FI66751B - FOERFARANDE FOER FRAMSTAELLNING AV ETT STABILT INSULINPREPARATMED LAONGTIDSVERKAN - Google Patents

Description

---'- "" Ί Γβ1 ANNOUNCEMENT,. _ _ * f & T & utlAggningsskkipt 66751 C (45) Γηt: η ΐ 11 cyZnn jt ty 10 12 1984 Patent noddolat (51) K ^ ftm.CL A 61 K 37/26, C 07 C 103/52 SUO MINFIN LAND («) 760058 (21) Hilmnhptlyf — ΑΐϋΒΙοιΙριρίΗ 12.01.76 V1) (23) Altefiiyft-GIWtMatei 12.01.76 (41) TbNmHHklMM - BttvKeffaMlf 16.07.76

National Board of Patents and Registration / 44¾ | «'ui ΐιιΠιii, an pnw. -

Patent- och 1 efliterityralsen AmOtaw uchgd oeh mtsannew pnHtiml 31.08.84 (32) (33) (31) ί ^ ΠΤ4 · “Τ eteeMue · —aeglrd prtortut 15.01.75 Denmark-Danmark (DK) 83/75 (71) Nordisk Insulinlaboratorium , Ved Stadion 2, DK-2820 Gentofte, Denmark-Danmark (DK) (72) Bruno Andre Hansen, Rtfdovre, Finn Hede Andresen, Hillereid, Denmark-Danmark (DK) (7 * 0 Berggren Oy Ab (5 * 0 Method of stable , for the preparation of a long-acting insulin preparation - Förfarande för framstälIning av ett stabilt Insulin preparations with long-acting insulin

The present invention relates to a process for preparing a stable, long-acting insulin preparation having low antigenicity by reacting insulin with an amino base containing amino groups, preferably a polypeptide protamine.

Insulin preparations derived from porcine or bovine pancreas are commonly used to treat diabetes mellitus. Thus, about 30% of world insulin production is based on porcine insulin and approximately 70% on bovine insulin. It has also been proposed to make insulin from other animals, such as sheep, but to date it has not reached significant commercial importance.

Insulin therapy has in the past caused quite a number of disadvantages, e.g. have manifested as allergy and fatty metabolism.

It has long been known that conventional insulin treatment in most patients has resulted in the formation of insulin antibodies 66751, which in a few cases has resulted in increased insulin requirements due to unknown amounts of insulin binding to antibodies and ineffective insulin control as blood glucose control agents.

It has long been assumed that the main reason for the formation of antibodies and thus increased insulin consumption is the presence of various impurities in conventional commercial insulin.

Examples of such impurities are insulin dimer, proinsulin, intermediate insulin (proinsulin and insulin intermediate), arginine insulin, ethyl ester insulin, mono-desamido insulin and di-desamido insulin. The first three of these compounds are known to be highly antigenic. It is also known that the fourth or fifth or both of these are highly antigenic, while the sixth and seventh compounds and the insulin molecule itself are not or only slightly antigenic. Compare: Schlichtkrull, Monocomponent Insulin and its Clinical Implications, March 16, 1973, GB-PS 1,285,023; DE-OS 1 940 130; FI application 2276/69; FI-P 52 522; CA-PS 920 946.

According to these patents, all kinds of dosage forms of highly purified insulin known per se, such as suspensions of amorphous zinc protamine insulin and crystalline protamine insulin, can be prepared. The starting material used is purified insulin which has been gel-filtered, precipitated with salt, separated by centrifugation, redissolved, isoelectrically precipitated and recrystallized. For example, to prepare zinc protamine insulin, the purified insulin is redissolved in hydrochloric acid and reacted with zinc chloride and protamine sulfate in the presence of a preservative. However, urea or other anti-aggregation compounds are not used in the reaction medium at any stage in this reaction.

It is known to remove said impurities by gel filtration and / or ion exchange, whereby it is possible to obtain high-purity insulin which contains essentially only one component. Thus, as early as 1960 or earlier, R. David Cole, see "The Chromatography of Insulin 66751 in Urea-Containing Buffer," J. Biol.Chem. 235 (1960), pp. 2294-2299, to isolate highly purified insulin preparations by ion exchange chromatography using urea-containing buffer solutions as eluents. J. Arthur Mirsky and Kazunori Kawamura, "Heterogenity of Crystalline Insulin", Endocrinology 78 (1966) pp. 1115-1119, also studied the composition of insulin from many different animal species, including humans, and isolated highly purified insulin fractions by gel filtration and separation. using polyacrylamide gel electrophoresis (DISC PAGE).

GB 881 885 discloses a general method for purifying proteinaceous materials by a chromatographic method in which an alcoholic solution such as 70% ethanol in an aqueous buffer is preferably used as the eluent. Depending on the conditions, the chromatographic method can be used to isolate insulin in high yield or to purify insulin, since both cation exchange resins and anion exchange resins have been shown as ion exchange resins.

In recent years, insulin preparations have been marketed which have more or less completely removed the above-mentioned impurities and which, in many cases, have also led to a reduction in the formation of antibodies in diabetic patients.

It has been found that both porcine and bovine insulin can be rapidly formulated in such a pure form that they do not cause or cause only very small amounts of insulin antibodies to form. Compare: Schlichtkrull, Monocomponent Insulin and its Clinical Implications, 16.3.1973. It has further been shown that it is possible to prepare long-acting preparations with significantly reduced antigenicity when these preparations are formed using effectively purified porcine insulin, cf. T. Deckert et al. Diabetology, Vol. 10, pp. 703-708, 1974. In contrast, although bovine insulin has been prepared to be so pure that current analytical methods must be considered as pure as porcine insulin, which can currently be prepared 4 66751, and so on, as pure, as mentioned above, it does not cause the formation of antibodies or causes it only when used in a very small amount as a fast-acting preparation, however, long-acting, low-antigen preparations based on effectively purified bovine insulin have not been marketed so far. This is a significant drawback because long-acting bovine insulin-based preparations are the most widely used internationally.

The formation of antibodies in an animal against a chemical compound, such as insulin, may be due to such chemical differences in the primary structure, cf. Arquilla E.R. et al., Immunochemistry of Insulin, Handbook of Physiology, 9, p. 160, 1972, that a chemical compound has a steric structure which is incompatible with the organism and which the latter rejects, cf. Arquilla E.R. et al. Immunology Conformation and Biological Activity of Insulin Diabetes, vol. 18, p. 194, 1969, or that the chemical compound has such a size in suspension (aggregate) that it is incompatible with the organism and the latter rejects it, cf. Arquilla, ER et al., Immunochemistry of Insulin, Handbook of Physiology, 9, pp. 160, 1972.

In addition, it is possible to induce the formation of antibodies by co-administering with a chemical compound an ingredient capable of activating the immune mechanism, such as so-called excipients, for example Freund's complete excipient and Freund's incomplete excipient.

Several studies show that the physical state of insulin plays a significant role in the formation of antibodies. (Kumar et al., Horm. Metab. Res. 6, (1974) 175-177, and P. A. Priers et al., Neth J. Med. 17 (1974) pp. 234-238).

The reason why fast-acting, high-purity insulin preparations do not or only to a limited extent cause antibody formation is presumably that, in these cases, in addition to being very pure, the insulins are present in the form of a monomer or form only loose aggregates. whereas in previously prepared and marketed, long-acting, high purity insulin formulations, insulin was in the form of an aggregate and had properties that caused the formation of antibodies. This problem is very noticeable in the case of bovine insulin, which is probably due to the fact that bovine insulin is more prone to form aggregates compared to porcine insulin.

Bovine insulin is known to have a wider isoelectric precipitation zone or range without additives than porcine insulin. It is also known that this wide isoelectric precipitation range can be limited to the same magnitude as with known porcine insulin by the addition of certain substances such as phenol or m-cresol, vert. Danish Patent Publication No. 116,527. The wider isoelectric precipitation zone of said bovine insulin is assumed to be due to the fact that bovine insulin forms aggregates at pH values close to the neutral point.

The invention is based on the finding that it is possible to stabilize highly purified insulin so that no aggregates are formed during the preparation or storage or use of long-acting preparations which cause the formation of antibodies. Although highly purified insulin has been prepared in the past, it has not been previously observed that formulations prepared therefrom form aggregates or polymers that can cause antigenicity. The process according to the invention is characterized in that the reaction of highly purified insulin and an organic base containing amino groups is carried out in an aqueous buffer solution containing a stabilizer which during the reaction keeps the insulin dissolved and in a stabilized monomeric or loosely aggregated state, after which the insulin preparation is isolated.

Examples of suitable organic bases which can be used in the process of the invention are those commonly used for the preparation of long-acting insulin products, such as polypeptides, e.g. poly-arginine, somatostatin, protamine or its cleavage products or globin. An example of a useful base that is not a polypeptide is surfene, i.e., 1,2-bis- (4-amino-2-methyl-6-quinolyl) urea. The recommended base is protamine.

Stabilizers capable of keeping insulin (and other proteins) in a stable monomeric or loosely aggregated state are well known in the literature, see for example DE-OS 1 940 130; Cole, J., Biol. Chem. 235 (1960) and Elbaum et al., Biochemistry, vol. 13, no.

6, 1974, pp. 1268-1278. In particular, such protein dissociating stabilizers have been used in the chromatographic purification of proteins to prevent the formation of hydrogen ion bonds and the polymerization of protein molecules. In cases where stabilizers have been used in the purification of insulinii), see for example DE-OS 1 940 130, they have always been removed during the isolation of the purified insulin and before this can possibly be converted into a long-acting product.

According to the invention, the reaction is conveniently carried out in the presence of urea as a stabilizer. Examples of other suitable stabilizers include lower alkanols such as methanol and ethanol, dialkylformamide, e.g. dimethylformamide, acetamide, N-alkylacetamide such as N-methylacetamide, and acetonitrile. These stabilizers have a dissociating or depolymerizing effect on the protein and are therefore effective in stabilizing the insulin in a monomeric or loosely aggregated state. A loosely aggregated state is understood to mean a state from which insulin can return to a monomeric state, and equilibrium shifts from a monomeric state to a protein in the presence of a protein dissociating or depolymerizing stabilizer.

The invention can be used in connection with insulin obtained from many different animal species, such as pig, bull or sheep, but the invention is particularly relevant in connection with bovine insulin.

In all cases, all antigenic impurities are removed as far as possible or desired before the reaction with protamine is performed. This purification can be carried out in a manner known per se, such as gel filtration and / or ion exchange chromatography. In this way, highly purified insulin remains, as said, in a stabilized monomeric or loosely aggregated state during the reaction. This avoids the polymerization or aggregation that might otherwise occur in the absence of a stabilizer, such as during isolation and redissolution of highly purified insulin. After reaction with protamine, the insulin is attached to a stable monomeric state and cannot form antigenic polymers or aggregates, although the stabilizer is removed and the insulin-protamine complex is suspended in a conventional carrier medium for injection.

A preferred embodiment of the invention is characterized in that the insulin-containing fraction obtained by chromatographic purification of insulin using a buffered aqueous eluent containing a stabilizer which maintains the insulin in a stabilized monomeric or loosely aggregated state is mixed with a solution of the amino-containing base. ti is isolated. Thus, aggregate formation can be prevented with a high degree of certainty, since highly purified insulin is not first isolated as such, which would pose a risk of aggregate formation. Furthermore, this method is very simple, because immediately in connection with efficient purification, a long-acting insulin preparation is obtained in a stable state.

According to another preferred embodiment of the invention, the reaction of highly purified insulin and an organic base containing amino groups is carried out in an aqueous buffer solution containing dissolved urea, after which the reaction product is precipitated by diluting the reaction mixture. Suitable stabilization is achieved by using a buffer of about 7 molar relative to urea.

The invention is further illustrated by the following examples.

66751 8

Example 1 250 mg of recrystallized bovine insulin are dissolved in 5.2 ml of a stabilized buffer solution containing 7 moles of deionized urea and 0.02 moles of tris having a pH of 8.1.

The solution is mixed with 5.2 ml of a 7 molar urea solution. The pH of the mixture is adjusted to 8.1. The column, 5 cm in diameter, is packed with a 2.1 cm thick layer of DEAE cellulose ("Whatman DE 52") and equilibrated with a buffer solution having the above composition. The insulin solution is applied to the column and eluted at this rate at 75 ml per hour using the following scheme: 2.5 hours with a buffer having the above composition 3 hours with a buffer having the above composition added 0.0045 moles of sodium chloride per liter for 12 hours with a buffer having the above composition added to 0.011 moles of sodium chloride per liter.

The eluate is divided into fractions. Thoroughly purified fractions are collected and their insulin content is determined. In a 7 molar urea solution of the same volume as the mixture of thoroughly purified fractions, protamine is dissolved in the amount necessary to provide an isophane relationship between the highly purified insulin and the protamine.

The insulin solution is slowly added dropwise to the protamine solution with stirring, optionally precipitating the protamine-insulin complex in an amorphous state, possibly after diluting the urea concentration to 1 molar.

The precipitate is separated by centrifugation and shows essentially only one band when treated with 300 μg by isoelectric focusing on a polyacrylamide gel using a 2% "Ampholine ®" (series of amphoteric aliphatic polyamino-polycarboxylic acids) (pH 3-10) in 6-deionized urea. .

This precipitate can be used to prepare injectable insulin preparations by soaking in known carrier media.

9 66751

Example 2

The stabilized buffer solution is prepared by mixing the two solutions I and II in such a ratio that the pH is 6.0. Solution I comprises 7 molar deionized urea and 0.13 molar monosodium phosphate. Solution II comprises 7 molar deionized urea and 0.13 molar disodium phosphate. 400 mg of recrystallized bovine insulin are dissolved in 3 ml of this buffer, after which the pH is readjusted to 6.0 with a small amount of solution II and the resulting mixture is filtered.

A column 2.5 cm in diameter is filled with a 27 cm high layer of Amberlite ® resin CG-50 type II and equilibrated with a buffer solution of the above composition. The insulin solution is applied to the column and eluted at a rate of 40 ml per hour with the buffer solution of the above. composition shown.

The eluate is divided into fractions. The purified insulin fractions are combined and the insulin concentration is determined. In a 7 molar urea solution of the same volume as the purified fractions, protamine is dissolved in the amount required to obtain an isophane ratio of purified insulin to protamine.

The insulin solution is slowly added to the protamine solution with stirring, after which, possibly after dilution to a 1 molar concentration of urea, the protamine-insulin complex precipitates in an amorphous form. The precipitate is isolated by centrifugation and has the same purity as the product shown in Example 1.

This precipitate can be used to prepare injectable insulin preparations by slurrying it in known carrier media.

Example 3

The procedure of Example 1 or 2 is repeated, in which zinc chloride corresponding to 0.5% of Zn ions relative to the amount of insulin and 0.3% of m-cresol, based on the volume of the mixture, is added to the protamine solution. The protamine-insulin complex then precipitates in crystalline form.

10 66751

The precipitate is isolated by centrifugation and gives essentially only one band when 300 ug is focused isoelectrically on a polyacrylic (ίδ amide gel using 2% "Ampholine®" (pH 3-10) in 6-M deionized urea.

This precipitate can be used to prepare injectable insulin preparations by breastfeeding with known carrier media.

Example 4

The procedure of Example 1 is repeated using porcine insulin prepared from an aqueous crude extract formed in the manufacture of insulin by the addition of NaCl to a concentration of 3.5-M at pH 8.5. The salt is separated from the resulting salt cake in a known manner before ion exchange.

The preparation obtained has the same purity as the product described in Example 1.

Example 5

The procedure of Example 4 is repeated and the resulting salt cake (R) is gel filtered using "Sephadex® G-50".

The product has the same purity as the product described in Example 1.

Example 6

The procedure of Example 1 or 2 is repeated, but to the combined, thoroughly purified insulin fractions is added a 2% aqueous solution of bis- (4-amino-2-methyl-quinolyl-6 -) - urea hydrochloride in an amount corresponding to 10% by weight of the present. the amount of insulin. In this case, possibly after diluting the urea concentration to 1 m with 0.025 m sodium phosphate buffer, pH 7.3, an insulin complex precipitates, which after standing for some time can be separated by centrifugation. The product prepared has the same purity as the product described in Example 1.

Ί1 66751

Example 7

The procedure of Example 6 is repeated, but using recrystallized bovine insulin purified using gel filtration 11a "Sephadex ® G-50" as the starting material. The purity of the product prepared is the same as the purity of the product described in Example 1.

Example 8

The procedure of Example 6 is repeated, but starting from pork insulin prepared by separating, salting the aqueous crude extract formed in the manufacture of insulin and adding NaCl to a concentration of 3.5 m at pH 8.5. The salt is separated from the salt cake formed in a known manner before ion exchange. The purity of the product obtained is the same as that of the product described in Example 1.

Example 9

The procedure of Example 8 is repeated and the resulting salt cake is filtered through a gel using "Sephadex ® G-50".

The product has the same purity as the product described in Example 1.

To demonstrate the altered immunogenic properties of the protamine-insulin complex prepared according to the invention, comparative experiments have been performed in rabbits, which animals are commonly used to study the antigenic properties of insulin and insulin-like components.

The following preparations were used in the comparative experiments: 1. Conventional NPH bovine insulin prepared from recrystallized bovine insulin containing the above impurities.

2. Purified NPH bovine insulin prepared from recrystallized bovine insulin but from which the above impurities have been removed by column chromatography.

3. A novel NPH bovine insulin prepared according to the invention as described in Example 3.

12 66751 Insulin NPH is a term commonly used for insulin protamine to mean "neutral protamine Hagedorn insulin".

"Standard NPH insulin" was prepared aseptically. 150 mg (4000 IU) of recrystallized bovine insulin was dissolved in 40 ml of water by adding 2 ml of 0.1 N hydrochloric acid, and 75 mg of m-cresol, 30 mg of phenol, 800 mg of glycerin, 143 μg of protamine sulfate and 41 μg of zinc were added with constant stirring. - 2 + chloride solution with 10 mq Zn / ml.

The volume of the solution was adjusted to 50.0 ml, after which it was sterilized by filtration and labeled "solution I".

A solution of 75 mg of m-cresol, 30 mg of phenol, 800 mg of glycerin and 184 mg of Na 2 PO 4 in 45 ml of water was then prepared. The pH was adjusted to 7.7 with 1N sodium hydroxide, after which the volume of the solution was adjusted to 50.0 ml. The solution was sterilized by filtration and labeled "Solution II".

Solution II was added to solution I with stirring. The mixture was allowed to stand for 48 hours for complete crystallization.

The resulting suspension containing "regular NPH insulin" was used for immunization as described below (Figures 1 and 4).

"Purified NPH insulin" was prepared aseptically. The above procedure was repeated using chromatographically purified insulin crystallized with zinc and 35 μl of zinc chloride solution (10 mg Zn 2+ / ml).

The resulting suspension was labeled "purified NPH insulin" and used for immunization as shown below (Figures 2 and 5).

"New NPH insulin" was prepared aseptically at 4 ° C. To 550 ml of eluate from ion exchange chromatography of recrystallized bovine insulin containing 395 mg of insulin was added with stirring 900 mg of m-cresol, 330 mg of phenol and 281 μl of 13,6751 zinc chloride solution containing 9.9 mg of Zn 2+ / ml. The solution was sterilized by filtration and labeled A.

37.62 mg of protamine sulfate was dissolved in 50 ml of 7M deionized urea and the resulting solution was sterilized by filtration and labeled B.

Solution B was added to solution A with stirring, and the resulting solution was allowed to stand for one hour while stirring.

The mixture was continuously diluted with stirring with 3.6 liters of distilled water to which 5.4 g of m-cresol and 2.16 g of phenol had been added until its urea concentration was 1 molar, and sterilized by filtration. Dilution was performed within 5 hours and the mixture was allowed to stand for 24 hours for complete crystallization.

The resulting suspension was concentrated to a volume of about 300 ml by filtration through a membrane filter with a pore diameter of 3 μm. The NPH crystals were separated by centrifugation at 200 G and immediately resuspended as wet crystals in 300 ml of sterile standard NPH medium comprising 0.15% m-cresol, 0.06% phenol and 1.6% glycerin. In 0,133 molar sodium phosphate buffer at pH 7.3. The resuspended NPH crystals were allowed to stand for one hour.

The procedure was repeated six times, after which the volume of the suspension was adjusted to contain 1.5 mg of insulin corresponding to 40 IU / ml.

The insulin concentration of the suspension was determined from samples from which the crystals were separated by centrifugation and freed from phenol and m-cresol by washing with 70% ethanol. The washed crystals were then dissolved in a known volume of 1M acetic acid, after which the insulin concentration was determined photometrically by measuring the extinction at 276 nm.

The total yield was 110 ml of suspension, labeled "new NPH insulin" and used for immunization as shown below (Figures 3 and 6).

14 66751

Comparative experiments were performed by injecting rabbits subcutaneously every ten days with a standard dose of 20 IU of the test insulin preparation. As it was not possible to demonstrate any substantial antibody formation after injection of conventional NPH bovine insulin without excipient, it was mandatory for comparison to use the immunization method normally used to prepare antibodies, ie inject insulin preparations first emulsified in Freund's complete vehicle and subsequently emulsified. To Freund's incomplete excipient.

Insulin antibody formation was examined every 20 days using the method developed by Ortved Andersen et al. (Acta Endocr. (Kbh.) 69, 195-208, 1972).

The results obtained are shown in Figures 1, 2 and 3.

As the results show, it was possible to show antibody formation with conventional NPH bovine insulin and reduced antibody formation with purified NPH bovine insulin, while it was practically impossible to show any antibody formation with the novel NPH bovine insulin prepared according to the invention. It should be noted that Preparations 2 and 3 were purified using the same chromatographic purification, the only difference being that Preparation 2 was prepared by first isolating insulin and then reacting it with protamine in a known manner, whereas Preparation 3 was prepared by reacting in a urea-containing eluate without prior insulin isolation. .

In addition, a series of experiments have been performed using a milder immunization method in which the initial stimulation of the immune preparation with the killed bacteria has been omitted, i.e. the preparations have been injected each time emulsified in Freund's incomplete excipient. Rabbits were injected with a standard dose of 20 IU every 10 days.

Insulin antibody formation was examined every 10 days using the new method described below.

66751 PEG assay for antibody 1 100 ul of rabbit serum, 100 ul of insulin, about 2 ul / ml, 100 ul of insulin solution, 250 ul / ml and 700 ul of phosphate buffer, 0.04 ul. -M, pH 7.4 and 0.15-M NaCl and 0.5% human albumin are incubated for 2 days at 4 ° C. 500 [mu] l of PEG 6000 are added, 360 g / l are mixed in a rotary mixer and the samples are allowed to stand for one hour at 20 [deg.] C.

Centrifuge for 10 minutes at 3000 rpm, decant the supernatant and discard, counting the precipitate I.

A sample with an excess of guinea pig anti-insulin, max. Binding, and a sample of rabbits not immunized with rabbit serum, O sample, are included in each batch. The results are calculated as follows:

T - T

% B = - · 100 T - T M o where T = number of unknowns

Tq = "T,. = Max. Binding" of the O-sample

M

The results obtained are shown in Figures 4, 5 and 6.

As can be seen from the results, with the new NPH bovine insulin prepared according to the invention (Figure 6), it was not possible to detect the generation of an antibody, whereas other insulins caused a significant generation of an antibody (Figures 4 and 5).

Because the test results are determined relative to the standard, a few values are below the abscissa due to measurement uncertainty.

Claims (5)

16 66751 A process for preparing a stable long-acting insulin preparation having low antigenicity by reacting highly purified insulin with an organic amino-containing base, preferably a polypeptide protamine, characterized in that the reaction is carried out in an aqueous buffer solution containing a stabilizer which during the reaction to keep the insulin dissolved and in a stabilized monomeric or loosely aggregated state, after which the insulin preparation formed is isolated. Method according to Claim 1, characterized in that the insulin used is bovine insulin. Process according to Claim 1 or 2, characterized in that the insulin-containing fraction obtained by chromatographic purification of insulin using a buffered aqueous eluent containing a stabilizer which keeps the insulin dissolved in a stabilized monomeric or loosely aggregated state is mixed with an aqueous solution of an organic base containing amino groups. after which the insulin preparation formed is isolated. Process according to one of Claims 1 to 3, characterized in that the stabilizer is urea. Process according to Claim 4, characterized in that the reaction of highly purified insulin and an organic base containing amino groups is carried out in an aqueous buffer solution containing dissolved urea at a concentration of about 7 molar, after which the reaction product is precipitated by diluting the reaction mixture.