DK160880B - INSULIN DERIVATIVES AND INJECTABLE SOLUTIONS CONTAINING THESE - Google Patents

Description

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DK 160880B

The present invention relates to novel insulin derivatives of the kind set forth in claim 1 and to novel injectable solutions with retarded insulin action.

For the treatment of diabetes mellitus, 5 and many different insulin preparations have been proposed. Some of the preparations are fast-acting, and others are more or less retarded. Such a delayed effect can be obtained by administering the insulin as a suspension of insulin crystals. The crystalline preparations can be obtained by crystallizing insulin in the presence of zinc (such as Lente ^, see Schlichtkrull: Insulin Crystals, Chemical and Biological Studies on Insulin Crystals and Insulin Zinc Suspensions, Munksgaard, 1958) or by crystallization of insulin in the presence of zinc and protamine (such as NPH insulin, see Rep.Steno 15 Mem.Hosp. 1 (1946), 60).

A disadvantage of the use of the known suspensions of zinc insulin crystals or of zinc protamine insulin is the need to shake the vial to ensure that the correct amount of insulin is injected and to ensure that the insulin concentration in the vial remains constant during its use. In Penfill 1 vials, where no air may be present, insulin suspensions with a retarded effect necessitate incorporation of a solid body into the vial to allow shaking. The shaking of insulin suspensions and 25 insulin solutions with air is an undesirable process in itself, as insulin tends to denature to form fibrils at water-air interfaces. Insulin solutions with a delayed effect are therefore desirable.

Solutions of insulin derivatives with retarded action could be obtained from insulin modified in the amino groups by reaction with phenylisocyanate (so-called Isoinsulin, see Hallas-Møller: Chemical and Biological Insulin Studies based on the Reaction between Insulin and Phenylisocyanate, Copenhagen). ii DK 160880 B! 2 port 1945). Similarly, Al, B29-dij

Boc-substituted insulin (Boc denotes tertiary butyloxycarbonyl) shows retarded insulin action after subcutaneous administration (see Geiger & Enzmann: Proinsulin, Insulin, C-Peptide; Proceedings of the Symposium on Proinsulin, Insulin and C-Peptide,

Tokushima 1978; Amsterdam-Oxford 1979, 306 - 310). Al, B29-di-Boc-substituted insulin was found to exhibit too little retarded effect to be clinically useful.

Solutions of unmodified insulins require large amounts of zinc ions (e.g. 0.4-1 mg / U insulin) to exhibit retarded action (see J. Pharmacol. (1935), 206). Injection of such large doses of zinc ions is likely to be painful, and therefore such solutions have never been used for treatment purposes.

The insulin isoelectric point is approx. 5.5, and attempts have been made to decrease the solubility of insulin derivatives at neutral pH by changing the isoelectric point upwards, e.g. by introducing into the B-chain N-terminal basic amino acids such as lysine or arginine (see, for example, German Publication No. 2,042,299) or with the basic dipeptide, arginyl-arginine (see Geiger & Enzmann, which is listed above). However, the solubility of Arg '-Arg insulin near its isoelectric point was much higher than the solubility of the underlying insulin.

Japanese Patent Application No. 55-144032 relates to human insulin analogs wherein the B30 amino acid is replaced by an amino acid having at least five carbon atoms, and amides and esters thereof.

These insulin analogues should be used in patients who have produced antibodies to mammalian insulin. Japanese patent application discloses six specific compounds, none of which are stated to have retarded effect. No specific injectable compositions are disclosed in the Japanese patent application.

European Patent Application No. 84108442.9 (EP Publication No. 132,770) relates to insulin analogues in which a basic organic group is attached to the B30 amino acid, wherein

DK 160880B

3 by introducing a positive charge at neutral pH. In these analogues, the B30 amino acid is neutral and preferably threonine as in human insulin. German Patent Application No. 3,327,709.5 relates to a suspension of crystals of the derivatives disclosed in the above-mentioned European patent application and an aromatic hydroxy compound. German Patent Application No. 3,326,473,2 relates to a medicament containing a mixture of insulin compounds, at least one of which is described in the above-mentioned European patent application. European Patent Application No. 84110813.7 (EP Publication No. 140,084) relates to compounds which are very closely related to the compounds mentioned in the first European patent application.

European patent application publication no. No. 194,864 of March 12, 1985, relates to insulin derivatives wherein the 15 C-terminal B30 residue is always blocked by an amido or ester group and wherein the A21 amino acid is always asparagine as in human insulin.

None of the patent applications cited in the preceding two sections deal with the poor chemical stability of the compounds. In none of the cited patent applications is the particular substitution pattern underlying the present invention.

Surprisingly, it has now been found that insulin derivatives of the general formula I as claimed in claim 1 have very high chemical stability and a combined short and retarded insulin action.

The insulin derivatives of the present invention are different from human insulin in that: a) the presence of an unsubstituted amide residue in the B-chain C-terminal carboxyl group; b) it has at least one charge more than human insulin at pH 7, preferably no more than 4 charges more than human insulin at pH 7, j

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] ΐ i c) that the C-terminal asparagine residue in the A chain, |

A21 I

Asn, may be replaced by another naturally occurring amino acid which can be encoded by nucleotide sequences, jd) that if a change in charge is obtained by blocking the carboxyl group of the B30 amino acid, the A21 amino acid residue is different from and asparagine residue.

The change in charge is achieved by replacing one or more of the amino acid residues compared to human insulin and, if desired, by blocking the carboxyl group of the B30 amino acid.

Specifically, the compounds of interest in the practice of this invention can be characterized as follows:

One or more of the four glutamic acid residues in A4, A17, B13 and B21 are replaced by a glutamine residue and / or the threonine residue in B27 is replaced by the L-arginine or L-lysine residue and / or the 15 threonine residue in B30 is replaced by a lysine residue, the C-terminal carboxyl group in the B chain may be protected, and the asparagine residue in A21 may be replaced by one of the L-amino acid residues specified in claim 1.

The invention also relates to solutions of the following compounds of formula I, optionally containing a controlled zinc ion level. This improves and regulates the degree of retardation of insulin action. These injectable solutions of the invention are characterized in that they contain as active ingredient a compound according to claim 1.

25 The excellent chemical stability is shown in Tables IV and V below, which show that the stability is obtained by the replacement in the A21 position (W). The other amino acid replacements provide the desired retarded effect.

Accordingly, in this invention, several changes have been made to human insulin to increase both chemical stability and retarded action.

A subset of compounds of formula I are novel compounds of general formula I wherein A, B, E ^, 2 3 4 E, E, E, X and Y are as defined in claim 1, R represents an unsub-35. stituted amide residue blocking the C-terminal carboxyl group in the B chain, and W is different from an asparagine residue.

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Compared to human insulin, the change in charge is achieved by replacing threonine residue at the B27 position with an arginine or lysine residue and / or by replacing any of the four glutamic acid residues in the A4, A17, B13 and B21 positions with and a glutamine residue. Further, the C-terminal carboxyl group in the B chain may be blocked by an amino group, thereby eliminating the negative charge of the carboxyl group. Furthermore, a positive charge can be introduced by introducing a lysyl residue into the B30 position.

Since compounds of formula I can be used in the clinic as solutions of retarded action, a decrease in immunogenicity may occur in comparison with the commonly used solutions of porcine or human insulin.

The degree of retardation can be increased and regulated by the addition of zinc ions.

Essential parameters for controlling the degree of retardation of the insulin action are the concentration of zinc and the choice of the compound of formula I. The range of the preferred zinc content ranges from 2 µg to approx. 2 mg / ml, preferably 20 from 5 µg to 200 µg / ml zinc, with replacement in the B13 and / or B27 position, and preferably from ca. 20 to 200 µg / ml with other analogues in a composition containing 240 nmol of a compound of formula I ml. Using other concentrations of the compound of formula I, the zinc content 25 should be adjusted accordingly.

The retarded effect of solutions of compounds of formula I in the presence of zinc ions is attributed to the low solubility of such compounds at neutral pH.

Preferably, the pH of the injectable solution of this invention should be below the physiological pH, the upper limit being the pH at which precipitation occurs. At the physiological pH, compounds of formula I according to the invention have a low solubility. Stable solutions containing approx. 240 nmol / ml of 35 compounds of formula I at pH 5.5. The upper limit depends on the constituents of the solution, ie. isotonic, concert- DK 160880 B) i 6! j i! in the preservative and zinc concentration and of the choice of compound of formula I. There is no lower limit on the pH value of the solutions and the chemical stability of the compounds of formula ii I, where W is different from an asparagine residue, is high even at \ PH 3. The preferred pH range of the injectable solutions of this invention is from ca. 2.5 to 8.5, more preferred The pH range is from about 4.5 to 8. 2.5 to 5.5, especially from ca. 3 to 4.5.

It is a further aspect of this invention that it enables increased flexibility for patients. The patient may, with two aqueous solutions, one containing a compound of formula I and the other containing a zinc salt, obtain a desired degree of retarded action and a desired profile by appropriately mixing the two solutions. Accordingly, using 15 two stock solutions, the patient has the opportunity to choose one effect and profile for the morning injection and another effect and profile for the evening injection. The zinc solution according to the invention preferably contains between approx. 2 µg and 20 mg zinc per ml. Alternatively, both stock solutions may contain zinc, either at the same or different concentrations, and / or both stock solutions may contain a compound of formula I - either the same or different compounds.

The injectable solutions of this invention preferably have a strength of between about 60 and 6000 nmol / ml of the compound of formula I.

When W is not an L-asparagine residue, it may be glycine, serine, threonine, aspartic acid, arginine or histidine.

In a group of preferred compounds of formula I, Y is a lysine residue.

Another preferred embodiment of this invention are compositions containing a compound of formula 12 wherein E, E, E and / or E is a glutamine residue and / or X is Lys or Arg and W is Gly , Ser, Thr, His or Asp, and within this subgroup of compounds of formula I is one

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In another preferred embodiment, compositions containing a compound of formula I wherein the group -Y-R is -Thr-NH 2 or -Lys-NH 2.

Specific preferred compounds of formula I are each of the following: A21 non

Gly, Arg, Thr -NH 2 -human insulin, A21 R27 ^

Ser, Arg, Thr -NH2 human insulin,

HisA₂, ArgB₂, Thr B₂-NH₂ human insulin,

AspA ^, ArgB ^, ThrB ^-NHH-human insulin, A21 R77 Ώ7Π Arg Asp, Arg °, LysB -NH₂ human insulin,

GlyA₂, GlnB₂, ArgB₂, ThrB₂-NH₂ human insulin,

Ser ^^ Gln® ^ ArgB ^, ThrB ^ -NH2 human insulin and

ThrA ^, GlnB ^, ^ ArgB, thrB ^ -NH2 human insulin.

A further preferred embodiment of this invention is compositions containing a compound of formula I in which Y is Thr, R is hydroxy and W is Gly, Ser, Thr, His or Asp, and an example of such a compound is A21 B27.

His / Arg -humamnsulm. 1 2

a group of preferred compounds of formula I

2 3 20, E and E are a glutamine residue.

In another group of preferred compounds of formula I, X is an arginine or lysine residue.

In a further group of preferred compounds of formula I, W is Gly, Ser, Thr, His or Asp.

As is well known to those skilled in the art, not all amino acid residues in human insulin are essential for the action of insulin.

Swine and bovine insulin, which amino acid residues other than human insulin have also been used to treat diabetics. Significant variations in the insulin molecule occur from species to species. Thus, many amino acid residues in the human insulin molecule can be altered without unduly decreasing insulin activity, including some residues affecting the isoelectric point of the molecule.

• i 8

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It is obvious that the groups designated E, E, E, E4, R, W, X and Y must be selected such that the resulting compound of formula I is pharmaceutically acceptable. In the known biphasic insulin preparations, it is common to combine fast-acting dissolved insulin with 1 crystalline insulin with retarded effect in the same injection. i i

Using compounds of formula I according to this invention finding a correspondingly combined fast and re- | tardated effect with a solution of a single compound of formula I. The ratio of fast action to retarded effect

concentration decreases as the zinc ion concentration of the solution is increased. IN

Compounds of formula I can be prepared by a transpeptidation in which a biosynthetic precursor compound having the correct insulin disulfide bridges and having the one of general formula II: ii XB (26-22) -E4-B (20- 14) -E3-B (12-1) I (II)

B {28-29) - (Q G T) r A (1-3) -E 1-A (5-16) E2-A (18-20) -W

wherein Q is a peptide chain of q amino acids, q is an integer from 20 to 33, T is Lys or Arg, r is zero or one, and A, B, E1, 2 3 4 E, E, E, W and X is each as described in claim 1, reacted with an amino compound of general formula III: HYR (III) wherein Y and R are each as defined in claim 1, using trypsin or a trypsin-like enzyme as a catalyst in a mixture of water and organic solvents by analogy as described in U.S. Patent No. 4,343,898. A preferred compound of formula III for use in this method is Thr-Ni

Among the trypsin-like enzymes, lysylendopeptidase from 30 Achromobacter lyticus is useful.

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The compound of formula II can be expressed in a host organism such as yeast similar to the description in European patent application publication no. 163,529 using a gene with the correct codons for the amino acids concerned. The gene encoding the novel insulin derivative is then inserted into a suitable expression vector which, when transferred to yeast, is capable of expressing the desired compound. The expressed product is then isolated from the cells or culture fluid, depending on whether it is secreted from the cells or not.

Changes in the A4, A17, A21, B13, B21 or B27 positions may conveniently be made by genetic engineering, introducing the desired C-terminal residue in the B chain by trypsin-catalyzed semisynthesis.

The advantage of introducing the additional positive charges within the framework of the insulin molecule's 51 amino acids to make the novel compounds of formula I instead of extending the B chain beyond the mammalian insulin residues is that the preparation becomes easier. In the semisynthetic transpeptidation, a large molar excess of the amino acid amide or amino acid ester is used. If dipeptidamide or ester were to be used in the transpetidation reaction, either the cost or solubility, or both, would prevent the use of a large excess and the yield of the product would consequently be lower.

Insulin preparations of this invention are prepared by dissolving a compound of formula I in an aqueous medium under slightly acidic conditions, e.g. at a concentration of 240 or 600 nmol / ml. The aqueous medium is made isotonic, e.g. with sodium chloride, sodium acetate or glycerol. In addition, the aqueous medium may contain zinc ions at a concentration of up to ca. 30 µg Zn ++ pr. nmol of the compound of formula I, buffers such as acetate, citrate and histidine, and preservatives such as m-cresol, nipagen or phenol. The pH of the final insulin preparation depends on the number of charges changed in the compound of formula I, the concentration of zinc ions, the concentration of the compound of formula I and of which compound of formula I is selected. . The pH is adjusted to a value suitable for administration, e.g. ca.

2.5 - 4.5, thereby avoiding precipitation. The insulin preparation is sterilized by sterile filtration.

The insulin preparations of this invention are used analogously to the use of the known insulin preparations.

The abbreviations used for the amino acids herein are those set forth in J. Biol.Chem. 243 (1968), 3558. The amino acids described in this specification are in L configuration. In Formula I and elsewhere herein, A (1-3) is Gly-Ile-Val, A (5-6) is Gln-Cys, etc., cf. the amino acid sequence of human insulin. Unless otherwise indicated, the type of insulin in this specification is human insulin.

Synthesis of Insulin Compounds 15 The insulin starting material was an insulin precursor expressed in yeast as described in European Patent Application Publication no. 163,529.

The insulin precursors were recovered from the fermentation liquids by adsorption to LiChroprep® RP-18 as described in Example 7 in the same European patent application. The precursors were eluted from the column with 0.2 M. KCl, 0.001 M HCl in 33% (v / v) ethanol. The insulin precursors were crystallized from the collected fraction by successively adding water (1 volume per volume of the collected), solid trisodium citrate 25 to give a molarity of 0.05 M and lastly zinc acetate to give a molarity of 0.006 M. pH value was adjusted to 6.8 and the mixture was allowed to stand overnight at 4 ° C. The crystals were isolated by centrifugation, washed with water and dried in vacuo.

30 Protected amino acids and protected peptides for enzymatic semisynthesis were either prepared by standard methods or purchased (ordinary syntheses) from either Nova Biochem or Bachem, both Switzerland.

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The letters VM after a name indicate that it is a trademark.

In the starting material of Examples 1 through 14, (Q -T) r of Formula II was selected for Ala-Ala-Lys and prepared 5 as described for yeast plasmid pMT610 in Example 10 of European Patent Application Publication No. 163,529. Nucleotides encoding. B13 pi Al7, B27 τ B27. A21 rl "A21 A21 for Gin. / Gin / Ajtcj * List ψ Asp * Gly ^ His / A? 1 A21

Ser and Thr were replaced in pMT610 by site specific mutagenesis using the method of Nucl.Acids.Res.

10 11 (1983), 5103 - 5112.

Example 1 "B13" B27 ,. ,.

Synthesis of Gin, Arg -humamnsulm

25 ml of 25.5% (v / v) water in DMF (25.5 ml of water, DMF up to 100 ml) is added to a suspension of 5 g of D 1 o no η 15 Gin, Arg, B (1-29 ) -Ala-Ala-Lys-A (1-21) insulin precursor in 50 ml of 2 M Thr-OBut CH C00H (L-threonine tert-butyl ester hydroacetate salt). The suspension is cooled to 12 ° C with stirring. A solution of 0.5 g of pig trypsin in 12.5 ml of a 0.05 M aqueous solution of calcium acetate is added. Stirring is continued until dissolved. After 48 hours at 12 ° C, the proteins are precipitated by pouring the mixture into 600 ml of acetone. The precipitate is isolated by centrifugation, washed once with 200 ml of acetone, isolated by centrifugation and dried in a stream of nitrogen. The precipitate is dissolved in 100 ml of 0.04 N hydrochloric acid, the pH is adjusted to 2.5, 25 and the solution is put on a 5 x 30 cm preparative high pressure liquid chromatography column (hereinafter referred to as HPLC) which is packed with silica particles which are replaced by octadecyldimethylsilyl (average particle size 15 microns, pore size 100 Angstroms). The column is equilibrated with ethanol / 0.3 M aqueous solution of potassium chloride, 0.001 N hydrochloric acid in the ratio 35.5 / 64.5 (vol). The proteins are eluted from the column with the same buffer at a rate of 2 liters / h.

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12 «ι B13 * B27 _u B30« t.

Gin, Arg, Thr -OBu -humamnsulm are found in a peak emerging from the column between 55 and 100 minutes.

Gin, Arg, Thr. -OBu-humane sulfur is isolated from the collected material by successive additions of water to a 5% ethanol concentration of 15% (v / v), solid trisodium citrate to give a 0.05 M molarity of citrate and solid zinc chloride to obtain a molarity of 0.006 M with respect to zinc. The pH is adjusted to 6.8 and after 1 hour at room temperature, crystallization 10 is continued at 4 ° C for 24 hours with stirring. The crystals are centrifuged, washed twice with 20 ml of ice-cold water, centrifuged and dried in vacuo. Yield; 2.51 g of GlnB1, ArgB27, ThrB2 -OBu1 "human insulin.

ol q R9 7 R7H +

Gin, Arg, Thr -OBu human insulin is dissolved in 100 ml of trifluoroacetic acid and left for two hours at room temperature. The trifluoroacetic acid is removed by freeze-drying. The freeze-dried product is dissolved in 100 ml of water, the pH is adjusted to 2.5 and 20 g of sodium chloride are added. The salt cake consisting of B13 B27

Gin, Arg human insulin is isolated by centrifugation. The salt-20 cake is dissolved in 850 ml of water and Gin, Arg-human insulin is crystallized by successive addition of 150 ml of ethanol, 14.7 g of trisodium citrate dihydrate and 0.82 g of zinc chloride, followed by adjusting the pH to 6.8. After 1 hour at room temperature, crystallization is continued at 4 ° C for 24 hours with slight stirring. The crystals are centrifuged, washed twice with 20 ml of ice-cold water, centrifuged and dried in vacuo. Yield; 1.71 g B13 B27

Gin, Arg -human insulin equivalent to 36%.

The amino acid composition is in theory two arginine residues per molecule. The product is pure at 30 DISC PAGE electrophoresis, the migration rate is 55% of the human insulin migration rate, corresponding to a difference in charges of approx. 2. Details of DISC PAGE electrophoresis appear in Horm.Metab.Res.Supplement Series No. 5 (1974), 134. The zinc content of the crystals is 0.42% (w / w).

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13

Examples 2-6

Synthesis of GlnA ^ 7, GlnB ^ 3-human insulin, GlnA3-7, ArgB27-human insulin-B27 B27 lin. Arg -human insulin, Lys -human insulin and. A? 1 R97

His, Arg-Human Insulin __________________ 5 The 5 compounds mentioned are synthesized from the corresponding single-chain insulin precursors, viz.

Gin, GlnaiJ, B (1-29) -Ala-Ala-Lys-A (1-21),

Al 7 R77

Gin, Arg ", B (1-29) -Ala-Ala-Lys-A (1-21), B27 9, B (1-29) -Ala-Ala-Lys-A (1-21), B2 7 10 Lys, B (1-29) -Ala-Ala-Lys-A (1-21) and A21 B27

His, Arg, B (1-29) -Ala-Ala-Lys-A (1-21) using the methods described in Example 1. Yields, charges relative to human insulin, rate of movement relative to insulin by DISC PAGE electrophoresis at pH 8.9, and deviations in the 15 amino acid composition from human insulin are shown in Table I.

Examples 7-13

Synthesis of AspA21, ArgB27, ThrB30-NH2 human insulin,

GlyA2 ^, Arg B27, ThrB3B-NH2 human insulin,

HisA2 ^, ArgB27, ThrB3B-NH2 human insulin, GlyA2 ^, GlnB ^ 3, ArgB27, ThrB3B-NH2 human insulin, SerA21, GlnB13, ArgB27, ThrB30-NH2 human insulin,

ThrA21, GlnB13, ArgB27, ThrB30-NH2 human insulin and SsrA2 ^, Arg827, ThrB39-NH2 human insulin

The 7 named compounds are synthesized from the corresponding single-chain insulin precursors, namely A21 B27

Asp, Arg, B (1-29) -Ala-Ala-Lys-A (1-21), A21 no7

Glyph, Arg1 4, B (1-29) -Ala-Ala-Lys-A (1-21), A21 B27

His, Arg / B (1-29) -Ala-Ala-Lys-A (1-21), A21 Ri ^ R97

Gly, Gln, Arg, B (1-29) -Ala-Ala-Lys-A (1-21), A21 Bi3 b27 Ser, Gln, Arg, B (1-29) -Ala-Ala-Lys-A (1-21), DK 160880 B! 14 A21 B13 B27

Thr, Gln, Arg / B (1-29) -Ala-Ala-Lys-A (1-21) and i

A21 B27 I

Ser, Arg, B (1-29) -Ala-Ala-Lys-A (1-21) by tryptic trans-peptidation in organic aqueous solution in the presence of Thr-NH2 as described in European Patent Application Publication no. j 5 194,864 / Examples 4 and 6. Yields, charges relative to |

human insulin, rates of movement relative to insulin at I

DISC PAGE electrophoresis at pH 8.9 and deviations from human insulin with respect to the amino acid composition are shown in Table I.

Example 14 Synthesis of AspA2 ^, ArgB ^^, LysB30-NH4-human insulin

The title compound is synthesized from the corresponding single-chain insulin precursor, viz

AspA ^, ArgB₂, B (1-29) -Ala-Ala-Lys-A (1-21), by tryptic trans-peptidation in organic aqueous solution in the presence of B3 0 15 Lys (Boc) -NH₂, purification of the intermediate, Lys (Boc) -N 2 -human insulin, followed by removal of the Boc protective group by TFA as described in European Patent Application Publication no. 194,864, Examples 5 and 7. Yield and analysis data are shown in Table I.

l5 DK 160880 B

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Example 15 i

Preparation of injectable solutions of compounds of formula I

For the purpose of analyzing the degree of retarded action, sterile injectable solutions of the compounds of formula I are prepared using 1.6% (w / v) glycerol as isotonic with 0.3% (w / v) m-cresol as a preservative and buffered with 0.01 M sodium acetate. The concentration of zinc ions is 8 or 80 µg / ml. The pH value of the solutions is adjusted far enough from the isoelectric point of the compounds of formula I to keep the solutions clear by storage at 4 ° C. The solutions contain 240 nmol / ml of the compounds of formula I. The concentration of 240 nmol / ml is established by measuring the absorption at 276 nm of a 15 more concentrated stock solution free of m-cresol using the 6100 swine insulin molar extinction coefficient for these derivatives (see Handbook of Inner Medicine, vol.

7 / part 2A, editor: Oberdisse, 1975, 113). For monocomponent swine insulin, the recognized strength is 28.5 U / mg dry matter (see 20 Diabetes Care, Vol. 6 / Supplement 1 (1983), 4), ie. 1 U corresponds to 5.95 nmol.

Injectable solutions containing 240 nmol / ml of the compounds of formula I are prepared as shown in Table II and having pH values and zinc content as shown therein.

25 Insulin effect retardation test

The retardation of the hypoglycemic effect obtained with the injectable insulin solutions is tested according to British Pharmacopoeia 1980, A 142, on fasting rabbits. Each sample solution is administered subcutaneously at a dose of 14.3 nmol to 30 rabbits in a group of 12 animals weighing 3 to 4 kg and the course of hypoglycemia is monitored for 6 hours. For comparison

17 DK 160880B

For example, the fast-acting preparation, ActrapidVM swine insulin, and the fast-acting Monotard® human insulin are included in the experiments. The test results are shown in Table II.

Table II

Compound Zn, pH% glucose of the original_ of formula I_µ9 / πι1_1 hour 2 hours 4 hours 6 hours

Gin 7, ArgB27 human insulin 80 4.5 53 47 50 66

GlnB2, ArgB2 ^ human insulin 80 4.5 55 46 61 91

Glri 3, 7, GlnB 3 human insulin 80 4.5 53 47 55 82 10 ArgB27 human insulin 80 4.5 45 34 51 91 DO 7

Lys -human insulin 80 4.5 .47 40 55 93. A21. B27 B30

Asp, Arg, Thr human insulin 80 4 60 54 58 60. A21. B27 τ B30 Asp, Arg, Lys -N ^ - 15 human insulin 80 4 72 67 61 59

Gly, Arg, Thr -NH_- human insulin 8 4 59 62 71 74

Gly "W2W30 - (<V

human insulin 80 4 72 73 74 74 20 His ^ 2 ^, ArgB27 human insulin 80 4 65 53 66 88

His, Arg, Thr human insulin 80 4 61 52 52 72

Gly ^ Gln813, ^ 7, ^ 630- NH '-human insulin 80 4 82 86 85 90 25 SerA21, GlnB13, ArgB27, ThrB3 ° - NH' -human insulin 80 4 90 91 88 92 ^ '\ GlnB13, W * V30- ΝΕ , -human insulin 80 4 90 90 88 93

SeHW ^ .Thr »0 - ^ - 30 human insulin 80 4 60 62 64 68

Actrapid ^ swine insulin 15 7 46 44 74 91

Monotard ^ human insulin_80 7_54_43_50_74

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18 j

The strength of the insulin compounds is determined by the mouse blood glucose depletion test (British Pharmacopoeia 1980, A 141 -A 142). To minimize the problem of assessing the strength of the insulins with a timing different from the standard, insulin solutions are prepared for strength determinations without the addition of zinc. Solutions are prepared to contain 240 nmol / ml based on the absorption at 276 nm. The zinc content of the solutions is 8 - 10 µg / ml derived from the crystalline derivatives. The estimated potency of some insulin compounds 10 is shown in Table III.

Table III

19

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Strength in Confidence Ratio to Limits ______ insulin,% _ (p = 0.05),% A17 B27 5 Gin, Arg -human insulin 69 79 - 62

P3 B27

Gin, Arg -human insulin 78 88 - 69 A17 _ Bl3.

Gin, Gln -human insulin 50 63 - 40 B27

Arg -human insulin 87 95 - 80 B27

Lys Human Insulin 88 97 - 80 10 AspA21, ArgB27, ThrB30-NH2- Human Insulin 83 92 - 74

AspA21, Arg82 7, Lys83 O-NH2 ~ human insulin 69 77 - 62

GlyA21, ArgB27, ThrB30-NH2-human insulin 75 83 - 68 API RP7

His, Arg human insulin 71 79 - 63 H isA21, Arg82 7, Thr83 0-NH2 ~ human insulin 72 81 - 64

GlyA21, GlnBl3, ArgB27, Thr830-20 NH_ human insulin 49 54 - 44 * A21 r, B13 B27 B30

Ser, Gln, Arg, Thr NHp human insulin 47 54 - 40 ^ A21 B13 B27, B30

Thr, Gin, Arg, Thr - NH - human insulin 28 32 - 24 __ A21, B27 B30 Ser, Arg, Thr ~ NH2 ~ human insulin 76 83 - 68

Example 16

The marked improvement of the undesirable formation of

"API

monode s ami doproducts obtained by replacing Asn with 30 other amino acids are shown in Table IV below. The obtained

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20 improvement at a pH of 3 - 5 is a factor of 10 - 100 times and is highest at pH 3. The table shows the rate at which monodesamidoproducts are formed as a percentage per per week for various insulins when stored as dissolved, sterile preparations.

Table IV

Formation of monodesamido products,% per week

Temperature, ° C 45 37 25 pH pH di_3.0 5.0 3.0 5.0 3.0 5.0 10 Human insulin 45 2 * 90 2 * - B27, B30

Arg, Thr -NH2 ~ human insulin 40 7 - 10 10 2. A21 Λ B27 B30

Asp, Arg, Thr -NH2 human insulin - 1.5 l GlyA21, ArgB27, ThrB30-NH2 human insulin 1 1 1 2 0.20.2

HisA21, ArgB27, ThrB30-NH2 ~ human insulin_2 _0.5 _0.2 * partially precipitated 20 The marked improvement in the undesirable formation of di- and polymerization products obtained by replacement of AsnA2 with other amino acids is shown in the table below V.

Table V

21

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Formation of di- and polymerization products,% per week

Temperature, ° C 45 37 25 pH 3.0 3.0 5.0 5.0 3.0 5.0 5 Human insulin 2.4 7.9 * 2.2 8 *

ArgB27, ThrB30-NH2-human insulin 1.6 5.1 - 0.15 1.3

AspA21, Arg827, ThrB3 O-NH2 human insulin - 0.1 0.3 GlyA21, ArgB27, ThrB30-NH2 human insulin 0.1 0.4 0.06 0.27 0.03 0.17

HisA21, ArgB27, ThrB30-NH2-human insulin_0.1 -_-_-0.02 0.02 Partially precipitated 15 The high stability achieved by replacing A21

Asn with other amino acids may allow storage of dissolved insulin preparations at room temperature, whereas the previously used insulin preparations must be stored in a cool place.

Example 17 20 The excellent protracted action of the compounds of this invention is further illustrated in the following Table vi, where the index of protracted action is calculated as given in Protein Engineering 1 (1987), page 211, upper left column. In Table VI, the first 3 compounds 25 are covered by patent claims in previously published patent applications.

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Table VI

22

Index for Protracted

Compound effect / 3 Zn ++ / hexamer O O 1

Lys -NH 2 -human insulin -7 5 Arg -Arg -pig insulin 4 B31 332

Lys -Arg -human insulin -9 A21 _ A27 B30 "TT,

Gly, Arg / Thr NH ~ human insulin 41 A21 B13 B27 B30 .

Gly #Gln / Arg, Thr -NH_-humanxnsulxn 37 A21 Ri ^ R27 R ^ O ^

Ser / Gin / Arg, Thr -NH-Human Insulin 38 A? 1 Ri2 R77 R ^ H ^ 10 Thr »Gin / Arg, Thr -NH-Human Insulin 29 A21 B27 B13 ^

Ser / Arg, Thr -NH_-human insulin 44. A21 B27 __ B30 *. ,. . .

Arg / Arg, Thr -NH 2 -humanxnsulxn_14_ The following Table VII gives the index of protected effect for some additional compounds of this invention at higher zinc addition.

Table VII

Index for Protracted

Compound_ effect / 3 Zn ++ hexamer

AspA ^, ArgB ^, ThrB ^ ° -NH₂-human insulin 56 AspA ^, ArgB ^, Lys53 ° -NH_-human insulin 70

Hxs, Arg -human insulin ___ 25_

Claims (10)

1. Insulin Derivatives of General Formula I A (1-3) -E1-A (5-6) -Cys-A (8-16) -E2-A (18-19) -Cys-W (A-Chain) I I Compound according to claim 1, characterized in that X is a lysine or arginine residue. DK 16088QB Compounds according to any one of the preceding claims, characterized in that E and EJ are each a glutamine residue. Compounds according to any one of the preceding claims, characterized in that Y is lysine. Compounds according to any one of the preceding claims, characterized in that R is -NH 2. 5. SII s S (I) IIB (1-6) -Cys-B (8-12) -E3-B (14-18) -Cys 10 I (B chain) RY-Lys-Pro-XB (26 -22) -E4-Gly wherein the letters A and B followed by numbers in brackets denote the peptide fragments indicated by the numbers in brackets in the A and B chains of human insulin, E, E, E "3 and E4 respectively, respectively. or different and each represents a residue of glutamic acid or glutamine, X represents an L-threonine, L-arginine or L-lysine residue, W represents a residue of arginine, histidine, glycine, aspartic acid, asparagine, serine or threonine, Y represents a residue of threonine or lysine and R represents hydroxy or 20 -NH 2 / with the proviso that if W is an asparagine residue, R is hydroxy and if W is an asparagine residue, X and -YR are a threonine residue, and E3 is a glutamic residue, not both E1, E2 and E4 are glutamic acid residues, and further provided that at least 12 of 4 of the six amino acid residues E, E, E, E, W and X are different from the amino acid residues, who are present in the corresponding posts in human insulin and at least one of the seven amino acids residues E, E, E, E, W, X and Y and the group R is selected such that the compound of formula I has at least one charge more than human insulin at a pH at 7. Compounds according to any one of the preceding claims, characterized in that W is an aspartic acid, histidine, glycine, serine or threonine residue. Compound according to claim 1, characterized in that it is GlyA21, ArgB27, Thr53 ° -NH - human insulin, "A21. B27 B30 1TT /. Ser, Arg, Thr -NH - human insulin, K A21 B27 B30 *. . His, Arg, Thr -NH ~ human insulin, Λ01 R97 ^ Asp, Arg, Thr -NH2 human insulin, AspA23 ", ArgB27, LysB3 ^ -NH - human insulin, Gly, Gln, Arg, Thr -NH2 ~ human insulin, SerA2 \ GlnB3-3, ArgB2 7, ThrB3 ^ -NH2 human insulin, ThrA2 ^, GlnB ^^, ArgB27, ThrB3 ^ -NH2 'human insulin or H ice, ArgB2 7 -human insulin. Injectable solutions with retarded insulin action, characterized in that they contain an insulin derivative of the general formula I as claimed in claim 1. Injectable solutions according to claim 8, characterized in that they contain a compound of formula I according to any one of claims 2-7. A composition according to claim 8 or 9, characterized in that it contains zinc ions, preferably from about 10 to about 10. 2 pg to approx. 2 30 mg zinc per ml, most preferably from ca. 5 µg to 200 µg zinc per ml.