DE2459515B2 - Medicinal preparations with an antidiabetic effect - Google Patents

Description

The invention relates to insulin preparations made from crystalline insulin in aqueous suspension which are characterized in that they are dissolved des-B1-phenylanaline-insulin of the same species and contain a total of 25-80 meg zinc ions per 1001 units of insulin.

The ratio of crystallized insulin to dissolved Des-Phe-insulin determines the time-effect profile. It can be between 60:40 and 90:10 and is preferably 80:20 to 90:10. The total insulin content of the preparation is expediently 40-100 IU / ml. The suspension is brought to a pH of 6.8-7.2, preferably pH 6.8-7.0, adjusted and contains 25-80 meg zinc ions per 100 I.U.

From DE-OS 22 56 215 it is known that crystalline insulin can be contained in insulin preparations in addition to amorphous Des-B1-phenylalanine-insulin (Des-Phe ^BI -insulin) of the same species. It is a suspension of the amorphous Des-Phe ^BI insulin.

In contrast to this, the subject matter of the present invention is a combination of crystalline insulin with dissolved Des-Phe ^B1 insulin, which surprisingly has to be kept in solution due to the claimed zinc ion content.

It was not to be expected without further ado that the claimed solution would be stable, since there were two Substances that are capable of co-crystallization, so that the observed shelf life all physico-chemical expectations of the skilled person contradicts.

While so far no zinc concentration has been found in insulins of the same species at which the dissolved one and crystalline form are stable next to each other for a long time (with lower zinc concentration Crystallized insulin in solution, dissolved insulin precipitates at a higher zinc concentration) was used in the case of the invention Combination surprisingly found that within the claimed zinc concentration both phases are stable next to each other. This has been the case in observation periods of more than 18 or 10 Months for bovine or pig insulin without the slightest change.

The insulin combination according to the invention also has advantages over previously known insulin preparations Insulins of the same species have distinct advantages. In particular, the insulin combination according to the invention has an ideal time-effect profile with a relatively high initial concentration and long, but not too long Duration of action.

The insulin preparation is made as follows:

1. To produce the suspension of insulin crystals (crystalline component of the finished preparation), insulin is first crystallized from a buffer containing zinc ions. Insulin crystals of a uniform size of 15-35 μ with a zinc content of 03-1.0% are obtained.
After the mother liquor has been separated off, the crystals are suspended in a buffer solution, the volume of which is such that the suspension is adjusted to a defined insulin content in the range of 40-100 LEVmI. The buffer solution contains so much zinc ions, e.g. B. in the form of zinc chloride that the total zin content of the crystal suspension adjusted to 40-100 IEL / ml is 30-80 mcg / 100 IU. The buffer solution also contains salts such as sodium acetate, expediently in a concentration of 0.1- 0.2% sodium chloride, expediently in a concentration of 0.5-1%, as well as preservatives such as 4-Hydroxybcnzoic acid methyl ester, expediently in a concentration of about 0.1% and is adjusted to a pH value in the range of pH 6 with sodium hydroxide solution , 8-7.2, preferably pH 6.8-7.0

2. To prepare the solution of Des-Phe ^B1 insulin of the same species (dissolved component of the finished preparation), as much crystallized, dry Des-Phe ^BI insulin as has a zinc content of 0.3-0.7% is dissolved in a solution liquid that the solution has a defined Des-Phe ^B1 insulin content in the range of 40-100 I. EVmI. B. in the form of zinc chloride that the total zinc content of the solution is 25-40 mcg / 100 IU. The solution liquid also contains sodium chloride and 4-hydroxybenzoic acid methyl ester as well as sufficient amounts of acetic acid and hydrochloric acid ^{to dissolve the Des-Phe B1 insulin.} After sterile filtration, the Des-Phe ^B1 insulin solution is adjusted to a pH value in the range of pH 6.8-7.2, preferably pH 6.8-7.0, with sodium hydroxide solution. It then has the same sodium chloride content, sodium acetate and 4-hydroxybenzoic acid methyl ester like the buffer solution described under 1.

3. To produce the ready-to-fill depot insulin preparation, mix the crystalline component described under 1. and the dissolved component described under 2., using insulin crystals and dissolved Des-Phe ^BI -insulin of the same species. The volume ratio of crystalline component: dissolved component is 60:40 to 90:10, preferably 80:20 to

so 90:10. The total insulin content is 40-100 I. E./ml. The zinc content of the finished preparation depends on the mixing ratio of the two components dependent and is 25-80 mcg / 100 I.U. “The pH value is in the range of pH 6.8-7.2 preferably 6.8-7.0.

The preparation according to the invention has a longer-lasting and balanced insulin depot effect. It is also after one year of storage at 4 ° - 8 ° C and at 25 ° C in terms of its physical Condition and biological effect unchanged

The preparation takes place as a depot insulin at the

Treatment of diabetes mellitus use. It is particularly useful when recruiting new patients appropriate when the aim is to prevent the formation of antibodies against multiple insulin species.

The following examples explain the production of the preparations according to the invention.

example 1

A. Manufacture of Bovine insulin crystal suspension

16 g of pure bovine cherry insulin with a minimum activity of 251. R / mg, calculated on the dry substance, are dissolved in 750 ml of 0.02 N hydrochloric acid, the Contains 0.013% zinc ions in the form of zinc chloride, dissolved. 250 ml of a Solution containing 0.95% glacial acetic acid and 2.8% sodium chloride was added. The clear mixture will then made sterile by sterile filtration using a membrane layer. The sterile filtered solution is added so much of a sterile 4 N sodium hydroxide solution is added that is a pH value between pH 5.4-5.8 is reached. The amorphous insulin suspension obtained is briefly below Stirred under sterile conditions at room temperature and then in the refrigerator at +4 to + 7 ° C Crystallization stopped

After a maximum of 36-40 hours, evenly shaped rhombohedral crystals of a uniform size between 15 and 35 microns have formed. The crystals are thrown off in sterile beakers at a low number of revolutions, with any remaining small amorphous portions are separated. The centrifuged crystal is then placed in a sterile buffered, neutral insulin dilution solution (composition see Table 1) suspended again (Stammsuspen! Ion).

Table 1 Composition of the insulin dilution solution 101 solution contains:

10g 4-hydroxybenzoic acid 0.1% methyl ester = 0.14% 14g Sodium acetate 3 H2O = 0.75% 75 g Sodium chloride = 5.0 mcg ⁺ Zn + / ml 105 mg Zinc chloride (ZnCl ₂ ) Caustic soda up to to pH 6.9 The solution is filtered sterile. Preparation of the 401st EVmI containing Depot component

According to the determined insulin content in the stock suspension, this is now made by adding the sterile dilution solution adjusted to the desired insulin content of 40 I. EVmI. yield maximum 101.

Determination of the insulin crystal content in the stock suspension

To determine the insulin crystal content in the stock suspension, the crystal suspension is after Intensive mixing with the help of a magnetic stirrer 4 times 10 ml each (volumetric pipette) removed and the content on insulin determined by the following methods:

1. Gravimetrically after drying with acetone and ether and weigh out.

2. After the crystals have dissolved, spectrophotometrically in the UV at 280 nm.

3. By determining the N content.

50

55

B. Preparation of the dissolved component of Des-Phe bovine insulin

6304 g of crystallized Des-Phe ^B1 insulin from beef are dissolved in 3.5 liters of a dissolving liquid of the composition shown in Table 2

Table 2 Composition of the Des-Phe ^Bi solution liquid 5 liters of solution contain:

5g 4-hydroxybenzoic acid 0.1% + / ml methyl ester = 2.85 ml acetic acid (gives 0.14% Sodium acetate 3 H2O) 0.75% 37.5 g Sodium chloride = 21 mg Zinc chloride 2.0 mcgZn + (anhydrous) = 5 ml 4 η hydrochloric acid

30th

35

40

45 The clear solution of the Des-Phe ^BI insulin is then sterilized by means of a membrane filter and brought to a volume of 3.9 l by adding 400 ml of liquid which was passed through the same sterile filter after the insulin filtration -Value of 6.9 set (consumption approx. 13 ml). A final volume of 3.9471 and an insulin content of 401 EVmI is set by adding 34 ml of a sterile, neutral insulin dilution solution (composition see Table 1), ^{based on an activity of Des-Phe B1} insulin of 251 EVmg.

C. Production of the preparation ready for filling

By mixing the depot component prepared according to A., which contains bovine insulin crystals, with the component prepared according to B., which contains dissolved Des-Phe ^B1 bovine insulin, in a volume ratio of 80:20, a preparation is obtained which has 32 I.EVml crystalline insulin and 8 I. EVmI dissolved Des-Phe ^BI -insulin, so a total of 40 I. EVmI insulin contains. The suspension is filled in sterile bottles of 10 ml each.

Example 2

A. Preparation of the porcine insulin crystal suspension

A sterile suspension containing 16 g of pig insulin crystals of 15-35 μ in size, which are made at pH 5.4-5.8 a suitable buffer analogous to Example 1, were crystallized, is centrifuged. After decanting the With the supernatant, the crystals are resuspended in 7 l of a sterile, buffered, neutral insulin dilution solution (composition see Table 3) (Stock suspension).

Table 3 Composition of the insulin dilution solution 101 solution contains:

60

65

10g 4-hydroxybenzoic acid 0.1% methyl ester = 0.14% 14g Sodium acetate ■ 3H ₂ O = 0.75% 75 g Sodium chloride = 10 mcg Zn + 210 mg Zinc chloride (ZnCl ₂ ) Caustic soda up to to pH 6.9 The solution is filtered sterile

> 7ml

Determination of the insulin crystal content
in the stock suspension

To determine the insulin crystal content in the The stock suspension becomes the crystal suspension after thorough mixing with the aid of a magnetic stirrer 4 times 10 ml (full pipette) are removed and the insulin content is determined using the following methods:

1. Gravimetrically after drying with acetone and ether and weigh out.

2. After the crystals have dissolved, spectrophotometrically in the UV at 280 nm.

3. By determining the N content.

Preparation of the 401st EVmI containing
Depot component

According to the ascertained insulin content in the stock suspension, this is now adjusted to the desired insulin content of 40 I. EJm] by further addition of the sterile dilution solution. Yield maximum 101.

B. Preparation of the dissolved component
by Des-Phe ^B! -Pork insulin

6.304 g of crystallized pork des-Phe insulin are dissolved in 3.5 l of a dissolving liquid of the composition shown in Table 4.

Table 4

Composition of the Des-Phe ^B1 solution liquid

5 liters of solution contain:
5g

0.1%

0.75%

4.0 mcg ⁺ Zn + / ml

4-hydroxybenzoic acid methyl ester
2.85 ml of acetic acid

(gives 0.14%
Sodium acetate 3 H ₂ O)
37.5 g sodium chloride
42 mg zinc chloride
(anhydrous)
5 ml of 4 η hydrochloric acid

The clear solution of Des-Phe ^B1 insulin is then sterilized by means of a membrane filter and brought to a volume of 3.9 l by adding 400 ml of solution liquid, which was passed through the same sterile filter after the insulin filtration. A pH of 6.9 is set by adding 4 η sterile sodium hydroxide solution (consumption approx. 13 ml). A final volume of 3.9471 and an insulin content of 401 U / ml is set by adding 34 ml of a sterile, neutral insulin dilution solution (composition see Table 1), based on ^{an activity of the Des-Phe BI} insulin of 251 U / ml will.

C. Production of the preparation ready for filling

By mixing the depot component produced according to Au, which contains pig insulin crystals, with the

ίο A component prepared according to B., which contains dissolved Des-Phe ^B1 pig insulin, in a volume ratio of 80:20, results in a preparation containing 32 I. U / ml of crystalline insulin and 8 1. U / ml of dissolved Des-Phe - ^BI -insulin, so altogether 40 I U / ml insulin contains The suspension is sterile filled into vials of 10 ml each

Example 3

75 parts of the depot component prepared according to A., which contains insulin crystals, are mixed with 25 parts of the component prepared according to B., which contains dissolved Des-Phe ^B1 insulin of the same species, and a preparation containing 30 I. EVmI crystalline insulin and 10 I. U / ml of dissolved Des-Phe ^B1 insulin, i.e. a total of 401 U / ml of insulin. The suspension is filled in sterile bottles of 10 ml each.

Example 4

70 parts of the depot component prepared according to A., which contains insulin crystals, are mixed with 30 parts of the component prepared according to B., which contains dissolved Des-Phe ^B1 insulin of the same species, and a preparation is obtained which has 28 IU / ml crystalline Insulin and 12 IU / ml of dissolved Des-Phe ^B1 insulin, so a total of 401. EVmI contains insulin. The suspension is filled in sterile bottles of 10 ml each.

B e i s ρ i e 1 5

90 parts of the depot component produced according to A., which contains insulin crystals, are mixed with 10 parts of the component produced according to B., which contains dissolved Des-Phe ^BI -insulin of the same species, and a preparation is obtained which has 36 IU / ml crystalline Contains insulin and 4 IU / ml of dissolved Des-Phe ^B1 insulin, so a total of 401 U / ml insulin. The suspension is filled in sterile bottles of 10 ml each.

Claims (2)

Patent claims: 1. Insulin preparation from crystalline insulin in aqueous suspension, characterized by a content of dissolved Des ^B1 -phenylalanine insulin of the same species and a content of 25-80 meg zinc ions per 1001 units of insulin. 2. Insulin preparation according to claim 1, characterized in that the insulin content is 40-100 LEVmI is