DK152832B - Process for preparing an aqueous insulin solution which is physically stabilized against boundary surface polymerization - Google Patents

Description

! DK 152832B

The present invention relates to a process for the preparation of aqueous insulin solutions physically stabilized against interface polymerization, characterized in that insulin is mixed with a phospholipid of the general formula I

H-CH-OR 'CH-OR "(I) H-CH-OP (O) (OH) -OR" 1 wherein R' and R "are the same or different and each represents hydrogen, alkylcarbonyl, alkenylcarbonyl, alkadienylcarbonyl, alkatrienylcarbonyl or alkatetraenylcarbonyl, with the proviso that not both R 'and R "simultaneously represent hydrogen, 15 and R" 1 represent a hydrophilic group, and with water and optionally with a zinc salt, a preservative, an agent which makes the solution isotonic, and a buffer substance wherein the weight ratio of the phospholipid to the insulin is in the range of 1: 5 to 1: 10,000, and the concentration of the phospholipid is in the range of about 10 to about 200 µg / ml.

Insulin dissolved in a liquid medium, e.g. water, can be stored for several years at room temperature, and the preparations are stable within that period. However, if an insulin solution is heated to ca.

25 80 ° C, the insulin denatures within minutes, which is referred to as heat denaturation or heat polymerization.

Shaking an insulin solution for a few days at a lower temperature where no or substantially no heat denaturation occurs, e.g. at 41 ° C, another kind of polymerization will occur, and this kind of polymerization is referred to herein as non-covalent interface polymerization.

In insulin manufacturers, insulin dealers and patients, insulin preparations are usually stored at approx.

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2 5 ° C, and there does not appear to be any interface polymerization in such compositions, although the compositions are inevitably shaken from time to time during transport.

Over the past few years, a steady effort has been made to develop portable or implantable systems for continuous infusion of insulin. The mechanical portion of a continuous insulin delivery apparatus comprises, in principle, components such as an insulin reservoir, a pump system, and a suitable catheter for delivering insulin to the patient. If the insulin solution is supplied by a pump, it can also act as an insulin reservoir.

Unfortunately, when insulin in commercially available solutions is placed in such systems, it has been found that the insulin has the ability to undergo interface polymerization at body temperature and thus clog both mechanical parts and supply catheters. This characteristic of insulin solutions has proven to be a major obstacle to further development and clinical use of continuous infusion apparatus.

It is clear that insulin solutions of any kind of continuous delivery apparatus are subjected to motions that provide interface polymerization. The general imperfection of known insulin preparations in this context is richly documented in the literature, cf.

25 e.g. Diabetologia 19 (1980), 1-9.

To solve this problem, it has been proposed to use acidic insulin solutions containing glutamic acid or aspartic acid, cf. Diabetes 30 (1981), 83. However, insulin is chemically unstable in acid, even at temperatures below body temperature. It has further been proposed to use insulin preparations containing nonionic surfactants, cf. DE Publication No. 2,952,119. However, nonionic surfactants may be considered undesirable in drugs for parenteral use.

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such genes are overcome according to the present invention, which relates to a process for preparing aqueous insulin solutions in which the insulin is substantially less likely to undergo non-covalent interface polymerization under the conditions of continuous insulin delivery apparatus than is the case with conventional insulin preparations.

It has now surprisingly been found that aqueous insulin solutions can be stabilized against interface polymerization when at least one phospholipid of formula I is present in the solution.

In phospholipids of formula I, the hydrophilic group (R, n) is preferably 2- (trimethylammonio) ethyl, 2-aminoethyl, 2-carboxy-2-aminoethyl, 2,3-dihydroxypropyl or 2,3,4,5 , 6-pentahydroxycyclohexyl, the phosolipids concerned having particularly advantageous properties and of the substituents in question, 2- (trimethylammonio) ethyl is preferred. The aforementioned alkylcarbonyl, alkenylcarbonyl, alkadienylcarbonyl, alkatrienylcarbonyl and alkatetraenylcarbonyl groups may contain from ca. 8 to approx. 22 carbons, and in one embodiment of this invention, they contain from ca. 12 to approx. 22 carbon atoms.

The aqueous insulin solution prepared by the process of the present invention may contain 25 one or more phospholipids of formula I and optionally a zinc salt, a preservative, an agent which is isotonic, a buffer and other physiologically acceptable compounds known as additives for insulin solutions.

A preferred subgroup of compounds of formula I are compounds wherein R 1 and R "each represent alkylcarbonyl. A further preferred subgroup of compounds of formula I are compounds wherein R" 1 represents 2- (trimethylammonio) ethyl, which compounds are known like 35 lecithins. Further preferred are compounds of formula I wherein R 1 and R "each represent alkylcarbonyl having from about 8

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to approx. 16 carbon atoms or from approx. 12 to approx. 16 represents carbon (2) and R "represents 2- (trimethylammonio) ethyl. The most preferred subgroup of compounds of formula I are compounds wherein R 1 and R" each represent octanoyl. Compounds of formula I, wherein R 'and R "each represent octanoyl, are preferred because they do not appear to form liposomes and, moreover, because they do not appear at lower concentrations, e.g., below about 160 µg / ml. forming micelles.

The amount of a phospholipid of formula I required to stabilize an insulin solution is in the range of about 10 to approx. 200 µg / ml, more preferably from ca. 10 to approx. 100 µg / ml, preferably from ca. 25 to approx. 75 µg / ml, most preferably from ca. 30 to approx. 50 µg / ml, insulin solution. The concentration of dissolved insulin in the solutions prepared by the process of the present invention is in the range of about 5 to approx. 1000 international units (I.U.) per ml or even higher.

Some of the stabilized insulin solutions prepared according to the following examples may contain 20 liposomes. However, such liposomes are unlikely to contain insulin as they are formed before the addition of insulin. If liposomes are present in the insulin solutions prepared by the process of the present invention, it is preferred that the insulin be substantially outside such liposomes. The term essentially denotes that preferably more than 90%, but preferably more than 99%, of the insulin is outside any liposomes present.

Known liposomes containing insulin, cf. eg. 30 European Patent Application No. 32622 can be distinguished from this invention both by their purpose and by the fact that the presence of insulin within an liposomal vesicle present in the insulin solutions prepared by the process of the present invention is purely random. While the insulin solutions prepared by the process of the present invention are calculated

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for parenteral administration, oral administration is the primary reason for encapsulating insulin in liposomes. One purpose of liposomes containing insulin is to protect the insulin from unwanted chemical attack, e.g. chemical breakdown 5 of the insulin in the stomach if the insulin is administered orally.

The articles on liposomes containing insulin do not deal with the physical stabilization of insulin solutions against interface polymerization. In known liposomes containing insulin, the weight ratio of the phospholipid 10 to insulin is e.g. in the range of 1: 0.01 to 1: 0.001, while the weight ratio of the insulin solutions prepared by the process of the present invention is in the range of 1: 5 to 1: 10,000, preferably 1:10 to 1: 1000.

West German Publication No. 2,652,636 discloses a method for stabilizing sensitive proteins by the addition of protective compounds with amphophilic structure. Contrary to the present invention, the stabilization of the aforementioned disclosure is achieved by encapsulating the sensitive protein to avoid contact with water. In addition, according to the terminology of the aforementioned publication, insulin is not to be considered a sensitive protein.

It is an object of this invention to keep the insulin in contact with water, i.e. in solution, the insulin 25 being excluded from contact with other interfaces.

The insulin solutions prepared by the process of the present invention preferably contain bovine, porcine or human insulin.

In a preferred embodiment, the insulin solutions prepared by the process of the present invention contain zinc. However, the amount of zinc must be chosen so that no precipitation occurs. Good stability to interface polymerization is achieved in insulin solutions where the ratio of the molar concentration of zinc ions available to the insulin to the molar concentration of insulin calculated as hexamer insulin

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is in the area from approx. 1.5 to approx. 4.6, preferably from ca. 3 to approx. 4.5, most preferably from ca. 3.6 to approx. 4.3. Preferred zinc salts are soluble zinc salts such as zinc acetate or chloride. If the insulin solutions prepared by the process of the present invention contain compounds which form complexes with insulin, such as an amino acid, e.g. glycine or histidine, or a hydroxy carboxylic acid, e.g. citric acid, is only part of the total zinc content available for the insulin.

An example of the preparation of insulin solutions according to the invention comprises dissolving insulin, e.g. crystalline zinc insulin, e.g. high purified insulin such as "monocomponent" insulin, cf. British Patent Specification No. 1,285,023, in water in the presence of acid, e.g. hydrochloric acid. Separately an aqueous solution of a preservative, e.g. phenol, an alkyl phenol such as cresol, or p-hydroxybenzoic acid methyl ester, and the solution also contains, if desired, an agent which renders the solution isotonic, such as sodium chloride or glycerol. The preservative solution may further contain a buffer such as sodium orthophosphate, sodium citrate, sodium acetate or TRIS (tris (hydroxymethyl) aminomethane). The resulting preservative solution is then added, if desired, to the acidic insulin solution followed by addition of a base, e.g. a sodium hydroxide solution to adjust the pH to neutral. In the context of this invention, the term neutral indicates a pH in the range of about 6.5 to approx. 8. The phospholipid of formula I may be added to the insulin solution as a solution or colloidal solution prepared by dissolving or suspending the phospholipid of formula I in water and subjecting, if necessary, to ultrasound treatment prior to admixture with the insulin solution. The phospholipid solution may, if desired, contain a buffer and a preservative. After admixture of the phospholipid, the pH of the insulin preparation can be set to neutral. Make up the resulting insulin solution

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add up to the calculated volume by adding water, then sterilize by filtration and then transfer aseptically to sterile vials, which are then closed.

The process of the present invention 5 can be carried out by mixing insulin with a compound of formula I and optionally. with a zinc salt, a preservative, an agent that makes the solution isotonic, and a buffer in the presence of water.

Some compounds of formula I are known and the remaining 10 compounds of formula I can be prepared analogously to the preparation of known compounds.

Further details of the practice of the present invention are given in the following examples. The insulin starting material used in the Examples contains from about 15 20 to approx. 35 µg zinc per mg of nitrogen. The determination of the stability factor is shown below.

To determine the stability of the insulin solutions against interface polymerization, the solutions are subjected to a stability test under forced conditions as follows: 12.5 ml vials containing 10 ml of test sample are each provided with a rubber stopper and placed vertically in a shaker (sold by Heto, Denmark) in total immersed in a water bath maintained at 41 ° C +/- 0.1 ° C. The cap-25 slides are subjected to horizontal, rocking movements with a frequency and amplitude of 100 rpm and 50 mm, respectively.

The opalescence of the test samples is determined at regular time intervals on a "Fischer DRT 1000 nephelometer" (negotiated by Fischer, Canada). It is estimated that 30 interface polymerization has occurred when the cloudiness exceeds 10 NTU (nephelometric turbidity units).

The stability factor is calculated as the ratio of time elapsed before interface denaturation in the test sample to the corresponding time for a control sample prepared in the same manner as the test sample, with the proviso that no compound of formula I is added. .

Example 1

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Dissolve 8,500 mg of semisynthetic human insulin in 10 ml of 0.045 N hydrochloric acid and add 359 mg of p-hydroxybenzoic acid methyl ester, which is dissolved in 300 ml of distilled water.

Further, 476 mg of sodium acetate trihydrate, 2.46 g of sodium chloride and 4.73 ml of a 0.2 N sodium hydroxide solution dissolved in 15 ml of distilled water are added. 9 mg of dimyristoyl, L-alpha-phosphatidylcholine are suspended in 10 ml of a solution of 70 mg of sodium chloride, 13.6 mg of sodium acetate and 10 mg of p-hydroxybenzoic acid methyl ester in distilled water. Nitrogen is bubbled through the slurry, which is treated with ultrasound in an ultrasonic bath for 2 hours. The resulting colloidal solution is stirred into the insulin solution. The pH is adjusted to 7.45 with 0.2 N hydrochloric acid or a 0.2 N sodium hydroxide solution, and distilled water is added to a volume of 350 ml. The resulting insulin solution stability factor is above 125.

Example 2 9.65 g of porcine insulin is dissolved in 400 ml of 0.02 N hydrochloric acid, 5.0 g of crystalline phenol and 40 g of anhydrous glycerol are added, and further distilled water is added to a volume of 2200 ml. The pH is adjusted to 7.45 with a 0.2 N sodium hydroxide solution. Dissolve 125 mg of distearoyl, L-alpha-phosphatidylcholine by gentle heating in 25 ml of ethanol (96%) and spray vigorously, through a cannula, into 100 ml of distilled water at a temperature of 70 ° C. The resulting cloudy solution is treated for 15 minutes with ultrasound with a high-energy ultrasound probe, the resulting collide solution is stirred into the insulin solution and distilled water is added to a volume of 2500 ml. If necessary, adjust the pH to 7.45. The stability factor is over 30.

Examples 3-8

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Insulin solutions are prepared in an analogous manner as described in Example 1, with the proviso that the phospholipids used are lecithins, wherein the hydrophobic moieties, i.e. R 'and R "are identical and are as given in Table I. The results are also shown in Table I.

Table I _______

Example R 'and Insulin Stability No. __Article Factor_ 10 3 myristoyl pigs above 120 4 palmitoyl pigs 104 5 stearoyl pigs above 117 6 lauroyl human over 133 7 myristoyl human over 133 15 8 palmitoyl human 75

Example 9

An insulin solution is prepared in an analogous manner as described in Example 1, with the proviso that the phospholipid used is egg lecithin and the insulin used is porcine insulin. The stability factor is 96.

Examples 10-14

Insulin solutions are prepared in an analogous manner as described in Example 1, with the proviso that swine insulin is added in an amount which gives the final concentrations indicated in Table II. The results are also shown in Table II.

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Table IX__

Example Insulin, Stability No._I. U. / ml_factor_ 10 20 over 120 5 11 40 over 120 12 100 97 13 200 79 14 500 53

Example 15 10 1.50 g of porcine insulin is dissolved in 6.5 ml of 0.2 N hydrochloric acid and water is added to a volume of 50 ml. Dissolve 1.0 g of p-hydroxybenzoic acid methyl ester and 1.78 g of sodium phosphate in 900 ml of distilled water and add the insulin solution. The pH is adjusted to 7.45 with a 0.2 N sodium hydroxide solution. A colloidal dimyristoyl, L-alpha-phosphatidylcholine solution prepared in an analogous manner as described in Example 2 is added together with distilled water to a volume of 1000 ml to obtain a phospholipid lute concentration of 50 µg / ml. The stability factor is 20 over 30.

Example 16

An insulin solution is prepared in an analogous manner as described in Example 15, with the proviso that the final solution contains 20 I.U. insulin per ml. The stability factor is over 17.

Example 17

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An insulin solution is prepared by analogy as described in Example 15, with the proviso that the final insulin concentration is 500 I.U./ml and the final 5 content of dimyristoyl, L-alpha-phosphatidylcholine is 50 µg / ml. The stability factor is over 30.

Examples 18-20

Insulin solutions are prepared in an analogous manner as described in Example 2, with the proviso that dimyro-10oyl, L-alpha-phosphatidylcholine is used in an amount to obtain the final concentration indicated in Table III. The results are also shown in Table III.

Table III________

Example Dimyristoyl Compound, Stability 15 No. _µg / ml ml factor 18 1 1.3 19 10 1.8 20 50 over 33

Example 21 Dioctanoyl, L-alpha-phosphatidylcholine is dissolved in distilled water and added, in a sufficient amount to reach a final concentration of 30 µg / ml, to an insulin solution prepared analogous to the insulin solution of Example 1. The stability factor is above 63 .

Claims (10)

3.65 g of semi-synthetic human insulin are dissolved in 100 ml of 0.02 N hydrochloric acid and 2.0 g of crystalline phenol. 16 g of anhydrous glycerol and 0.3 ml of zinc chloride solution containing 4% zinc are added and further distilled water is added to a volume of 900 ml. The pH is adjusted to 7.45 with a 0.2 N sodium hydroxide solution. Dissolve 50 mg of dioctanoyl, L-alpha-phosphatidylcholine in distilled water and add to the solution. Distilled water is added to a volume of 1000 ml. The stability factor is 65. Example 23 3.65 g of semi-synthetic human insulin are dissolved in 100 ml of 0.02 N hydrochloric acid and 2.0 g of crystalline phenol. 16 g of anhydrous glycerol and 0.3 ml of zinc chloride solution containing 4% zinc are added and further distilled water is added to a volume of 900 ml. The pH is adjusted to 7.45 with a 0.2 N sodium hydroxide solution. Dissolve 40 mg of dioctanoyl, L-alpha-phosphatidylcholine in the insulin solution, and distilled water is added to a volume of 1000 ml. The stability factor is over 30. A process for preparing an aqueous insulin solution physically stabilized against interface polymerization, characterized in that insulin is mixed with a phospholipid of the general formula I-CH-OR 'CH-OR' (I) Process according to claim 1, characterized in that the hydrophilic group is 2- (trimethylammonio) ethyl, 2-aminoethyl, 2-carboxy-2-aminoethyl, 2,3-dihydroxypropyl or 2,3,4,5,6-. pentahydroxycyclohexyl. Method according to claim 1 or 2, characterized in that the insulin is substantially outside any liposomes present. Process according to any one of the preceding claims, characterized in that R '' represents 2- (trimethylammonio) ethyl. Process according to claim 4, characterized in that R1 and R "each represent alkylcarbonyl containing from about 8 to about 16 carbon atoms. H-CH-OP (O) (OH) -OR "wherein R 'and R" are the same or different and each represents hydrogen, alkylcarbonyl, alkenylcarbonyl, alkadienylcarbonyl, alkatrienylcarbonyl or alkatetraenylcarbonyl, with the proviso that not both R' and R "simultaneously represents hydrogen, 10 and R" represent a hydrophilic group, and with water and optionally with a zinc salt, a preservative, a solution which isotonic, and a buffer, the weight ratio of the phospholipid to the insulin being in the range 1: 5 to 1: 10,000 and the concentration of the phospholipid is in the range of about 15 10 to approx. 200 µg / ml. Process according to claim 5, characterized in that R 1 and R "each represent alkyl carbonyl containing from about 12 to about 16 carbon atoms. Process according to any one of claims 1-5, characterized in that R 'and R "each represent octanoyl. 14 DK 152832 B Process according to any one of the preceding claims, characterized in that the concentration of phospholipid of the formula I in the solution in question is in the range of approx. 10 to approx. 100 µg / ml. A method according to any one of the preceding claims, characterized in that the weight ratio of the phospholipid of formula I to insulin is in the range of approx. 1:10 to approx. 1: 1000. Process according to any one of the preceding claims, characterized in that the weight ratio of the molar concentration of zinc ions available for the insulin to the molar concentration of insulin calculated as hexameric insulin is in the range of about 10%. 1.5 to approx. 4.6. 15