KR790000807B1 - Process for manufacturing alkali metal or ammonium insulin - Google Patents

Abstract

Title compds. having both rapid onset and prolonged duration of hyperglycemic activity were prepd. by crystallizing from an aq. soln. contg. a divalent metal (Mn, Fe, Co, Ni, Cu, Zn, Cd) insulin (10-1, 000U/ml) 0.0002-0.05 M chelating agent of formula (I; R1, R5 = carboxy methyl, C1-6 hydroxyalkyl, R2, R6 = carboxy(m)ethyl, R3 = H, OH, C1-6 alkyl, R4 = H, carboxy(m)etyl, m = 0,1, p = 0,1, r = 2,6, s = 0,2) and 0.2-0.1 M ammonium or alkali metal ions. pH was 7.6-8.6 and molar ratio 1 to divalent metal was 3:1.

Description

Method for preparing alkali metal or ammonium insulin

1: Electrophoresis analysis result of sodium insulin as a starting material

Figure 2: Electrophoresis of the product sodium insulin

Figure 3: Results of electrophoresis of sodium insulin as a starting material

Figure 4: Results of electrophoresis analysis of the product sodium insulin

The present invention relates to a method for preparing alkali metal and ammonium insulin, which have a dual effect on diabetes.

According to the present invention, the divalent metal insulin has a concentration of about 10 to 1,000 u / ml, and the divalent metal is selected from the elements of Group I, Group IB and IIB of the periodic table, and the concentration of alkali metal or ammonium ion is about 0.2 to 1.0 mole, the pH of this solution is about 7.6 to 8.6, the concentration of chelating agent is about 0.0002 to 0.05 mole, and the molar ratio of chelating agent to divalent metal of divalent metal metal insulin is at least about 3: 1, An alkaline aqueous solution composed of divalent metal insulin, a divalent metal chelating agent of the following structural formula and an alkali metal or ammonium ion is prepared to crystallize the alkali metal or ammonium insulin.

In the above structural formula

R ₁ and R ₅ are monovalent organic radicals independently selected from carboxymethyl, carboxyethyl, C ₁ -C ₆ alkyl and C ₁ -C ₆ hydroxy alkyl,

R ₂ and R ₆ each represents a monovalent organic radical selected from carboxymethyl and carboxyethyl,

R ₃ is a monovalent radical selected from hydrogen, hydroxy and C ₁ -C ₆ alkyl,

R ₄ is a monovalent radical selected from hydrogen, carboxymethyl and carboxyethyl,

m is an integer from 1 to 6,

n is 0 or 1,

p is 0 or 1,

r is an integer from 2 to 6,

s is an integer of 2 in 0.

Commercially available insulin preparations currently effective as therapeutic agents for diabetes are usually classified as having a short, medium or long duration of hypoglycemic effect duration. In most cases of diabetes, particularly in pediatric patients, the insulin preparation with a long lasting so-called dual effect is preferred while the hypoglycemic effect is rapid.

Recently, at least two different insulin preparations have been mixed to produce a dual effect insulin preparation. Typical mixtures include zinc insulin solutions from the pig pancreas and high concentrations of zinc insulin suspensions from cattle. Another formulation consists of a mixture of protamine insulin or propamine zinc insulin suspension and zinc insulin solution.

However, these dual effect insulin preparations have several problems, even if they succeed in some degree double effect. These problems usually refer to storage, integrity, purity, stability, color and contamination of hypoglycemic factors (glucagon) and high molecular weight antigenic proteins.

These problems with commercially available insulin preparations having a dual effect are solved, if not all, by the use of preparations containing alkali metal or ammonium insulin and protamine (US Pat. No. 3,758,683).

Alkali metal and ammonium insulins are generally prepared according to the method of U.S. Patent No. 3719655, which adjusts the pH of the solution containing insulin to about 7.2 to 10.0 and the concentration of alkali metal or ammonium ions to about 0.2 to 1.0 mole. Allowing the alkali metal or ammonium insulin to crystallize. Even if the problem related to zinc crystallization is solved, the non-insulin protein component removed only by zinc crystallization remains in the alkali metal or ammonium insulin. Therefore, insulin preparations having a dual effect should use an alkali metal or ammonium insulin which often contains non-insulin protein components not contained in zinc insulin, and thus should use the above-mentioned mixture having this problem.

The accompanying drawings are polyacrylamide disk-gel electrophoresis results of sodium insulin as a starting material and sodium insulin as a product of Examples 2 and 3.

The term "alkali metal" used herein refers to elements of Group 1A of the Periodic Table of the Elements up to 5 cycles (Robert C. Wist, Editor, "Chemistry and Molly Handbook", 53rd Edition, Chemical Labor Co., Cleveland, Ohio, 19 68, PB-3). Lithium, sodium and potassium are preferred, with sodium being the best.

"Divalent metal" refers to elements of groups VI _B , X, I _B and II _{B of the} Periodic Table of the Elements. Suitable divalent metals are manganese, iron, cobalt, nickel, copper, zinc, cadmium. In commercial importance, zinc is most preferred.

As mentioned above, the process according to the invention requires an alkaline aqueous solution of divalent metal insulin, which also contains a chelating agent and an alkali metal or ammonium ion. The pH of the solution is about 7.6 to 8.6 with a preferred pH of about 8.2.

The concentration of divalent metal insulin is about 10 to 1,000 u / ml (international units per ml of solution), preferably about 500 u / ml.

The chelating agent used in the present invention can be represented by the following general formula.

Examples of the chelating agent having the above general structural formula are as follows.

Ethylenediamine-N, N, N ', N'-tetraacetic acid,

1,2-diaminopropane-N, N, N ', N'-tetraacetic acid,

1,3-diamino-2-hydroxypropane-N, N, N ', N'-tetraacetic acid,

Diethylene triamine tetraacetic acid,

Diethylene triamine pentaacetic acid,

Hexamethylenediamine-N, N, N ', N'-tetraacetic acid,

N-butylethylenediamine-N, N ', N'-triacetic acid,

N, N'-dimethyltetrameritylenediamine-N, N'-diacetic acid

N- (2-hydroxyethyl) ethylenediamine-N, N ', N'-triacetic acid,

Ethylenediamine-N, N, N ', N'-tetrapropionic acid and the like. Preferred chelating agents are polymethylene diamine tetraacetic acids and the most preferred chelating agent is ethylene diamine-N, N, N ', N'-tetraacetic acid.

Generally, chelating agents can be used in their free acid form or in the form of alkali metal or ammonium salts. When used as a salt, the cation of the salt must be the same as the cation of the desired alkali metal or ammonium insulin. The amount of cation added should be taken into account when determining the amount of alkali metal or ammonium ions.

The molar ratio of chelating agent to divalent metal in the divalent metal insulin must be at least about 3: 1 to remove the divalent metal from the insulin and to rapidly crystallize the alkali or ammonium insulin. At lower rates, the rate at which alkali metal or ammonium insulin crystallizes from the alkaline solution is reduced. Also at low rates, at least, there is an increased likelihood that some divalent metal remains bound to insulin.

Therefore, the concentration of the chelating agent should be at least about 0.0002 moles and can be increased to about 0.05 moles. A concentration of about 0.01 mole is suitable at a concentration of about 500 u / ml of divalent metal insulin.

The alkaline solution of the divalent metal insulin used in the preparation process according to the invention must contain alkali metal or ammonium ions. Generally, the concentration of alkali metal or ammonium ions is about 0.2 to 1.0 mole.

Alkali metal or ammonium ions can serve as water-soluble compounds that do not have a deleterious effect on insulin under the manufacturing process conditions. Alkali metal or ammonium ions can also be provided as one or more such water soluble compounds.

Some of the alkali metal or ammonium ions required as described above may be provided in the form of alkali metal salts or ammonium salts of chelating agents, or may be provided from basic alkali metal or ammonium compounds necessary to adjust the pH of the solution to the desired degree of alkalinity. . Ingone may also be provided in the form of salts with anions such as chlorine, bromine, fluorine, iodine, acetate, sulfate, phosphate. Chloride and phosphate salts are preferred, with chloride being the best.

As noted above, the alkali metal and ammonium ions added must match the cations of the desired alkali metal or ammonium insulin. Generally these ions are provided in combination of the above three methods.

An example of a method of preparing a solution of divalent metal insulin is as follows. That is, the divalent metal insulin is dissolved in an acidic aqueous medium and the pH is adjusted to alkaline with an appropriate base. In general, chelating agents and alkali metal or ammonium ions can be added at any time in the form of a solid or an aqueous solution. When using a solution, the increase in total volume of the solution must be taken into account when calculating the final component concentration.

The acidic solution of divalent metal insulin should have a pH of about 2.5 to 3.5. The acidity of the solution can be controlled using inorganic acids, with hydrochloric acid being suitable. Since the preferred pH is about 3, water dissolves the divalent metal insulin in hydrochloric acid to obtain the most suitable pH.

If desired, this acidic solution may contain other agents such as preservatives and / or isotonic agents. Suitable preservatives include phenol, methylparaben and the like, and suitable isotonic agents include glycerin, glucose and the like.

The amount of preservative used can be increased to about 0.4%, about 0.2% is a suitable concentration. Suitable preservatives are phenols, which when used, promote the rate of crystallization of alkali metal or ammonium insulin.

The acidic solution of divalent metal insulin is made alkaline with the appropriate base. Generally the base is a hydroxide, carbonate or other basic compound of an alkali metal or ammonium. Thickened solutions of suitable hydroxides are preferred because solid alkali metal hydroxides prevent exothermic by any compound or extreme dilution of divalent metal insulin solutions.

Chelating agents or alkali metal or ammonium ions can be added to the divalent metal insulin solution at any time, for example, by dissolving the divalent metal insulin in dilute aqueous hydrochloric acid solution containing the chelating agent or chelating in an acidic aqueous solution of divalent metal insulin. Add topical agent. Or make the divalent metal insulin solution alkaline and then add the chelating agent.

Usually, the chelating agent is added to the acidic solution in the form of an alkali metal or ammonium salt, and the neutralization of all the carboxy groups in the chelating agent increases the water solubility. A flat chelate agent is added to the alkaline solution in the form of the free acid.

A more preferred method for preparing alkaline solutions is to add alkali metal or ammonium ions together with the chelating agent. The added ions are supplied to the base used to make the acidic solution alkaline. The remainder of the necessary ions are supplemented by adding an appropriate amount of alkali metal or ammonium salt. These salts are usually added before alkalizing the solution, but can be added at any time, such as chelating agents.

Another method of preparing an alkaline solution of divalent metal insulin is to dissolve the divalent metal insulin in an alkaline aqueous medium of pH about 8 to 9.5 and adjust the pH of this solution to about 7.6 to 8.6. The alkalinity of the initial aqueous solution is provided by the addition of alkali metal or ammonium salts, and hydrochloric acid or phosphoric acid is suitable for adding a dilute inorganic acid when adjusting the pH later. It should be noted that solutions with higher concentrations of divalent metal insulin are more difficult to prepare than dilute solutions because the dissolution of divalent metal insulin in alkaline solutions is lower.

If desired, chelating agents, alkali metal or ammonium ions may be added at any time, but usually chelating agents and alkali metal or ammonium ions are added before final adjustment of pH.

Alkali metal or ammonia insulin is crystallized from the solution after making an alkaline solution of divalent metal insulin. Crystallization occurs at or above ambient temperature, but the crystallization temperature should generally be about 30 ° C or below.

Crystals of the alkali metal or ammonium insulin thus obtained are usually separated according to methods well known to those skilled in the art. Typically the crystals are separated by centrifugation and successively washing with a dilute solution of alkali metal or ammonium salt (usually about 0.5 mole), acetone, anhydrous alcohol and ether and then drying under vacuum.

Example 1

8 g of zinc insulin, consisting of about 70% bovine insulin and 30% porcine insulin, is dissolved in 400 ml of acid-phenol water made by dissolving 0.8 g of phenol in 400 ml of 0.01 N hydrochloric acid. The solution is then divided equally into four parts and 2.0 g of sodium chloride is added to each part. Four parts are called samples 1,2,3,4 and ethylenediamine tetraacetic acid and tetrasodium salt are added to three samples as shown in the following table.

Each solution is added with 40% aqueous sodium hydroxide solution to adjust the pH to 8.2. The determination of sodium insulin immediately occurs in sample 2-4. However, in Sample 1, no crystals formed after stirring for 3 hours.

Sample 1 was titrated with 0.1 mol of ethylenediamine tetraacetic acid (with tetra sodium salt) and crystallization of sodium insulin started when the EDTA concentration reached about 0.006M. After stirring each sample overnight, the sodium insulin crystals are collected by centrifugation, washed once with acetone, twice with anhydrous alcohol and once with ether. Drying under vacuum yields sodium insulin as follows.

The titer of sodium insulin (and all insulin obtained in the examples listed below) was determined by G.W. Probst, et al., J. Pharm. Sci. 55, 1408 (1966), measured by immunological analysis. In this method the standard deviation is ± 15-25%.

Example 2

2 g of pure sodium insulin obtained from bovine pancreas, with a titer of 27.0 u / mg and containing a small amount of acidic protein impurities, are dissolved in 400 ml 0.5 N acetic acid. To this solution is added about 20 ml of anhydrous alcohol and 0.4 ml of 20% zinc chloride solution. This 0.4 ml zinc chloride solution is added. This 0.4 ml of zinc chloride corresponds to 40 mg of zinc chloride per gram of insulin. The pH of the solution is adjusted to 5.95 with 20% ammonium hydroxide and refrigerated at about 5 ° C. for 72 hours. The resulting zinc insulin crystals are obtained by centrifugation and washed once with distilled water and then dissolved in 50 ml of acid-phenol water prepared in Example 1. Dilute the solution with acid-phenol water to make 100 ml, and add 2.0 g (0.34 mole) of sodium chloride and 0.208 g (0.005 mole) of EDTA (as tetrasodium salt). When the solution is adjusted to pH 8.2 using a 40% soda solution, crystals of sodium insulin begin to form. Crystallize overnight at ambient temperature. The crystals are separated by centrifugation and washed and dried as in Example 1. The yield is 1.864 g and the yield is 93.2%. The potency of sodium insulin is 27.0 u / mg.

Sodium insulin, a sample of starting material, and final sodium insulin of Example 2 were added to R.L. Jackson et al. Analysis was performed by polyacrylamide disk-gel electrophoresis according to the method used in Diabetes, 21, 235 (1972).

1 is a result of electrophoresis analysis of the starting material sodium insulin and FIG. 2 is a result of electrophoresis analysis of the final sodium insulin, in which 1 is a polyacrylamide gel, 2 and 3 are non-insulin proteins, 4 and 5 are insulin and It is a pseudo insulin protein.

Example 3

As a starting material, the procedure of Example 2 is repeated using sodium insulin isolated from porcine pancreas with a titer of 25.0 u / mg. The yield of sodium insulin was 1.862 g, yield 93.1% and titer 28.9 u / mg.

The starting material (Figure 3) and final product (Figure 4) of Example 3 were analyzed by polyacrylamide disk-gel electrophoresis as in Example 2. In these figures, 1 is a polyacrylamide gel, 6,7,8 is a non-insulin protein, and 9 and 10 are insulin or similar insulin proteins. In Figure 1-4 it can be seen that the non-insulin protein is removed by this manufacturing process. Furthermore, the significant improvement in the purity of this final alkali metal or ammonium insulin is not apparent from titer or other data alone.

Example 4

6 g of zinc insulin crystals (70% in the pancreas and 30% in the pig pancreas) were dissolved in 200 ml of acid-phenol water prepared by Example 1. Take 100 ml of this solution and add 2.5 g (0.033 mol) of potassium chloride. The pH of this slightly turbid solution is adjusted to 8.2 with 3.5 ml of 10% potassium hydroxide, and 0.3 g (0.001 mol) of ethylenediamine tetraacetic acid is added to the turbid solution, and the pH of the solution is adjusted to 8.2 using 10% potassium hydroxide. Readjust until stable. The solution is stirred at ambient temperature (about 25 ° C.) for 48 hours to initiate crystallization for the first 12 hours. The resulting potassium insulin is centrifuged, washed twice with anhydrous alcohol, once again with ether and dried in vacuo. Yield of potassium insulin 1.736 g (86.8%), titer 24.9 u / mg potassium content 1.48%, water content 10.1%

Example 5

1.4 g (0.033 mol) of lithium is added to 100 ml of the zinc insulin solution prepared in Example 4. The solution was adjusted to pH 8.2 with 6.2 ml of 1N lithium hydroxide, ethylenediamine tetraacetic acid was added and readjusted until the pH was stabilized to 8.2 with 1N lithium hydroxide and the procedure of Example 4 was repeated. Repeating the crystallization process of Example 4 yielded lithium insulin having a yield of 1.234 g (61.1%), a titer of 27.8 u / mg, a lithium content of 2.37%, and a water content of 3.6%.

Example 6

The same procedure as in Example 5 is repeated except that 1.8 g of ammonium chloride is used instead of lithium chloride and 60 ml of 1N ammonium hydroxide is used instead of lithium hydroxide. After adding ethylenediamine tetraacetic acid and adjusting the pH with 1N ammonium hydroxide, crystallization does not occur even after 24 hours. 1.8 g of ammonium chloride was added, followed by further stirring for 24 hours to cause crystallization. The resulting ammonium insulin is isolated according to the method of Example 4 to yield 1.018 g (50.9%). Titer 27.6u / mg, moisture content 7.3%, sulfur containing ash 0.08%

Claims (1)

The divalent metal insulin has a concentration of about 10 to 1,000 u / ml, and the divalent metal is selected from manganese, iron, cobalt, nickel, copper, zinc, and cadmium, and the concentration of alkali metal or ammonium ion is about 0.2 to 1.0 mol. The pH of the alkaline solution is from about 7.6 to 8.6, the concentration of the chelating agent is from about 0.0002 to 0.05 mole, and the molar ratio of the chelating agent to the divalent metal of the divalent metal insulin is at least about 3: 1, 2 A method for producing an alkali metal or ammonium insulin, characterized by reacting a hydrated metal insulin, a divalent metal ion chelating agent having the following structural formula and an aqueous alkali solution composed of alkali metal or ammonium ions.

In the above structural formula R ₁ and R ₅ each represents a monovalent organic radical selected from carboxymethyl, carboxyethyl, C ₁ -C ₆ alkyl, C ₁ -C ₆ hydroxy alkyl, R ₂ and R ₆ each represent a monovalent organic radical selected from carboxymethyl and carboxyethyl, R ₃ is a monovalent radical selected from hydrogen, hydroxy, C ₁ -C ₆ alkyl, R ₄ is a monovalent radical selected from hydrogen, carboxymethyl, carboxyethyl, m is an integer from 1 to 6, n is 0 or 1, p is 0 or 1, r is an integer from 2 to 6, s is an integer of 2 in 0.

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