HK1006171B - Insulin derivatives, their use and a pharmaceutical composition containing same - Google Patents

Description

It is well known that insulin and insulin derivatives are required in significant quantities for the treatment of diabetes mellitus and are sometimes produced on a large scale.Despite the considerable number of existing insulin preparations and variations with different action profiles, there is still a need for further insulin products with different properties and action characteristics due to the diversity of organisms with their inter- and intra-individual variations.

For example, insulin derivatives with delayed action are described in EP-B-132 769 and EP-B-132 770. Other where R1 means H or H-Phe, R30 stands for the residue of a neutral, genetically coded L-amino acid and R31 is a physiologically sound organic group of basic character with up to 50 C atoms, which is composed of 0 to 3 α-amino acids and may have a free, ester, amide, lactone or CH2OH reduced terminal carboxyl function.

These insulin derivatives have an isoelectric point between 5.8 and 8.5 (as measured in isoelectric focus) and the isoelectric point, which is shifted into the neutral range relative to the isoelectric point of the unmodified native insulin or proinsulin (at pH = 5.4), is due to the additional positive charges on the surface of the molecule due to the basic modification, which makes these basically modified insulin derivatives less soluble in the neutral range than, for example, native insulin or proinsulin, which are normally soluble in the neutral range.

The delay or deposit effect of the basically modified insulin derivatives of formula I is due to their solubility at the isoelectric point. The resolution of the insulin derivatives under physiological conditions is to be achieved according to the two abovementioned formulae by cleaving the additional base groups, which, depending on the derivative, results in triptyl or trypsin-like and/or carboxypeptidase B-like and/or esterase activity. The cleavage groups are either purely physiological metabolites or readily metabolizable but physiologically harmless substances.

The above-mentioned principle of storage as a result of basic modification of insulin has been further exploited by the provision and corresponding use of other basically modified insulin derivatives, mainly within the A and B chains; see e.g. EP-A-0194 864 and EP-A-0254 516.

The insulin derivatives described in EP-A-0194 864 contain a basic amino acid at position B27 and/or a neutral amino acid at positions A4, A17, B13 and/or B21 and the C-terminal carboxyl group of the B-chain is blocked by an amide or ester residue.

The insulin derivatives according to EP-A-0254 516 are very similar to those according to the above-mentioned EP-A; however, in order to increase stability at the weak acid pH of the corresponding pharmaceutical preparations, the amino acid asn in position A21 can be replaced by other amino acids which are more stable in acidic media, such as Asp. Asn (= asparagine) differs from Asp (= asparagine acid) as is known by the blocking of one of the two carboxyl groups by the amide group: Other

Another modification of the insulin molecule in the A and B chains, in particular by replacing the amino acid His in the B10 position with zinc, which is responsible for the complex formation and thus has a certain delaying effect, is to produce rapidly acting insulin derivatives; see EP-A-0214 826.

All insulin derivatives according to the last three forms are mainly modified within the A and B chains and are produced by genetic engineering.

In the effort to increase the stability of the basically modified insulin derivatives at the C-terminal end of the B chain in acidic media, as described in the abovementioned EP patents EP-B-0132 769 and EP-B-0132 770, and to modify their action profile, if necessary, this objective has been found to be achieved in a favourable way. by substitution of Asn21 by other genetically coding amino acids not containing an amide group and, where appropriate, by substitution of HisB10 by other genetically coding amino acids.

The invention is therefore based on insulin derivatives of formula II Other in which R1H or H-Phe means,R2 means a genetically coded L-amino acid without an amide group,R30 means the rest of a neutral genetically coded L-amino acid,R31 means a physiologically safe organic group of up to 50 C-atoms of basic character, in which 0 to 3 α-amino acids are involved and where available the final carboxyl function may be free, as ester function, as amide function, as lactone or reduced to CH2OH, andX means a genetically coded L-amino acid with an isoelectric point between 5 and 8.5 and its physiologically compatible salts.

The new insulin derivatives and their physiologically compatible salts are stable over long periods at the low acid pH of corresponding pharmaceutical preparations and have a modified (shorter) action profile compared to the known unchanged basically modified insulin derivatives of formula I mentioned at the beginning, especially if HisB10 is still replaced by other amino acids.

In formula II, R1 is preferably H-Phe.

Genetically coded L-amino acids not containing an amide group - for R2 - are The following shall be added to the list of products: Gly, Ala, Ser, Thr, Asp and Glu, especially Asp, are preferred.

Neutral, genetically coded L-amino acids - for R30 - are Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe and Pro, with Ala, Thr and Ser being preferred.

R31 is a physiologically safe organic group of basic characteristics with up to 50 C atoms, in the construction of which 0-3 α-amino acids are involved. Amino- ((C2-C6-) alkoxy, C1-C4) alkylamino- ((C2-C6) alkoxy, Di- ((C1-C4) alkylamino- ((C2-C6) alkoxy, Tri- ((C1-C4) ammonium- ((C2-C6) alkoxy, Amino- ((C2-C6) alkylamino, [ ((C1-C4) alkylamino- ((C2-C6) alkylamino, Di- ((C1-C4) alkylamino- ((C2-C6) alkylamino or [Tri- ((C1-C4) alkylamino]- ((C2-C6) alkylamino, in particular -O- ((CH2) p-O- (O- ((CH2) p-O- (CH2) p-NR3, -NH- (NH-NH-NR2) p-NH-NR2 or p-NH-R2 or R-NR3, or is a different substance and is equal to or equal to R-C1 or R-C2 or R-C4 or R-C1 or R-C2 or R-C4 or R-C2 or R-C4 or R-C1 or R-C2 or R-C2 or R-C2 or R-C2 or R-C2 or R-C2 or R-C2 or R2 or R-C2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2 or R2

If up to 3 α-amino acids are involved in the construction of R31, these are primarily neutral or basic naturally occurring L-amino acids and/or their corresponding D-amino acids. Neutral naturally occurring amino acids are in particular Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro and Hyp. Basic naturally occurring amino acids are in particular Lys, Hyl, Orn, Cit and His. If only neutral α-amino acids are involved, their final carboxyl blocking function - R31 has a basic character - cannot be free; the carboxyl function must be much more in this case with a base group or a diethyl ester, whereby such groups are preferable to the base groups - in the case that neutral α-amino acids such as α-amino acids (C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-C1-

As a lactone, the final carboxyl function can of course only be present if the final amino acid is a hydroxy amino acid.

In addition, the final carboxyl function can also be reduced to CH2OH.

Preferably R31 consists of 1, 2 or 3 of the above basic naturally occurring amino acids; particularly R31 = Arg-OH or Arg-Arg-OH.

The same amino acids as for R2 are used as genetically coded L-amino acids for X, but the genetically coded L-amino acids containing an amide group - i.e. asn and gln - are also possible; the latter - asn and gln - are even preferred here.

Sequences (A1 - A20) and (B1 - B9, B11 - B29) are preferably human, porcine or bovine insulin sequences, in particular human insulin sequences.

Examples of insulin derivatives of formula II are:

The production of the insulin derivatives of formula II is mainly genetic engineering by site-directed mutagenesis using standard methods.

This is done by constructing a gene structure coding for the desired formula II insulin derivative and seeking expression in a host cell, preferably a bacterium such as E. coli or a yeast, in particular Saccharomyces cerevisiae, and, if the gene structure is coding for a fusion protein, releasing the formula II insulin derivative from the fusion protein; analogous methods are described, for example, in EP-A-0 211 299, EP-A-0 227 938, EP-A-0 229 998, EP-A-0 286 956 and DE patent application P 38 21 159.9 of 23 June 1988 (HOE 88/F 158).

The breakdown of the fusion protein component after cell excretion is carried out either chemically by halogen cyanide (see EP-A-0 180 920) or enzymatically by lysostaphin (see DE-A-37 39 347).

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The C-peptide is removed by trypthic cleavage - e.g. according to the method of Kemmler et al., J.B.C. (1971), p. 6786 - 6791 and the insulin derivative of formula II purified by known techniques such as chromatography - see e.g. EP-A-0 305 760 - and crystallization.

The preparation of the insulin derivatives of formula II with R2 = Asp and X = His is preferably carried out by hydrolysis of the known basically modified insulin derivatives of formula I in an aqueous acid medium (since only the amide group of asparagine at position A21 needs to be hydrolysed here), preferably at pH values between about 2 and about 4, in particular about 2.5, and at temperatures between about 0 and about 40 °C, preferably at room temperature.

The insulin derivatives of formula II and/or their physiologically compatible salts (such as the alkaline or ammonium salts) according to the invention are mainly used as active substances in a pharmaceutical preparation for the treatment of diabetes mellitus.

The pharmaceutical preparation is preferably a solution or suspension for injection and is characterised by a content of at least one insulin derivative of formula II and/or at least one of its physiologically compatible salts in dissolved, amorphous and/or crystalline, preferably dissolved, form.

The preparation should preferably have a pH between approximately 2,5 and 8,5, in particular between approximately 4,0 and 8,5, contains an appropriate isotonic agent, a suitable preservative and, where appropriate, an appropriate buffer, and preferably a certain concentration of zinc ions, All the ingredients except the active substance form the carrier.

The isotonic agents are e.g. glycerol, glucose, mannite, NaCl, calcium or magnesium compounds such as CaCl2, MgCl2 etc.

The choice of isotonic agent and/or preservative influences the solubility of the insulin derivative or its physiologically compatible salt at the low acid pH.

The use of the product in the final product shall be limited to the following:

For example, sodium acetate, sodium citrate, sodium phosphate, etc. can be used as buffers, especially for setting a pH between about 4.0 and 8.5. Otherwise, physiologically harmless diluted acids (typically HCl) or alkalis (typically NaOH) are also suitable for setting the pH.

If the preparation contains zinc, 1 to 2 mg, in particular 5 to 200 μg zinc/ml, is preferred.

In order to vary the profile of action of the preparation according to the invention, unmodified insulin, preferably bovine, porcine or human insulin, especially human insulin, may also be added.

The preferred concentrations are approximately 1 to 1500, preferably 5 to 1000, and in particular 40 to 400 international units/ml.

The invention is now explained in more detail by the following examples.

A) Genetically engineered production Example 1 Design of a plasmid for the production of Gly (A21) human insulin Arg (B31-OH)

The plasmid pSW3 was described in DE patent application P 38 21 159.9 (HOE 88/F 158). The plasmid DNA is translated with the restriction enzymes Pvu2 and Sal1 and then treated with alkaline bovine phosphatase. The two resulting fragments are separated by gel electrophoresis and the large fragment is isolated. This fragment is linked in a T4 DNA ligase reaction with the following synthetic DNA sequence:

Competent E. coli W3110 cells are transformed by the ligation approach. The transformation mixture is flattened on NA plates containing 20 μg Ap (=ampicillin)/ml and incubated overnight at 37°C. An overnight culture is obtained from individual colonies and extracted from this plasmid DNA. This DNA is characterised by restriction analysis and DNA sequence analysis. Correct plasmids encoding the altered A-chain are given the designation pIK100. The expression is analogous to e.g. 3 of the above-mentioned DE patent application P 38 21 159.9.

Example 2 Design of a plasmid for the production of human insulin Ser ((A21) (Arg B31-OH)

The structure is similar to the path described in the example above, but the synthetic DNA sequence is modified as follows:

The result is the plasmid pIK110, which is characterized by an additional BspH1 recognition sequence.

Example 3 Design of a plasmid for the production of Gly ((A21) -Asn ((B10) -Human Insulin-Arg ((B31-OH)

DNA from the plasmid pIK100 is cleaved with the restriction enzymes Hpal and Dra3 and treated with alkaline bovine phosphatase. The two fragments are separated by gel electrophoresis and the larger of the two fragments is isolated. The resulting plasmid pIK101 is further characterised as described in example 1.

Example 4 Design of a plasmid for the production of Ser ((A21) -Asn ((B10) -human insulin

The design is similar to the cloning described in example 3, but is based on DNA from the plasmid pIK110. The newly designed plasmid is designated pIK111.

Example 5 Design of an expression plasmid for monkey proinsulin

Monkey proinsulin differs from human proinsulin only in that it replaces a single amino acid in the C peptide (B37-Pro instead of Leu in this position of human proinsulin).

The plasmid pSW3 is opened with Hpa1 and Sal1 and the residual plasmid DNA is isolated. From the plasmid pK50 described in EP-A-0 229 998 the Dra3-Sal1-monkeyproinsulin fragment is isolated. The result is the plasmid pSW2, whose DNA is then used as the starting material for the construction of the expression plasmids encoding the di-Arg human insulin derivatives.

Example 6 Design of a plasmid for the production of Gly (A21) human insulin Arg (B31) -Arg (B32) -OH

DNA from the plasmid pSW2 is split with Pvu2 and Sal1 in example 1 and ligated with the synthetic DNA from example 1 to form the plasmid pSW21.

Example 7 Design of a plasmid for the production of Ser ((A21) human insulin-Arg ((B31)-Arg ((B32)-OH)

The plasmid pSW22 is constructed from pSW2 DNA in the same way as in example 2.

Example 8 Design of a plasmid for the production of Gly ((A21) -Asn ((B10) -Human Insulin-Arg ((B31) -Arg ((B32) -OH)

The plasmid pSW23 is constructed from pSW21 DNA in the same way as in example 3.

The following sequence is used as synthetic DNA sequence:

Example 9 Design of a plasmid for the production of Ser ((A21) -Asn ((B10) -human insulin B31 ((Arg) -B32 ((Arg) -OH)

The plasmid pSW24 is constructed from pSW22 DNA using the synthetic DNA sequence described in Example 8 in analogy to Example 4.

(b) Preparation of AspA21-Human Insulin-ArgB31-ArgB32-OH from human insulin-ArgB31-ArgB32-OH by hydrolysis

1 g human insulin ArgB31-ArgB32-OH is slurried in 100 ml H2O. By adding HCl, the pH is set to 2.5 and the solution is left at 37°C. After a week, about half of the material is converted into AspA21 human insulin ArgB31-ArgB32-OH. The product is separated from the starting material by means of an anion exchanger, cut from the eluate in a known way and crystallized in a buffer containing 10,5 g citric acid, 1 g of phenol and 5 ml of a 1% zinc chloride solution at a protein concentration of 5 g/l at pH 6,0. The amount is 390 mg of AspA21H2O31B3H2O3

C) Preparation of a solution for injection

The insulin derivative according to B is dissolved in a sterile carrier solution of the following composition (per ml) at a concentration of 1,4 mg/ml: The test chemical is a mixture of 18 mg glycerol, 10 mg benzyl alcohol, 80 μg Zn2+, pH 4.0.

D) Profile of action of an AspA21 human insulin ArgB31-ArgB32-OH preparation in dogs compared to human insulin ArgB31-ArgB32-OH and basal H insulin Hoechst (R) = an NPH (Hagedorn-neutral protamine) preparation with about 10 μg Zn2+.

This example shows that AspA21-Human Insulin-ArgB31-ArgB32-OH has the same beneficial baseline profile as Human Insulin-ArgB31-ArgB32-OH. In addition, AspA21-Human Insulin-ArgB31-ArgB32-OH has the beneficial property of being long-term stable under the chosen conditions.

Claims (12)

1. A process for the preparation of an insulin derivative of the formula II in which

   R¹   denotes H or H-Phe,

   R²   denotes a genetically encodable L-amino acid which contains no amide group,

   R³⁰   represents the residue of a neutral genetically encodable L-amino acid,

   R³¹   represents a physiologically acceptable organic radical from the group: amino-(C₂-C₆)-alkoxy, (C₁-C₄)-alkylamino-(C₂-C₆)-alkoxy, di-(C₁-C₄)-alkylamino-(C₂-C₆)-alkoxy, tri-(C₁-C₄)-alkylammonio-(C₂-C₆)-alkoxy, amino-(C₂-C₆)-alkylamino, [(C₁-C₄)-alkylamino]-(C₂-C₆)-alkylamino, di-(C₁-C₄)-alkylamino-(C₂-C₆)-alkylamino or [tri-(C₁-C₄)-alkylamino]-(C₂-C₆)-alkylamino, in particular -O-[CH₂]_p-NR₂, -O-[CH₂]_p-N^⊕R₃, -NH[CH₂]_p-NR₂ or -NH-[CH₂]_p-N^⊕R₃, in which p = 2 to 6 and R is identical or different and is hydrogen or (C₁-C₄)-alkyl, or represents 1 to 3 α-amino acids, whose terminal carboxyl functionality which is present where appropriate can be free, in the form of an ester functionality, an amide functionality, a lactone or reduced to CH₂OH, and

   X   represents a genetically encodable L-amino acid,

   which has an isoelectric point between 5 and 8.5, and the physiologically tolerated salts thereof wherein the expression of a gene structure coding for this insulin derivative is brought about in a host cell, preferably in a bacterium or in a yeast, and - if the gene structure codes for a fusion protein - the respective insulin derivative is liberated from the fusion protein obtained and converted if required into a physiologically tolerated salt.
2. The process as claimed in claim 1, which comprises preparing an insulin derivative of the formula II in which R¹ represents H-Phe.
3. The process as claimed in claim 1 or 2, which comprises preparing an insulin derivative of the formula II in which R² represents Gly, Ala, Ser, Thr, Asp or Glu, in particular only Asp.
4. The process as claimed in one or more of claims 1 to 3, which comprises preparing an insulin derivative of the formula II in which R³⁰ represents Ala, Thr or Ser.
5. The process as claimed in one or more of claims 1 to 4, which comprises preparing an insulin derivative of the formula II in which R³¹ represents Arg-OH or Arg-Arg-OH.
6. The process as claimed in one or more of claims 1 to 5, which comprises preparing an insulin derivative of the formula II in which X denotes Asn or Gln.
7. The process as claimed in one or more of claims 1 to 6, which comprises preparing an insulin derivative of the formula II in which the sequence (A1 to A20) and (B1 to B9, B11 to B29) are the sequence of human, porcine or bovine insulin, in particular the sequence of human insulin.
8. A process for the preparation of an insulin derivative of the formula II as defined in claim 1, but where

   R²   is only Asp and

   X   is His,

   and of the physiologically tolerated salts of this insulin derivative, which comprises subjecting an insulin derivative of the formula I in which R¹, R³⁰ and R³¹ have the same meaning as in formula II (as defined in claim 1), to hydrolysis in aqueous acidic medium, and converting the insulin derivative of the formula II produced thereby if required into the physiologically tolerated salts thereof.
9. The use of insulin derivatives of the formula II as defined in claim 1 and of the physiologically tolerated salts of these insulin derivatives as active substances for pharmaceutical compositions for the treatment of diabetes mellitus.
10. A process for the preparation of a pharmaceutical composition, which comprises converting an effective amount of at least one insulin derivative of the formula II and/or at least one of its physiologically tolerated salts with a physiologically acceptable vehicle and, where appropriate, with further additives and/or auxiliaries into a suitable pharmaceutical presentation.
11. The process as claimed in claim 10, wherein at least one zinc compound is also added, corresponding to a content of 1 µg to 2 mg, preferably 5 µg to 200 µg, of zinc/ml to the pharmaceutical composition.
12. The process as claimed in claim 10 or 11, wherein an unmodified insulin, preferably unmodified human insulin, is also added to the pharmaceutical composition.