a carbohydrate that is generally not able to interact with the amino groups of insulin in the preparations of this invention to form glycated insulin. This definition includes carbohydrates in which the carbonyl group (s) are inactivated (s) or blocked (s), for example, by the formation of anhydride or derivatization.

In one aspect, the present invention relates to an aqueous insulin preparation, comprising:
dissolved and / or precipitated human insulin or its analogue and / or derivative, and
from 100 to 400 mm, preferably from 150 to 250 mm, more preferably from 180 to 230 mm water-soluble reduced or non-reducing carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate, or water-soluble non-reducing ester and / or ester derivative of carbohydrate or a reduced carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate, or a mixture thereof.

In another aspect, the present invention relates to an aqueous insulin preparation, comprising:
dissolved and precipitated human insulin or an analogue and / or derivative thereof, and
a water-soluble reduced or non-reducing carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate, or a water-soluble non-reducing non-reducing ester and / or ether derivative of a carbohydrate or reduced carbohydrate containing at least 4 carbon atoms in the basic structure of a carbohydrate mixtures.

In another aspect of the embodiment, this invention relates to an aqueous preparation of an insulin analogue, including:
a dissolved and / or precipitated human insulin analogue and a water-soluble reduced or non-reducing carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate, or a water-soluble non-reducing ester and / or ester derivative of a carbohydrate or reduced carbohydrate containing at least 4 carbon atom in the basic structure of carbohydrate, or mixtures thereof.

Preferred Embodiments
The carbohydrate or carbohydrate derivative used in the insulin preparation of the present invention preferably contains from 5 to 18 carbon atoms in the basic structure of the carbohydrate and is preferably selected from the following compounds:
i) monosaccharides selected from the group of non-reducing aldoses or ketoses, preferably non-reducing aldotetrosis, ketotetrosis, aldopentosis, ketopentosis, aldohexoses and ketohexoses, more preferred non-reducing aldopentoses, ketopentoses, ketohexoses and
ii) reduced monosaccharides, i.e. polyhydric alcohols, such as alditols, preferably selected from the group consisting of reduced forms of aldotetrosis, ketotetrosis, aldopentosis, ketopentosis, aldohexoses and ketohexoses, more preferably from reduced forms of aldopentoses, ketopentoses, aldohexoses and ketohexoses and (i) pentos;
iii) non-reducing disaccharides, preferably selected from non-reducing dihexoses.

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

 Preferred compounds i) to iii) are mannitol and sorbitol, with mannitol being most preferred.

Preferred ester and ester derivatives are ester derivatives of C ₁ -C ₄ fatty acids and C ₁ -C ₄ alkyl ester derivatives, respectively.

 In a particular embodiment of the invention, the insulin preparation further comprises a halide, preferably chloride, more preferably sodium chloride. It has been shown that the presence of a halide leads to preparations having higher physical stability.

Since insulin preparations, including high-speed analogues of human insulin, usually exhibit very low physical stability, this invention, in particular, has advantages in connection with preparations comprising such analogues. Thus, the insulin preparation according to this invention preferably includes one or more high-speed analogues of human insulin, in particular, analogues in which position B28 represents Asp, Lys, Leu, Val or Ala and position B29 represents Lys or Pro; or des (B28-B30), or des (B27) or des (B30) human insulin. The insulin analog is preferably selected from human insulin analogs in which position B28 represents Asp or Lys and position B29 represents Lys or Pro. Most preferred analogs are Asp ^B28 human insulin or Lys ^B28 Pro ^B28 human insulin.

In another embodiment, the insulin preparation according to 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 insulins described in WO 95/07931 (Novo Nordisk A / S), for example, human insulin derivatives in which the ε-amino group of Lys ^B29 contains an acyl substituent that includes at least 6 carbon atoms.

Preferred derivatives of insulin are as follows:
B29-N ^ε -Mistyloyl des (B30) -human insulin, B29-N ^ε- myristoyl human insulin, B29-N ^ε -palmitoyl human insulin, B28-N ^ε -mistoyl Lys ^B28 Pro ^B29 -human insulin, B28-N ^ε- palmitoyl Lys ^B28 Pro ^B29 -human insulin, B30-N ^ε- myristoyl-Thr ^B29 Lys ^B30 -human insulin, B30-N ^ε -palmitoyl-Thr ^B28 Lys ^B30 -human insulin, B29-N ^ε - (N-palmitoyl -γ-glutamyl) -des (B30) -human insulin, B29-N ^ε - (N-lithocholyl-γ-glutamyl) -des (B30) -human insulin and B29-N ^ε - (ω-carboxyheptadecanoyl) -des ( B30) -human insulin; most preferred is B29-N ^ε -Mirstoyl des (B30) -human insulin.

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

 In this embodiment of the invention, the insulin preparation advantageously comprises crystals comprising insulin or an insulin analog and protamine, and optionally 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 from 0.20 to 0.40 mg of protamine base / 100 ME of insulin or an insulin analogue. The ratio of protamine to insulin in crystals more preferably corresponds to the isophan ratio. Zinc is preferably present in an amount of from 10 to 40 μg Zn / 100 ME of insulin, more preferably from 15 to 35 μg Zn / 100 ME of insulin. Phenol and m-cresol, respectively, are preferably present in an amount corresponding to from 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.

A preferred insulin preparation according to the invention includes:
a) from 60 to 3000 nmol / ml, preferably from 240 to 1200 nmol / ml, human insulin or an insulin analog and / or an insulin derivative;
b) a reduced or non-reducing carbohydrate, preferably mannitol, in a concentration of from 100 to 400 mm, preferably from 150 to 250 mm, more preferably from 180 to 230 mm;
c) chloride, preferably sodium chloride, in a concentration of from 0 to 100 mm, preferably from 5 to 40 mm, more preferably from 5 to 20 mm; and
d) a physiologically tolerant buffer, preferably a phosphate buffer, such as disodium phosphate dihydrate, in an amount of 1 to 4 mg / ml.

 The preparation of the present invention may also contain one or more compounds commonly used as isotonicity agents such as glycerin.

 The pH of the insulin preparation is preferably in the range of 7.0 to 7.8.

In addition, this invention relates to a method for producing an insulin preparation containing both dissolved and precipitated insulin analogs, and this method includes the following steps:
a) obtaining an acidic solution comprising an analog of human insulin, zinc, and a subisophanic (subisophanic) amount of protamine;
b) obtaining an alkaline solution comprising a substance that acts as a buffer at physiological pH;
where at least one of the above solutions further includes a phenolic compound;
c) mixing the acidic and alkaline solutions and optionally adjusting the pH to 6.5 to 8.0, preferably 7.0 to 7.8; and
d) maintaining the resulting suspension for precipitation.

 Using this method, an insulin preparation containing both dissolved and precipitated insulin analogs can be obtained in a very simple way. In addition, the precipitate of the suspension obtained usually contains rod-shaped crystals, which are useful in the so-called PreMix insulin preparations.

 The mass ratio of the insulin analog to protamine in the solution of step a) is preferably selected so as to obtain a mass ratio of insulin dissolved to the precipitated analog of the insulin in the final product in the range from 1:99 to 99: 1, preferably from 20:80 to 80:20, more preferably from 30: 70 to 79:30. In particular, the solution of step a) preferably comprises from 120 to 6000 nmol / ml of the insulin analogue and from 0.01 to 5.0 mg / ml of protamine.

 The solution of step a) further comprises zinc, preferably in an amount corresponding to 10-40 μg of zinc / 100 ME of insulin, more preferably 15-35 μg of zinc / 100 ME of insulin.

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

 The pH of the acidic solution of step a) is preferably below 5, more preferably from 2 to 3.5.

The insulin analog is preferably human insulin in which position B28 is Asp, Lys, Leu, Val or Ala and position B29 is Lys or Pro; or des (B28-BZ0), des (B27) or des (B30) human insulin, more preferably Asp ^B28 human insulin or Lys ^B28 Pro ^B29 human insulin, even more preferably Asp ^B28 human insulin.

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

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

 Said carbohydrate or carbohydrate derivative preferably contains from 5 to 18 carbon atoms in the basic structure of the carbohydrate.

 In a particular 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, more preferred mannitol.

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

 The precipitated insulin analog is preferably in the form of crystals, including the insulin analog and protamine.

The resulting suspension in step d) is preferably left at a temperature in the range from 5 to 40 ^{° C.} , preferably from 20 to 36 ^{° C.} , even more preferably from 30 to 34 ^{° C.} for precipitation.

 This invention is further illustrated by the following examples, which, however, should not be construed as limiting.

EXAMPLE I
Drug 1
An insulin preparation containing both dissolved and crystalline Asp ^B28 human insulin is prepared as follows:
Solution A:
A solution of Asp ^B28 human insulin at a concentration of 200 IU / ml is obtained by dissolving 76.5 mg of Asp ^B28 human insulin in water, adding 326 μl of 0.2 N. hydrochloric acid and 163 μl of zinc chloride solution (0.4 mg / ml). Then 6.35 mg of protamine sulfate in the solution is added to the insulin solution with stirring, and a mixture of 17.2 mg of m-cresol, 15 mg of phenol and 455 mg of mannitol is added to this solution with stirring. The pH of the resulting clear solution was adjusted to 2.6-2.9 and water was added to 10 ml. The solution is balanced at 28-32 ^o C.

Solution B:
25 mg of disodium phosphate dihydrate is dissolved in water for injection. 17.2 mg of m-cresol, 15 mg of phenol and 455 mg of mannitol are added with stirring. The pH of the resulting clear solution was adjusted to 9, and water was added to 10 ml. The solution is balanced at 28-32 ^o C.

Mixing solution A and B:
Solution B was added to solution A, and the pH was re-adjusted to 7.30. The resulting suspension is left at 30 ^o C for 6 days for crystallization.

 In the resulting preparation, the mass ratio of precipitated to dissolved insulin is 70:30.

The drug is administered in 1.5 ml Penfill ^® cartridges.

EXAMPLE II
Drug 2
An insulin preparation containing both dissolved and crystalline Asp ⁸²⁸ human insulin is prepared as follows:
i) Crystalline fraction
Solution A:
A solution of Asp ⁸²⁸ human insulin at a concentration of 200 IU / ml was prepared by dissolving 190.3 mg of Asp ⁸²⁸ human insulin in water, adding 813 μl of 0.2 N. hydrochloric acid and 410 μl of a solution of zinc chloride (0.4 mg / ml). Then, 16.1 mg of protamine sulfate in the solution was added to the insulin solution with stirring, and a mixture of 43.0 mg of m-cresol, 37.5 mg of phenol and 909 mg of mannitol, and 14.6 were added to this solution with stirring. mg of sodium chloride. The pH of the resulting clear solution was adjusted to 2.6-2.9 and water was added to 22 ml. The solution is balanced at 32 ^o C.

Solution B:
62.4 mg of disodium phosphate dihydrate is dissolved in water. 43.0 mg of m-cresol, 37.5 mg of phenol and 909 mg of mannitol and 14.6 mg of sodium chloride are added with stirring. The pH of the resulting clear solution was adjusted to 9 and water was added to 22 ml. The solution is balanced at 32 ^o C.

Mixing solution A and B:
Solution B was added to solution A and the pH was re-adjusted to 7.30 and water was added to 50 ml. The resulting suspension was left at 32 ^{° C.} for 4 days for crystallization.

 In the resulting preparation, the mass ratio of precipitated to dissolved insulin is 70:30.

The drug is administered in 1.5 ml Penfill ^® cartridges.

EXAMPLES III
Drugs 3-6
Insulin preparations containing both dissolved and crystalline Asp ^B28 human insulin were prepared as described in Example II, except that the amount of mannitol used in solution A and B was 818, 1005, 1047 and 1137 mg, respectively.

Drugs are injected into 1.5 ml Penfill ^® cartridges.

EXAMPLES IV
Drug 7
An insulin preparation containing both dissolved and crystalline Lys ^B26 Pro ^B29 human insulin is prepared as follows:
1) Crystalline fraction
Solution A:
A solution of Lys ^B28 Pro ^B29 human insulin at a concentration of 200 IU / ml is obtained by suspending 69.7 mg of Lys ^B28 Pro ^B29 human insulin in water. To this solution, a mixture of 16.0 mg of m-cresol, 6.5 mg of phenol and 364 mg of mannitol and 25.1 mg of disodium phosphate dihydrate is added with stirring. Then add 50 μl of a solution of zinc chloride (10 mg / ml). The pH of the resulting clear solution was again adjusted to 7.40 and water was added to 10 ml. The solution is balanced at 15 ^o C.

Solution B:
A protamine sulfate solution is prepared by dissolving 7.61 mg of protamine sulfate and 25.1 mg of disodium phosphate dihydrate in water. 16.0 mg of m-cresol, 6.5 mg of phenol and 364 mg of mannitol are added during mixing. This solution is added to the protamine sulfate solution with stirring. The pH of the resulting clear solution was again adjusted to 7.40 and water was added to 10 ml. The solution is balanced at 15 ^o C.

Mixing solution A and B:
Solution B was added to solution A and the pH was re-adjusted to 7.30. The resulting suspension is left at 15 ^o C for 3 days for crystallization.

ii) Dissolved fraction
A solution of Lys ^B28 Pro ^B29 human insulin is obtained by dissolving 34.9 mg of Lys ^B28 Pro ^B29 human insulin in water, adding 33 μl of 1 N. hydrochloric acid and 25 μl of a solution of zinc chloride (10 mg / ml). Then, a mixture consisting of 26.1 mg sodium phosphate dihydrate, 6.5 mg phenol, 6.0 mg m-cresol and 364 mg mannitol is added to the insulin solution with stirring. The pH of the resulting clear solution was adjusted to 7.3 and water was added to 10 ml.

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

 In the resulting preparation, the mass ratio of precipitated to dissolved insulin was 70:30.

The drug is administered in 1.5 ml Penfill ^® cartridges.

EXAMPLE V
Drug 8
An insulin preparation containing both dissolved and crystalline human insulin is prepared as follows:
i) Crystalline fraction
Solution A:
A solution of human insulin is obtained by dissolving 69.7 mg of human insulin in water, adding 65 μl of 1N. hydrochloric acid and 26 μl of zinc chloride solution (10 mg / ml). Then 6.0 mg of protamine sulfate in solution is added to the insulin solution with stirring, and a mixture of 15.0 mg of phenol and 17.2 mg of m-cresol is added to the solution with stirring. The pH of the resulting clear solution was adjusted to 2.7-3.2, and water was added to 10 ml.

Solution B:
24.9 mg of sodium phosphate dihydrate is dissolved in water. 15 mg of phenol, 17.2 mg of m-cresol, 728 mg of mannitol and 11.7 mg of sodium chloride are added during mixing. The pH of the resulting clear solution was adjusted to 9 and water was added to 10 ml.

Solution B was added to solution A and the pH was adjusted to 7.30. The resulting suspension is left at 22-24 ^o C until the next day.

ii Dissolved fraction
A solution of human insulin is obtained by dissolving 34.9 mg of human insulin in water, adding 33 μl of 1 N. hydrochloric acid and 13 μl of zinc chloride solution (10 mg / ml). Then, a mixture consisting of 12.5 mg of sodium phosphate dihydrate, 15 mg of phenol, 17.2 mg of m-cresol and 364 mg of mannitol and 5.8 mg of sodium chloride is added to the insulin solution with stirring. The pH of the resulting clear solution was adjusted to 7.3 and water was added to 10 ml.

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

 In the resulting preparation, the mass ratio of precipitated to dissolved insulin was 70:30.

The drug is administered in 1.5 ml Penfill ^® cartridges.

EXAMPLE VI
Drug 9
An insulin preparation containing both dissolved and crystalline Asp ^B28 human insulin is prepared as follows:
Solution A:
A solution of Asp ^B28 human insulin at a concentration of 200 IU / ml is obtained by dissolving 189.9 mg of Asp ^B28 human insulin in water, adding 163 μl of 1 N. hydrochloric acid and 163.5 μl of zinc chloride solution (10 mg / ml). Then, 11.5 mg of protamine sulfate in solution is added to the insulin solution with stirring, and a mixture of 44.3 mg of m-cresol, 38.6 mg of phenol and 1048 mg of mannitol, and 7.3 mg are added to this solution with stirring. sodium chloride. The pH of the resulting clear solution was adjusted to 2.6-2.9 and water was added to 25 ml. The solution is balanced at 22-24 ^o C.

Solution B:
62.3 mg of disodium phosphate dihydrate is dissolved in water. 44.3 mg of m-cresol, 38.6 mg of phenol and 1048 mg of mannitol and 7.3 mg of sodium chloride are added during mixing. The pH of the resulting clear solution was adjusted to 9 and water was added to 25 ml.

The solution is balanced at 22-24 ^o C.

Mixing solution A and B:
Solution B was added to solution A, and the pH was re-adjusted to 7.30. The resulting suspension is left at 32 ^o C for 2 days for crystallization.

 In the resulting preparation, the mass ratio of precipitated to dissolved insulin is 50:50.

The drug is administered in 1.5 ml Penfill ^® cartridges.

EXAMPLE VII (comparative)
Drug 10
Insulin preparations containing both dissolved and crystalline Asp ^B28 human insulin are prepared as follows:
A solution of Asp ^B28 human insulin at a concentration of 200 IU / ml was prepared by dissolving 76.5 mg of Asp ^B28 human insulin in water, adding 326 μl of 0.2 N. hydrochloric acid and 163 μl of zinc chloride solution (0.4 mg / ml). Then 6.35 mg of protamine sulfate in the solution is added to the insulin solution with stirring, and a mixture of 17.2 mg of m-cresol, 15.0 mg of phenol and 160 mg of glycerol is added to this solution with stirring. The pH of the resulting clear solution was adjusted to 2.6-2.9 and water was added to 10 ml. The solution is balanced at 28-32 ^o C.

Solution B:
25 mg of disodium phosphate dihydrate is dissolved in water for injection. 17.2 mg of m-cresol, 15.0 mg of phenol and 160 mg of glycerol are added during mixing. The pH of the resulting clear solution was adjusted to 9 and water was added to 10 ml. The solution is balanced at 28-32 ^o C.

Solution B was added to solution A and the pH was re-adjusted to 7.30. The resulting suspension is left at 28-32 ^o C for 2 days for crystallization.

 In the resulting preparation, the mass ratio of precipitated to dissolved insulin is 70:30.

The drug is administered in 1.5 ml Penfill ^® cartridges.

EXAMPLE VIII
PHYSICAL LOAD TESTS
5 samples of each insulin preparation are introduced into Penfill ^® cartridges and subjected to the following exercise test:
Penfill ^® cartridges are fixed on the rotor, which is placed in the incubator, and rotated around the axis (360 ^o ) for 4 hours per day at a frequency of 30 rpm and at a constant temperature of 37 ± 2 ^o C. Penfill ^® cartridges are stored at 37 ± 2 ^o C when they are not rotated.

Penfill ^® cartridges are examined macroscopically 5 times a week and changes in the appearance of the composition are noted according to the following principles:
i) cartridges containing a white suspension, which is capable of resuspending with stirring and free of lumps and granules, are classified as “unfibrillated”;
ii) cartridges containing a suspension with lumps and / or granules that are not resuspended with stirring and / or deposited on the wall of the cartridge are called “fibrillated”. This is confirmed by adding 6 μl of 6 N. Hcl per cartridge: fibrillated cartridges do not visually become transparent after acid addition.

 Cartridges are rotated until all samples are fibrillated.

 The results are summarized in the table.

Claims (57)

 1. An aqueous preparation of insulin, including dissolved and / or precipitated human insulin, or an analogue thereof, and / or its derivative, and from 100 to 400 mm, preferably from 150 to 250 mm, more preferably from 180 to 230 mm water-soluble reduced or non-reducing carbohydrate containing at least 4 carbon atoms in the basic structure of a carbohydrate, or a water-soluble non-reducing ester and / or ester derivative of a carbohydrate, or a reduced carbohydrate containing at least 4 carbon atoms in about the basic structure of carbohydrate, or a mixture thereof.  2. An aqueous insulin preparation according to claim 1, characterized in that said carbohydrate or carbohydrate derivative contains from 5 to 18 carbon atoms in the basic structure of the carbohydrate.  3. The aqueous insulin preparation according to claim 2, characterized in that it includes mannitol, sorbitol, xylitol, inositol, trehalose, sucrose or any mixture thereof, preferably mannitol and / or sorbitol, more preferably mannitol.  4. An aqueous insulin preparation according to any one of the preceding paragraphs, characterized in that it further comprises a halide, preferably chloride, more preferably sodium chloride.  5. An aqueous insulin preparation according to any one of the preceding paragraphs, characterized in that it includes a human insulin analogue, wherein position B28 is Asp, Lys, Leu, Val or Ala and position B29 is Lys or Pro; or des (B28-B30), des (B27) or des (B30) human insulin. 6. The aqueous insulin preparation according to claim 5, characterized in that it comprises a human insulin analogue, wherein position B28 is Asp or Lys and position B29 is Lys or Pro, preferably Asp ^{B28 is} human insulin or Lys ^B28 Pro ^{B29 is} human insulin.  7. The aqueous preparation of insulin according to any one of paragraphs. 1-4, characterized in that it includes a derivative of human insulin having one or more lipophilic substituents, preferably acylated insulin. 8. The aqueous insulin preparation according to claim 7, characterized in that it comprises a human insulin derivative selected from the group consisting of B29-N ^ε -mirstoyl des (B30) -human insulin, B29-N ^ε -myristoyl human insulin, B29 -N ^ε -palmitoil-human insulin, B28-N ^ε -mipictoil Lys ^B28 Pro ^B29 -human insulin, B28-N ^ε -palmitoyl Lys ^B28 Pro ^B29 -human insulin, B30-N ^ε -miristoil-Thr ^B29 Lys ^B30 -human insulin, B30-N ^ε -palmitoyl-Thr ^B29 Lys ^B30 -human insulin, B29-N ^ε - (N-palmitoyl-γ-glutamyl) -des (B30) -human insulin, B29-N ^ε - (N-lithoxolyl- γ- lytamil) -dez (B30) -human insulin and B29-N ^ε - (ω-kapbokcigeptadekanoil) -dez (B30) -human insulin, preferably B29-N ^ε -mipictoil-dez (B30) -human insulin.  9. The aqueous preparation of insulin according to any one of the preceding paragraphs, characterized in that it includes crystals, including human insulin, or its analogue, or its derivative and protamine.  10. The aqueous insulin preparation according to claim 9, characterized in that the crystals further include zinc and, optionally, a phenolic compound, preferably phenol, m-cresol or a mixture thereof.  11. The aqueous insulin preparation according to any one of the preceding paragraphs, characterized in that it comprises dissolved and precipitated, preferably crystalline, insulin or an insulin analogue in a mass ratio of from 1: 99 to 99: 1, preferably from 20: 80 to 80: 20, more preferably from 30: 70 to 70: 30.  12. An aqueous insulin preparation 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 of human insulin, or an insulin analog, or derivative.  13. The aqueous insulin preparation according to any one of the preceding paragraphs, characterized in that it comprises from 0 to 100 mm, preferably from 5 to 40 mm, more preferably from 5 to 20 mm chloride, preferably sodium chloride.  14. An aqueous insulin preparation according to any one of the preceding paragraphs, characterized in that it further comprises a physiologically tolerant buffer, preferably a phosphate buffer. 15. An aqueous preparation of insulin, including dissolved and precipitated human insulin, or an analogue thereof, and / or its derivative and a water-soluble reduced or non-reduced carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate, or a water-soluble non-reduced ester and / or an ether derivative of a carbohydrate or a reduced carbohydrate containing at least 4 carbon atoms in the basic structure of a carbohydrate, or a mixture thereof,
16. The aqueous insulin preparation according to claim 15, characterized in that said carbohydrate or carbohydrate derivative contains from 5 to 18 carbon atoms in the basic structure of the carbohydrate.  17. The aqueous insulin preparation according to claim 16, characterized in that it includes mannitol, sorbitol, xylitol, inositol, trehalose, sucrose or any mixture thereof, preferably mannitol and / or sorbitol, more preferably mannitol.  18. The aqueous preparation of insulin according to any one of paragraphs. 15-17, characterized in that it further includes a halide, preferably chloride, more preferably sodium chloride.  19. An aqueous preparation of insulin according to any one of paragraphs. 15-18, characterized in that it includes an analogue of human insulin, where position B28 represents Asp, Lys, Leu, Val or Ala and position B29 represents Lys or Pro; or des (B28-B30), des (B27) or des (B30) human insulin. 20. The aqueous insulin preparation according to claim 19, characterized in that it comprises a human insulin analogue, wherein position B28 is Asp or Lys and position B29 is Lys or Pro, preferably Asp ^{B28 is} human insulin or Lys ^B28 Pro ^{B29 is} human insulin.  21. The aqueous insulin preparation according to any one of paragraphs. 15-18, characterized in that it includes a derivative of human insulin having one or more lipophilic substituents, preferably acylated insulin. 22. The aqueous insulin preparation according to p. 21, characterized in that it comprises a derivative of human insulin selected from the group consisting of B29-N ^ε -mistoyl des (B30) human insulin, B29-N ^ε -myristoyl human insulin, B29-N ^ε -palmitoyl-human insulin, B28-N ^ε -mistyloid Lys ^B28 Pro ^B29 -human insulin, B28-N ^{ε- palmitoyl} Lys ^B28 Pro ^B29 -human insulin, B30-N ^ε -myristoyl-Thr ^B29 Lys ^B30 - human insulin, B30-N ^ε - ^palmitoyl -Thr ^B29 Ls ^B30 -human insulin, B29-N ^ε - (N-palmitoyl-γ-glutamyl) -dez (B30) -human insulin, B29-N ^ε - (N-lithoxolyl -γ-glutam sludge-des (B30) -human insulin and B29-N ^ε - (ω-carboxyheptadecanoyl) -des (B30) -human insulin, preferably B29-N ^ε- myristoyl des (B30) -human insulin.  23. The aqueous insulin preparation according to any one of paragraphs. 15-22, characterized in that it includes crystals comprising insulin, an insulin analogue or an insulin derivative, and protamine.  24. The aqueous insulin preparation according to claim 23, wherein the crystals further include zinc and, optionally, a phenolic compound, preferably phenol, m-cresol, or a mixture thereof.  25. The aqueous insulin preparation according to any one of paragraphs. 15-24, including dissolved and precipitated, preferably crystalline, insulin or insulin analogue in a mass ratio of from 1: 99 to 99: 1, preferably from 20: 80 to 80: 20, more preferably from 30: 70 to 70: 30.  26. The aqueous preparation of insulin according to any one of paragraphs. 15-25, in which the reduced or non-reducing carbohydrate, preferably mannitol, is present in an amount of from 100 to 400 mm, preferably from 150 to 250 mm, more preferably from 180 to 230 mm.  27. The aqueous insulin preparation according to any one of paragraphs. 13-26, characterized in that it comprises from 60 to 3000 nmol / ml, preferably from 240 to 1200 nmol / ml of human insulin, or an insulin analogue or derivative.  28. The aqueous insulin preparation according to any one of paragraphs. 15-27, characterized in that it comprises from 0 to 100 mm, preferably from 5 to 40 mm, more preferably from 5 to 20 mm chloride, preferably sodium chloride.  29. The insulin preparation according to any one of paragraphs. 15-29, characterized in that it further comprises a physiologically tolerant buffer, preferably phosphate buffer.  30. An aqueous insulin analogue preparation comprising a dissolved and / or precipitated human insulin analogue and a water-soluble reduced or non-reducing carbohydrate containing at least 4 carbon atoms in the basic structure of a carbohydrate, or a water-soluble non-reducing ester and / or ester derivative of a carbohydrate or reduced carbohydrate containing at least 4 carbon atoms in the basic structure of a carbohydrate, or mixtures thereof.  31. The aqueous insulin preparation according to claim 30, wherein said carbohydrate or carbohydrate derivative contains from 5 to 18 carbon atoms in the basic structure of the carbohydrate.  32. An aqueous insulin preparation according to claim 31, characterized in that it includes mannitol, sorbitol, xylitol, inositol, trehalose, sucrose or any mixture thereof, preferably mannitol and / or sorbitol, more preferably mannitol.  33. The aqueous insulin preparation according to any one of paragraphs. 30-32, characterized in that it further comprises a halide, preferably chloride, more preferably sodium chloride.  34. The aqueous preparation of insulin according to any one of paragraphs. 30-33, characterized in that the human insulin analogue is human insulin, in which position B28 represents Asp, Lys, Leu, Val or Ala and position B29 represents Lys or Pro; or des (V28-VZO), des (V27) or des (VZO) human insulin. 35. The aqueous insulin preparation according to claim 34, wherein the human insulin analogue is human insulin in which position B28 is Asp or Lys and position B29 is Lys or Pro, preferably Asp ^{B28 is} human insulin or Lys ^B28 Pro ^{B29 is} human insulin. 36. The aqueous insulin preparation according to claim 35, wherein the analogue of human insulin is Asp ^B28 -human insulin.  37. The aqueous preparation of insulin according to any one of paragraphs. 30-36, characterized in that it includes crystals, including an insulin analog and protamine.  38. The aqueous insulin preparation according to claim 37, wherein the crystals further include zinc and optionally a phenolic compound, preferably phenol, m-cresol or a mixture thereof.  39. The aqueous preparation of insulin according to any one of paragraphs. 30-38, characterized in that it includes dissolved and precipitated, preferably crystalline, an insulin analogue in a mass ratio of from 1: 99 to 99: 1, preferably from 20: 80 to 80: 20, more preferably from 30: 70 to 70: 30 .  40. The aqueous insulin preparation according to any one of paragraphs. 30-39, characterized in that it comprises from 60 to 3000 nmol / ml, preferably from 240 to 1200 nmol / ml, of a human insulin analogue.  41. The aqueous insulin preparation according to any one of paragraphs. 30-40, characterized in that it comprises from 0 to 100 mm, preferably from 5 to 40 mm, more preferably from 5 to 20 mm chloride, preferably sodium chloride.  42. The aqueous preparation of insulin according to any one of paragraphs. 30-41, further comprising a physiologically tolerant buffer, preferably a phosphate buffer.  43. A parenteral pharmaceutical formulation comprising an insulin preparation according to any one of the preceding claims.  44. A method of obtaining an insulin preparation containing both dissolved and precipitated insulin analogue, comprising the steps of: a) obtaining an acidic solution comprising human insulin analogue, zinc and a sub-isophan amount of protamine; b) obtaining an alkaline solution comprising a substance that acts as a buffer at physiological pH; where at least one of the above solutions further includes a phenolic compound; c) mixing the acidic and alkaline solutions and, optionally, adjusting the pH to a value of from 6.5 to 8.0, preferably from 7.0 to 7.8; and d) maintaining the resulting suspension for precipitation.  45. The method according to p. 44, characterized in that the mass ratio of the insulin analog to protamine in the solution of stage a) is chosen to obtain in the final product the mass ratio of the dissolved insulin analog to the precipitated insulin analog from 1: 99 to 99: 1, preferably from 20: 80 to 80: 20, more preferably from 30: 70 to 70: 30.  46. The method according to p. 45, characterized in that the solution of stage a) comprises from 120 to 6000 nmol / ml of an insulin analogue and from 0.01 to 5.0 mg / ml of protamine.  47. The method according to p. 46, wherein the solution of step a) further comprises zinc, preferably zinc chloride in an amount corresponding to 10-40 μg zinc / 100 ME insulin, preferably 15-35 μg zinc / 100 ME insulin.  48. The method according to any one of paragraphs. 44-47, characterized in that the solution a) and / or solution b) includes chloride, preferably sodium chloride in an amount; corresponding to from 0 to 100 mm, preferably from 5 to 40 mm, more preferably from 5 to 20 mm in the final product.  49. The method according to any one of paragraphs. 44-48, characterized in that the pH of the acidic solution of stage a) is below 5, preferably from 2 to 3.5.  50. The method according to any one of paragraphs. 44-49, characterized in that the insulin analogue is human insulin, in which position B28 represents Asp, Lys, Leu, Val or Ala and position B29 represents Lys or Pro; or des (V28-VZO), des (V27) or des (VZO) human insulin. 51. The method according to p. 50, wherein the insulin analogue is Asp ^B28 -human insulin or Lys ^B28 Pro ^B29 -human insulin, preferably Asp ^B28 -human insulin.  52. The method according to any one of paragraphs. 44-51, characterized in that the solution of stage a) and / or stage b) further comprises a phenolic compound, preferably phenol, m-cresol or a mixture thereof.  53. The method according to any one of paragraphs. 44-52, characterized in that the solution of stage a) and / or stage b) further includes a water-soluble reduced or non-reducing carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate, or a water-soluble non-reducing ester and / or ester derivative of carbohydrate or a reduced carbohydrate containing at least 4 carbon atoms in the basic structure of the carbohydrate, or a mixture thereof.  54. The method according to p. 53, characterized in that said carbohydrate or carbohydrate derivative contains from 5 to 18 carbon atoms in the basic structure of the carbohydrate.  55. The method according to p. 54, wherein the solution of stage a) and / or stage b) comprises mannitol, sorbitol, xylitol, inositol, trehalose, sucrose or any mixture thereof, preferably mannitol and / or sorbitol, more preferably mannitol.  56. The method according to any one of paragraphs. 44-55, characterized in that the buffer substance used in the alkaline solution of stage b) is a physiologically tolerant buffer, preferably a phosphate buffer, more preferably disodium phosphate dihydrate.  57. The method according to any one of paragraphs. 44-56, characterized in that the precipitated insulin analogue is in the form of crystals, including the insulin analogue and protamine. 58. The method according to any one of paragraphs. 44-57, characterized in that the suspension obtained in stage d) is left at a temperature of 5-40 ^° C, preferably 20-36 ^° C, more preferably 30-34 ^° C, for precipitation. Priorities by points and signs:
Paragraphs 1-45, 47-58 have priority on 06/20/1996. The sign of "des (B30) human insulin" in paragraphs. 4, 8, 34, 50 and pH the pH value in item 44 is below 7.0 above 7.8 priority from 06/19/1997. , p. 46 priority of June 19, 1997.

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