DE2459515C3 - Medicinal preparations with an antidiabetic effect - Google Patents

Description

15th

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

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 r, I.EVml. The suspension is adjusted to a pH of 6.8-7.2, preferably pH 6.8-7.0, 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 anior- »1 phem Des- ^B1 -phenylalanine-insulin (Des-Phe B1 -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 ^B 'insulin, which is surprisingly 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 it involves two substances which are capable of co-crystallization, so that the stability observed contradicts all physicochemical expectations of the person skilled in the art. A ^r >

While no zinc concentration has been found in insulins of the same species at which the dissolved and crystalline form are stable side by side for a long time (with a lower zinc concentration, crystalline insulin dissolves, with a higher zinc concentration, dissolved insulin precipitates) Combination according to the invention surprisingly found that both phases are stable next to one another within the claimed zinc concentration. This has been shown in observation periods of more than 18 and 10 ½ months for bovine and porcine insulin without the slightest change occurring.

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 one Initial concentration and long, but not too long, duration of action.

The insulin preparation is made as follows: M

1. To produce the suspension of insulin crystals (crystalline component of the finished preparation tung), insulin is first crystallized from a buffer containing zinc ions. One obtains Insulin crystals of uniform size from 15-35 μ, which have a zinc content of 0.3-1.0%. After the mother liquor has been separated off, the crystals are suspended in a buffer solution, whose volume is dimensioned so that the suspensions to a defined insulin content in the area from 40-100 IUml is set. 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 LEVmI The buffer solution is 30-80 meg / 100 I.U. 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 Hydroxybenzocsäuremethylester, expediently in a concentration of about 0.1% and is with Caustic soda to a pi I value in the range of pi I 6.8-7.2, preferably pH 6.8-7.0.

2. To prepare the solution of Des-Phe ^BI -insulin of the same species (dissolved component of the finished preparation), dissolve as much crystallized, dry Des-Phe ^BI -insulin as has a zinc content of 0.3-0.7%, in a solution liquid that the solution has a defined Des-Phe ^BI -insulin content in the range of 40-100 I.EVml. The solution liquid contains so much zinc ions, e.g. 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-Hydroxybcn / .ocsäuremethylester as well as ^{sufficient quantities of acetic acid and hydrochloric acid to dissolve the Des-Phe "1} -insulin. After sterile filtration, the Des-Phe ^III insulin solution is brought to a pH value in the range pH 6.8-7.2, preferably pH 6.8-7.0, which then has the same sodium chloride, sodium acetate and 4-hydroxybenzoic acid methylene content as the buffer solution described under 1.

3. To produce the ready-to-fill depot insulin preparation, mix the crystalline component described under I. and the dissolved component described under 2., using insulin crystals and dissolved DeS-PlIe ¹¹¹ InSuHn of the same species. The volume ratio of crystalline component: dissolved component is 60:40 to 90:10, preferably 80:20 to 90:10. The total insulin content is 40-100 IU / 111I. The zinc content of the finished preparation depends on the mixing ratio of the two components and is 25-80 mcg / 100 IU, 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 even after one year of storage at 4 ° -8 ⁰ C and at 25 ° C. unchanged with regard to their physical state and the biological effect.

The preparation is used as a depot insulin in the treatment of diabetes mellitiis. she is especially appropriate when re-setting patients, if it is to be prevented that themselves Build antibodies against several species of insulin.

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 beef cherry insulin with a minimum activity of 25 1. Eirng, calculated on the dry substance, are dissolved in 750 ml of 0.02 N hydrochloric acid containing 0.013% zinc ions in the form of zinc chloride. 95% glacial acetic acid and 2.8% sodium chloride containing, added. The clear mixture is then made sterile by sterile filtration using a membrane layer. Sufficient sterile 4N sodium hydroxide solution is added to the sterile-filtered solution so that a pH value between 5.4-5.8 is reached. The amorphous insulin suspension is stirred briefly under sterile conditions at room temperature and then adjusted in a refrigerator at +4 to +7 ⁰ C for crystallization

After a maximum of 36-40 hours, evenly shaped rhombohedral crystals have become more uniform Size between 15 and 35 μ 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 crystals are then placed in a sterile buffered, neutral insulin diluent (composition see table 1) resuspended (stock suspension).

Table 1

Composition of the insulin dilution solution

10 1 solution contains:

10g 4-1 hydroxybenzoic acid = 0.1% methyl ester = 0.14% Ed Sodium acetate · JH ₂ O = = 0.75% 75 g Sodium chloride = 5.0 mcg Zn ^f 105 mg Zinc chloride (ZnCI ₂ ) Caustic soda up to to pH 6.9 The i solution is filtered sterile. Production of the 40 IU / ml containing Depot component

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

6.304 g of crystallized Des-Phe ^B1 -! Nsulin from bovine -> are dissolved in 3.5 l of a dissolving liquid of the composition shown in Table 2

Table 2

Composition of the Des-Phe ^BI solution liquid

κι The 5 liter solution contains:

5 g of 4-hydroxybenzoic acid

methyl ester
2.85 ml of acetic acid

(gives 0.14%

Sodium acetate 3 H ₂ O)

Sodium chloride

Zinc chloride

(anhydrous)

4 η hydrochloric acid

According to the ascertained insulin content in the stock suspension, this is now determined by adding the "> '> sterile dilution solution adjusted to the desired insulin content of 40 1. U./ml. 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 (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. St.

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

3. By determining the N content.

37.5 g
21 mg

JO 5 ml

0.1%

0.75% Zn = 2.0 mcg ⁺ + / ml

The clear solution of 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 solution liquid, which was passed through the same sterile filter after the insulin filtration. A pH of 6.9 is set by adding 4N sterile sodium hydroxide solution (consumption approx. 1 3 ml). A final volume of 5.9471 and an insulin content of 401 U / ml is set by adding 34 ml of a sterile, neutral insulin dilution solution (for the addition, see Table 1), with an activity of the Des-Phe ^BI insulin of 25% I .E. / Mg tension is set.

C. 1 creation of the preparation ready for filling

By mixing the depot component prepared according to A., which contains Rirderinsulin crystals, with the component prepared according to U., which contains dissolved I) es-Phe ^ll1 -cattle insulin, in a volume ratio of 80:20, a preparation is obtained, which contains 32 IU / ml of crystalline insulin and 8 IU / ml of dissolved Des-Phe ^BI -insulin, a total of 40 IU / ml of insulin. The suspension is sterile filled into vials of 10 ml each.

Example 2

A. Preparation of the .Pork insulin crystal suspension

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

Table 3

Composition of the insulin dilution solution The 10 l solution contains:

10 g of 4-hydroxybenzoic acid

methyl ester = 0.1%

14 g sodium acetate ■ 3 H ₂ O = 0.14% 75 g sodium chloride = 0.75%

210 mg zinc chloride (ZnCl;) = 10 meg Zn ^f + / ml sodium hydroxide solution up to
to pH 6.9

The solution is filtered sterile.

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 determined insulin content in the stock suspension, this is now adjusted to the desired insulin content of 40 IU / ml by further addition of the sterile dilution solution. Yield maximum 101.

B. Preparation of the dissolved component
of Des-Phe ^B 'porcine insulin

6.304 g of crystallized pig des-Phe insulin are in 3.5 l of a solution liquid as shown in Table 4 reproduced composition resolved.

Table 4

Composition of the Dcs-Phe ^H1 solution liquid

5 liters of solution contain:

5 g of 4-hydroxybenzocic acid

methylestcr = 0.1%

2.85 ml of acetic acid

(gives 0.14%
Sodium acetate 3 HjO)
37.5 g sodium chloride = 0.75%

42 mg zinc chloride

(anhydrous) = 4.0 mcg Zn * ♦ / ml

5 ml of 4 η hydrochloric acid

The clear solution of the f) cs-Phc ^H1 -insilin 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 filter after the insulin infiltration. A pH of 6.9 is set by adding 4 η sterile sodium hydroxide solution (consumption ea. 13 ml). A final volume of 3.9471 and an insulin content of 401 EVmI are set by adding 34 ml of a sterile, neutral insulin dilution solution (composition see Table 1), with an activity of Des-Phe ^B1 insulin · -, of 25 liters. EVmg is used as a basis.

C I Creation of the preparation ready for filling

By mixing the depot component prepared according to A., which contains Schwcincinsulin crystals, with the

Hi component prepared according to B., which contains dissolved Des-Phe "'- Schwcincinsulin, in the volume ratio 80:20, a preparation is obtained which contains 32 IU / ml of crystalline insulin and 8 I of EVmI dissolved Dcs-Phc - "'- insulin, so altogether 40 I. EVmI insulin

r. contains. The suspension is sterile in vials of each 10 ml bottled.

Example 3

2i) 75 parts of the Dcpotkoniponcntc prepared according to A., which contains insulin crystals, are mixed with 25 parts of the component prepared according to B., which contains dissolved Des-Phc ^{l! 1} -insulin of the same species, and a preparation is obtained which contains 30 I. EVmI crystalline

r < insulin and 10 IU / ml of dissolved Des-Phe "'insulin, so a total of 401. [L / ml contains insulin. The suspension is sterile filled into vials 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 Dcs-Phc ^{l! l} -insulin of the same species, and a preparation is obtained which contains 28 I. EVmI crystalline

t '· insulin and 12 I. [' ../ml of dissolved Dcs-Phc ^m -! nsulin contains a total of 40 IU / ml of insulin. The suspension is filled in sterile bottles per 10 ml.

B c i s ρ i c I 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. and which contains dissolved Dcs-Phc ^HI -insulin of the same species, and a preparation containing 36 I. EVmI crystalline is obtained Insulin and 4 I. EVmI dissolved Dcs-Phe " ¹ insulin, so a total of 401 U / ml insulin. The suspension is sterile filled into bottles of 10 ml each.

Claims (2)

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