DE1900367A1 - Stable glucagon solution and process for its preparation - Google Patents

Description

9678

NOVO TERAPEUTISK LABORATORIUM A / S

Stable glucagon solution and process for its preparation

The invention relates to neutral or approximately neutral stable glucagon solutions and their production. In particular, has the invention stable aqueous, injectable glucagon solutions and a method for producing such solutions.

Glucagon supplements for injection come in the way of the Market that one container contains dry glucagon powder and another container contains an appropriate acidic injection medium. In view of the low stability of acidic glucagon solutions, the aforementioned powder and the The aforementioned medium was brought together only a short time before use. By mixing the powder and the injection medium, an acidic solution is obtained that can be used for injection purposes. Since the isoelectric point of the glucagon is at a pH of about 7 and the glucagon precipitates in a range around this pH value, it is necessary to To adjust the glucagon solution acidic.

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1 3UUOO / - 2 -

Eb often proves necessary after a very glucagon short notice to inject. Because of this, it turns out inconvenient if the glucagon powder and the injection medium are mixed together before use have to. Furthermore, the acidity of the solution when it is injected gives rise to increased discomfort. It has now been found that it is possible to obtain stable, neutral or nearly neutral glucagon solutions by adding a solution containing 0.1 to Contains 5 mg glucagon per ecm, certain additions of one or more detergents are incorporated.

The detergents used according to the invention can very generally are characterized as compounds which are bound to glucagon by means of hydrophobic groups and which hydrophilic groups in the surrounding water molecules have positions closest to aqueous solution.

Many detergents are suitable for the production of neutral or approximately neutral glucagon solutions, including other quaternary ammonium compounds with surface-active properties, including such quaternary ammonium compounds, in which the nitrogen atom is involved in a ring formation. The stabilizing and solubilizing anionic and cationic detergents can be characterized as compounds that use hydrophobic groups bound to the glucagon and which because of their negative '

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or positive charge result in the complex at a lower or higher isoelectric pH than in the case is formed by free glucagon, creating the point of the least Solubility of the glucagon is shifted from the neutral pH range to a lower or higher pH. The neutral ones Detergents can be viewed as compounds which are bound to the glucagon by means of hydrophobic groups and which, due to their hydrophilic groups, result in a soluble complex.

When 1 ecm of a glucagon slurry containing 2 mg of glucagon in 0.9 # NaCl solution (pH = 7.4) contained, with 1 ecm Detergent mixed in 0.9 # NaCl solution (pH = 7.4) there was a stabilizing and solubilizing effect when using anionic detergents, e.g. Alkylarylsulfonate G-33OO (Atlas), 'or of non-ionic Detergents such as a polyoxyethylene alkyl ether urea complex Renex 35 (Atlas), or of cationic detergents, e.g. cetyltrimethylammonium bromide (Cetrimonium), benzylcetyldimethylammonium chloride (Rodalon) and cetyl pyridine chloride.

Among the stabilizing and solubilizing compounds mentioned above, the ionic detergents are the most effective, especially those having a higher alkyl group such as a cetyl group as one of the substituents.

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According to this, the stable aqueous, injectable glucagon solutions according to the invention can be characterized in that they contain glucagon in an amount of 0.1-5 mg per ecm and at the same time a stabilizing and solubilizing amount of a detergent. In practice , glucagon solutions are preferably prepared and used which contain 1 to 2 mg glucagon per ecm.

It has been found that in general it is found necessary to use the detergent in an amount of at least one fifth the amount of glucagon. In many cases it has proven to be expedient to use an amount of detergent which corresponds to about half the amount of glucagon. However, even larger amounts of detergent can be used, for example up to about a hundred times the amount of glucagon.

It was also found that such

) Detergents suitable for the purposes of the invention whose molecule contains at least one aliphatic side chain which has at least 6 carbon atoms, preferably 12 to 20 carbon atoms.

Among the valuable used in the context of the invention. Detergents include cationic detergents, preferably

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quaternary ammonium bases in which at least one substituent is an aliphatic chain of at least 6 carbon atoms, preferably 12 to 20 carbon atoms. Of the cationic detergents is cetrimonium (cetyltrimethylammonium bromide) particularly effective. A valuable injectable and stable glucagon solution can contain about 1 mg of glucagon contain per ecm and 0.5 mg cetrimonium per ecm.

Among the anionic detergents, the alkyl aryl sulfonates are particularly preferred.

According to the invention, the stable aqueous, Injectable glucagon solutions can be obtained by adding glucagon, water and a stabilizing and solubilizing agent Amount of detergent are brought together, the glucagon being used in an amount of 0.1-5 mg per ecm.

In this process, certain types of the above-mentioned detergents are preferably used, in particular cetrimonium, especially in the production of glucagon solutions with about 1 mg glucagon per ecm and about 0.5 mg cetrimonium per ecm.

Depending on the storage temperature and the possibility that the stored glucagon solutions adsorb with the detergent Materials come into contact, e.g. plastic materials in available syringes or ampoules or rubber pistons in glass

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syringes, the g-lucagon solutions according to the invention are over stable for several months and also still biologically active. Storage of the solutions in contact with plastics and rubber materials should therefore be avoided.

Paper electrophoresis showed that a part of the glucagon in solution is converted into derivatives that have different electrical charges. at these derivatives can be desamidoglucagones. It has been found that deamination occurs with slightly acidic pH values is suppressed. Experiments with glucagon solutions, which contain cetrimonium showed that deamination takes place at a pH value of about 4-6.

Based on the following tables, which show experimental results, the stabilizing and solubilizing Effect of the aforementioned detergents can be illustrated. The first table gives the results that were obtained when neutral glucagon solutions with various concentrations were left to stand at room temperature, without a stabilizing or solubilizing addition being incorporated into them.

Table I. ,

Glucagon was dissolved in a 0.9 # NaCl solution at pH $ . A 0.9 # NaCl solution was used to dilute the diluted

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Different concentrations used. In every pail the pH then adjusted to 7.6. Each solution was observed after it was allowed to stand at room temperature.

Glucagon Observations Concentration in mg / ecm 1.00 Immediately after the 0.75 Setting the 0.50 pH values were 0.25 Crystals formed.

Table II

The effect of adding cationic detergents to glucagon slurries was examined. The final concentration was 1 mg glucagon per ecm in 0.9 # NaCl solution pH 7.4. The solutions were stored at 20 ° C and 4 ° C.

Detergent
concentration M. Cetyl trimethyl
ammonium bromide
(Cetrimonium) 4 ° C Benzylcetyl
dimethyl
ammonium chloride
(Rodalon) Cetylpyridine
chloride M. 20 ° C solution 20 ° C 4 ⁰ C 20 ° C 4 ⁰ C 0.1 M. solution solution Solution solution Solution fDe tergenz) 0.01 M. solution solution Solution solution Solution (insoluble 0.002 M. solution Kri
stalls Solution off
flocculation Solution solution 0.001 solution Kri
stalls From from
flocculation flocculation Solution solution 0.0001 Kri
stalls Kri- Kri
stalls stalls Kri- Kri
stalls stalls

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Table III

Variation of the glucagon and cetrimonium concentrations. 0.9% NaCl solution; pH = 7.4.
Storage temperatures of the solutions: 20 ° C and 4 ° O.

Glucagon Conc. 1 mg / ecm 0.5 mg / cc 0.2 mg / cc 0.1 mg / ccn Cetrimonium
conc. 20 ° C 4 ° C 20 ° C 4 ° C 2O ⁰ C 4 ⁰ O 2O ⁰ C 4 ⁰ C 2 per cent
1 per cent solution
solution solution
solution solution
solution solution
solution 0.5 per cent solution solution solution solution 0.2 per cent Crystals Solution few
Crystals solution solution 0.1 per cent Crystals Crystals solution solution

! Table IV

The effect of the addition of anionic detergents to Glucagonlösungen containing 1 mg of glucagon per cc ^ 0.9 NaCl at pH 7.4 containing LöBung * Storage temperatures of the solutions: 20 ⁰ C and 4 ⁰ C

concentration M. Sodium lauryl - concentration 196 G-3300 (Atlas) 4 ⁰ C sulfate Alkyl aryl sulfonate unsolvable M. 2O ⁰ C. 4 ⁰ C ), 5 # 20 ⁰ C lich 0.1 M. Deter- Deter- solution H genz borrowed 0.1Ji genz solution 0.01 M. Solution unclear C. O ₁ solution solution 0.002 Crystals C. O. solution Crystals 0.001 Crystals O ₁ solution Crystals 0.0001 Crystals , 5% o Crystals Crystals , 20 / co Crystals 1 per cent Crystals

The non-ionic detergent Renex 35 has been

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Slurry of glucagon at pH 7.4 was added to set a final concentration of glucagon of 1 mg / ccm and 1 $ detergent. Clear solutions were obtained at storage temperatures of 20 ° and 4 ° C.

With glucagon solutions containing cetrimonium, were in Experiments were carried out in the manner described below, with the following results being achieved:

A. There were 300 ecm of a neutral glucagon solution with the the following composition:

Glucagon 1 mg / cc

NaCl 0.9%

Cetrimonium $ 0.05

pH 7.1

The solution was placed in capped vials with a volume of 2 ecm (1 ecm solution per vial); the vials were sealed with membrane bodies of blue natural rubber and allowed to stand at 4 ⁰ C, 15 ⁰ C, -25 ⁰ C, 37 ⁰ C and 45 ° C. The vials were stored in such a way that the solutions could not come into contact with the rubber caps.

B. 300 ecm of a neutral glucagon solution having the following composition was prepared;

Glucagon 1 mg / cc
NaCl; $ 0.8

. ν sodium acetate 0.01 M. , Cetrimonium $ 0.05

.-.- ■., - ph - ■. 7.2 ... 'ι ■ ;

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The solution was to inject into available plastic syringes and G-las with a piston made of butyl rubber introduced · Both types of syringes were left lying in the aforementioned temperatures.

The aforementioned solutions were examined by the following methods:

1. The Cetrimoniumgehalt any glucagon was determined spectrophotometrically (see eg AV Few & RH Ottewill, J. Colloid Science, JJ _-., 34 (1956)). The attempts made with Glucagon and Cetriraonium in various known. Concentrations carried out indicated that the presence of glucagon does not interfere with the detection of cetrimonium.

2. The appearance of the solutions was determined visually (precipitation, Viscosity increase). . .,

3. The UV absorption at 276 nm and pH 2 was determined using the method in,> solution in vials after 10-fold dilution

The pH of the solutions was measured. -:; ^ - i =

5. The biological activity of the solutions was at

Rabbits for sure. · - ·. . \, _-, roi & gl ^ i - ^ t .--, t-distribution ,;

6. The solutions stored in vials were paper-electro-

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analyzed theoretically using an electrophoresis apparatus LZB 3276-50 and a buffer (pH 6.0) containing 0.13 M sodium phosphate and 7 M urea. The electrophoresis and subsequent operations were carried out in principle as suggested by F. Sundby, J. Biol. Chem, 237 t 3406 (1962). However, heat denaturation of the electrophoresis strips was omitted. Relatively clear protein bands (glucagon + derivatives) with a mutual distance of about 30 cm (350 volts, 22 hours) were found. In view of the interference of the cationic detergents, the glucagon was first isolated by precipitation with acetone.

The crystallizability was examined by using a method in which the glucagon is crystallized from a medium containing 0.08 # glucagon and 2 # urea and having a pH of 6.8. The glucagon usually crystallizes in the form of rhombododecahedral crystals with a size of about 10 / U.

Since the presence of cetrimony the crystallization of the To prevent glucagon, it is first necessary to isolate the latter. The glucagon was therefore by precipitation with Acetone separated.

The following results were obtained:

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1. Ce trimonium concentration

Vials;

Storage temperature
in ⁰ O Tr imonium content tng / ccm 4th
months 6th
months 4th 2
months 0.38 0.39 15th 0.40 .0.39 0.37 25th 0.40 0.41 0.38 37 0.40 0.41 0.40 45 0.39 Q »41 0.37 0.41

Glass Spritsens

Stores temperature Cetriraonium content mg / pcm months 3
months 4th
months 6th
months in ⁰ G Weeks 0.48 Q ₁ SI 0.48 0.47 4th 0.50 Ο, 4β. " 0 * 53 0.47 0.45 15th 0.50 0.47 0.51 0.44 0.38 25th 0.51 0.44 0.38 0.20 0.16 37 0.51 0 »33 0.25 0.05 0.08 45 0 * 50

Eunetstoffspritzen "

Storage temperature Grain content mg / . 1.5
months 3
months 4th
months β
months in ⁰ Q 3 "'■
Weeks Q, 50 0.49 0 »45 Q »47 4th ■ 0.47 Öf§2 0.48 Ö, 4 € " ¹ Q tW 15th 0.50? Ö, 5Ö o? p 0 * W If. ö »44 0.11 37 ö, iü '" t, 06 .W . 45 - Qi5§

I \ J \ J \ J + J KJ I

Vials:

- 13 2. Appearance of the glucagon solutions

After 9 months, the solutions were clear, colorless and slightly liquid for all of the used.
Temperatures (4, 15, 25, 37 and 45 ° 0).

Glass syringes:

Storage temperature
in ⁰ C 2 months 6 months 8 months 4th clear solution clear solution clear solution 15th clear solution clear solution clear solution 25th clear solution clear solution slight yellowish
clear solution 37 ' clear solution yellowish
clear solution yellowish solution
and
slight precipitation 45 clear solution yellowish discoloration and yellow
Precipitations on the sides
the syringe

Plastic syringes:

Storage temperature
in ⁰ C 2 months 6 months 8 months 4th clear solution clear solution clear solution 15th clear solution clear solution clear solution • 25 clear solution clear solution Precipitation.on the
Sides of the syringe 37 clear solution Precipitation on
the sides
the syringe Precipitation on
the sides
the syringe 45, "- ■ · - Precipitation
to the be
ten of
Syringe : Precipitation on
the sides
the syringe Precipitation on
the sides
the syringe \

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About the stability of the glucagon solutions under extreme conditions To find out, one vial was placed on a shaker table at room temperature and another vial was subjected to continuous rotation at the same temperature, so that the solution is in repeated contact with the The rubber cap of the vial came. After shaking it was over 6 days most of the glucagon failed and it became one Cetrimonium concentration of 0.19 mg / ccm was found. To, 35 days of continuous rotation, a very small precipitate was observed at a single point and the content Cetrimonium was 0.39 mg / cc.

3. UV absorption

The following values are absorbance values at 276 nm and pH 2; the values were in a 10-fold dilution the glucagon solution obtained :.

Vials:

Storage temperature
in ° C 4th months 7, 5 months 4th 0 , 187 0 , 203 15th 0 , 191 0 , 208 25th 0 , 192 0 , 208 37 0 , 203 0 , 223 45 0 , 210 -

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- - 15 -4. pH of the glucagon solutions

Vials:

Stores temperature
in ^Q Q pH As manufactured 4 months 9 months 4th 7.10 7 * 18 7 * 12 15th 6192 i _% m 25th 7 «Q§ 7.21 37 II 15 45 7.09

Storage temperature
in ° ο pi As manufactured 6 months 8 months 4th "7.IQ itm 15th 7t 13. 25th ItU 7133 37 7.01 7 * 30 45 6 * 94

Plastic syringes:

Storage temperature As manufactured pH 8 months in ° C 6 months 6.86 4th 6.87 6.95 15th 7.23 7.01 6.96 25th 7.01 6.74 37 7.08 6.53 45 6.75

5. Biological activity

The values shown in the table below are the activity ratios determined. The error limits (in fi) are given in brackets. A standard AB 66 glucagon lot was used.

I warehouse 1 month storage time 5 months 6 months. 9 months I υ σ Uly β Χ ei IjUX
in ° C 0.88
(76-101) 4 months 0.88
(66-117) 4th 0.79
(60-104) 15th 0.81
(68-97) 25th 0.63
(52-77) 37 0.32
(25-41) above
0.05 45

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6. Paper electrophoresis

The results shown in the table below represent percentages of the total eluted protein. The results were obtained from a spectrophotometric measurement (Eg ₂ c _nm ) of the protein chromium cresol, which is green in color.

Vials:

storage time temperature
in ° C Gang O
Glucagon Gang 1 Band 2 Band 3 5 months 4th 94 ° / $ 6 15th $ 91 $ 9 25th $ 85 $ 15 9 months 37 $ 49 $ 44 $ 5 $ 2 45 $ 16 $ 63 $ 17 $ 4 Glucagon
Lot AB 66
fresh
solution 4th $ 85 $ 14 $ 1 15th $ 81 $ 17 $ 2 25th $ 68 $ 30 $ 2 37 $ 23 $ 64 $ 13 $ 93.6 $ 6.4

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Claims (7)

- 18 claims 1. Stable aqueous injectable glucagon solution, marked 'is characterized by a content of glucagon in an amount of 0.1 - 5 mg / ccm and a stabilizing and solubilizing Amount of a detergent. ■. 2. glucagon solution according to claim 1, characterized in that the amount of detergent at least one fifth of the glucagon ~ amount is. 3. glucagon solution according to claim 2, characterized in that the molecule of the detergent is one or more aliphatic Has side chains each having at least 6 carbon atoms, preferably comprises 12-20 carbon atoms. 4. glucagon solution according to claim 2, characterized by a content of a cationic detergent, preferably one quaternary ammonium base in which at least one substituent f is an aliphatic chain having at least 6, preferably 12 to 20 carbon atoms. 5. glucagon solution according to claim 4, characterized by a content of cetrimonium (cetyltrimethylammonium bromide). 6. glucagon solution according to claim 5, characterized in that the content of glucagon is about 1 mg / ccm and the content of Cetrimonium is about 0.5 mg / ccm. 909836 / U2 3 ι a υ υ ό ο / 7. Glucagon solution after. To claim 2, characterized by a content of an anionic detergent, _s preferably an alkylaryl sulfonate. 8, method of making a stable aqueous, injectable glucagon solution according to claim 1, characterized in that that brought together glucagon, water and a stabilizing and solubilizing amount of detergent be »taking the glucagon in an amount of 0.1-5 mg / ecm is used. 909836 / U2 3