Field of this invention

The present invention relates to insulin analogues capable of being targeted to special organs after subcutaneous administration, pharmaceutical preparations containing such insulin analogues, and methods for making the insulin analogues.

Background of the art Insulin is a hormone which regulates the blood glu¬ cose level by decreasing glucose outflow from the liver and increasing glucose uptake in peripheral tissues, for example, muscles and adipose tissues. It exerts these effects by interacting with insulin receptors present on most cells. The insulin receptors on the hepatocytes bind most of the insulin to regulate the metabolism and synthesis of glucose in the liver cells. Insulin reaches the insulin receptors in the peripheral tissues after transendothelial transport, which is fully or partly receptormediated. After having reached the target organs in the periphery, insulin acts among other things by facilitating glucose uptake. Thus, the total reduction of blood glucose by insulin is due to such effects both in the liver and peripheral tissues.

In normal man, pancreatic insulin is secreted directly into the hepatic portal vein, thereby insulinizing the liver and avoiding persistent peripheral hyperinsulinaemia.

In diabetic patients, insulin is given subcu¬ taneously.

Many diabetic patients develop peripheral insulin resistance perhaps as a consequence of the exposure to con¬ stantly high insulin concentrations in the periphery. One way to circumvent this problem would be to develop an insulin which primarily has an influence on the glucose uptake in muscle tissue.

There is therefore a need in the art for insulin analogues which after subcutaneous injection are targeted to special organs, for instance the muscles.

Targeting of insulin to speficic organs may be pos- sible by altering the insulin receptor binding kinetics. The present invention is based on the surprising fact that certain insulin analogues have remarkably low association rate constants in the insulin receptor binding process.

We have examined a number of insulin analogues with regard to in vitro potency in the free fat cell assay (FFC) which measures the insulin analogue stimulated incorporation of ³ H from 3- ³ H-glucose into lipids. In order to further characterize the analogues, we examined their association rate to the human insulin receptor. In order to determine the association rates, Tyr ^A14 - ¹²⁵ I-analogue was added to the re¬ ceptor, and polyethyleneglycol precipitation of the receptor- analogue complex at different times yielded a pseudo first order curve from which the association rate constant could be calculated. Insulin analogues stated to be hepatospecific are described in International Patent Application having publi¬ cation No. WO 90/12814. In these known analogues, the amino acid residues in the positions A13, A14, A15, A19 and B16 have been exchanged with a hydrophobic group. No insulin analogues being targeted to organs dif¬ ferent from the liver are known.

It is the object of this invention to develop insulin analogues which are targeted to organs different from the liver. Such insulin analogues may be targeted to the muscles. The present invention is based on the surprising recognition that a certain, selected group of insulin analogues of the type described in EP application No. 214,826A are not targeted to the liver.

The forementioned EP application discloses insulin analogues wherein one or more of the amino acid residues present in positions A8, A9, A10, A13, A21, Bl, B2, B5, B9, BIO, B12, B14, B16, B17, B18, B20, B26, B27 and B28 may

be substituted with another naturally occuring amino acid residue.

Summary of the invention

The insulin analogues of this invention have in the A13 position and/or in the B17 position in the insulin molecule a naturally occurring amino acid residue different from leucine and/or have in the B18 position in the insulin molecule a naturally occurring amino acid residue different from valine. The insulin analogues of this invention have inter- esting properties. Examples of such interesting properties are their ability of being targeted to special organs after subcutaneous administration, for example, to the muscles. The insulin analogues of this invention can be used for the treatment of diabetes analogously with the treatment of dia¬ betic patients with similar insulin compounds. In addition, and surprisingly, the insulin analogues of this invention are of potential value in the treatment of insulin resistent dia¬ betics. Additionally and surprisingly, the insulin analogues of this invention are of potential value in the treatment of muscular dystrophy.

The insulin analogues of this invention are a selected, novel group of insulin analogues having additional advantageous characteristics over the known insulin analogues. A specific subclass of the present insulin analogues are such wherein leucine in position A13 or leucine in both position A13 and position B17 in the human insulin molecule has been replaced by another, naturally occurring amino acid residue.

The observed effect is most pronounced with a polar amino acid in the A13, B17 and/or B18 position, for example, glutamic acid and aspartic acid. Other suitable amino acids to be inserted in these positions are serine, threonine, asparagine, glutamine, alanine and glycine.

In addition to the changes made in the insulin molecule in the A13, B17 and/or B18 position, compared with human insulin, it may also be desirable to make other changes in the insulin molecule with the purpose of making the insulin analogue rapid acting as described in EP 214,826A or EP 383,472A. Further possible substitutions may be such as described in EP 129,864A wherein insulin analogues with a prolonged insulin action are disclosed. Examples of such exchanges, compared with human insulin, are substitution of the amino acid residue in the A17 position with glutamine, substitution of asparagine in the A21 position in the insulin molecule with another naturally occurring amino acid residue, deletion of the amino acid residue in the Bl position, substitution of the amino acid residue in the BIO position with glutamic acid, substitution of the amino acid residue in the B14 position with glutamine, substitution of the amino acid residue in the B27 position with glutamic acid, substitution of the amino acid residue in B28 with glutamic or aspartic acid and deletion of the amino acid residue in the B30 position. The present invention is also related to novel pharmaceutical preparations containing the novel insulin ana¬ logues according to the present invention in a solution with a conventional additive, adjuvant, carrier and diluent used for known insulin preparations. The present insulin analogues may be prepared by chemical synthesis by methods analogue to the method described by Marki et al. (Hoppe-Seyler's Z. Physiol.Chem. , 360 (1979), 1619 - 1632) . They may also be formed from separately .in vitro prepared A and B chains containing the appropriate amino acid residue substitutions, whereupon the modified A and B chains are linked together by establishing disulphide bridges according to known methods (for example, Chance et al., In: Rick, D.H. , Gross, E. (editors) Peptides: Synthesis - Structure - Function. Proceedings of the seventh American peptide symposium, Illinois, pp. 721 - 728) .

A more preferred method would be to make the insulin analogue biosynthetically. Thus, the insulin analogues

according to the present invention may be prepared by altering the proinsulin gene through replacement of codon(s) coding for the amino acid residue in position A13, B17 and/or B18 and any other of the positions where an exchange is desired in the native human proinsulin gene by codon(s) encoding the desired amino acid residue substitute(s) or by synthesizing the whole DNA-sequence encoding the desired insulin analogue. The gene encoding the desired insulin analogue is then inserted into a suitable expression vector which when transferred to a suitable host organism, for example, E. coli. Bacillus or yeast, generates the desired product. The expressed product is then isolated from the cells or the culture broth depending on whether the expressed product is secreted from the cells or not. The insulin analogues may furthermore be prepared by a method analogue to the method described in European patent application having publication No. 195,691, the disclosure of which is incorporated by reference hereinto. By such a method an insulin analogue precursor of human insulin wherein Lys ^B29 is connected to Gl ^ ¹ by means of either a peptide bond or a peptide chain of varying length is expressed and secreted by yeast and then converted into human insulin by the so-called transpeptidation reaction.

Accordingly, the present insulin analogues may be prepared by inserting a DNA-sequence encoding a precursor of the insulin analogue in question into a suitable yeast ex¬ pression vehicle which when transferred to yeast is capable of expressing and secreting the precursor of the insulin analogue in which Lys ^B29 is connected to Gly ^A1 by a peptide bond or a peptide chain with the formula I

-R _n -R ^» - (I)

wherein R is a peptide chain with n amino acid residues, n is an integer from 0 to 33, and R ^* is Lys or Arg when the trans¬ formed yeast strain is cultured in a suitable nutrient medium. The precursor is then recovered from the culture broth and

reacted with an amino acid derivative with the general formula II

Q-OR" (II)

wherein Q is the amino acid residue which is to be inserted in the B30 position, preferably threonine, and R" is a carboxy protecting group (for example, methyl or tert.butyl) , using trypsin or trypsin-like enzyme as a catalyst in a mixture of water and organic solvents analogously as described in US patent specification No. 4,343,898 (the disclosure of which is incorporated by reference hereinto) whereupon the carboxy protecting group is removed and the insulin analogue is isolated from the reaction mixture.

The insulin analogues may also be prepared by a method analogue to the method described in European patent application having publication No. 195,691 the disclosure of which is incorporated by reference hereinto. By this method, insulin analogue precursors of the type having a bridge between the A and B chain consisting of a single pair of basic amino acid (Lys, Arg) are produced in yeast and then converted into the insulin analogue by an enzymatic conversion.

The present insulin analogues may be used for the preparation of novel insulin preparations. Such novel insulin preparations may contain the insulin analogues according to the present invention or a pharmaceutically acceptable salt thereof in aqueous solution or suspension, preferably at neutral pH. The aqueous medium is made isotonic, for example with sodium chloride, sodium acetate or glycerol. Furthermore, the aqueous medium may contain zinc ions, buffers such as acetate and citrate and preservatives such as m-cresol, methylparaben or phenol. The pH value of the preparation is adjusted to the desired value. The insulin preparation is made sterile by sterile filtration.

Examples of insulin analogues according to the present invention are Ala ^B17 human insulin (herein designated X69) , Ala ^B18 human insulin, Asn ^A13 human insulin, Asn ^A13 ,Ala ^B17

human insulin, Asn ^A13 ,Asp ^B17 human insulin, Asn ^A13 ,Glu ^B17 human insulin, Asn ^B18 human insulin, Asp ^A13 human insulin, Asp ^A13 ,Ala ^B17 human insulin, Asp ^A13 ,Asp ^B17 human insulin, Asp ^A13 ,Glu ^B17 human insulin, Asp ^B18 human insulin, Gln ^A13 human insulin, Gln ^A13 ,Ala ^B17 human insulin, Gln ^A13 ,Asp ^B17 human insulin, Gln ^B18 human insulin, Glu ^A13 human insulin (herein designated X115) , Glu ^A13 ,Ala ^B17 human insulin, Glu ^A13 ,Asp ^B17 human insulin, Glu ^A13 ,Glu ^B17 human insulin, Glu ^B18 human insulin, Gly ^A13 human insulin, Gly ^A13 ,Ala ^B17 human insulin, Gly ^A13 ,Asn ^B17 human insulin, Gly ^A13 ,Asp ^B17 human insulin, Gly ^A13 ,Glu ^B17 human insulin, Gly ^B18 human insulin, Ser^ ^*13 human insulin, Ser ^A13 ,Gln ^A17 ,Glu ^B10 ,Gln ^B17 -des(Thr ^B30 ) human insulin (herein designated X74) , Ser ^A13 ,Ala ^B17 human insulin, Ser ^A13 ,Asn ^B17 human insulin, Ser ^A13 ,Asp ^B17 human insulin, Ser^ ¹³ ,Gln ^B17 human insulin, Ser^ ¹³ ,Glu ^B17 human insulin, _Ser A1 ³ _,Thr B17 human insulin, Ser ^B14 ,Asp ^B17 human insulin

(herein designated Y2) . Ser ^B18 human insulin, Thr^ ¹³ human insulin or Thr ^B18 human insulin.

Terminology The abbreviations used for the amino acids are those stated in J.Biol.Chem. 243 (1968), 3558. The amino acids are in the L configuration. Unless otherwise indicated, the species of insulins stated herein is human.

The replacement(s) made in the human insulin molecule according to the practice of the invention is (are) indicated with a prefix referenced to human insulin. As an example, Glu ^A13 human insulin herein is a human insulin analogue wherein the amino acid in the 13 position in the A chain of human insulin (leucine) has been replaced by glutamic acid. Herein, Glu ^A13 ,B(l-29)-Ala-Ala-Lys-A(l-21) human insulin is a precursor for the insulin analogue wherein the amino acid in the 13 position in the A chain (leucine) has been replaced by glutamic acid, and wherein the A chain (Al - A21) and the partial B chain (Bl - B29) are connected by the peptide sequence

Ala-Ala-Lys. Unless otherwise stated, the partial B chain (Bl - B29) and the A chain are connected by two disulphide bridges, namely between Cys ^A7 and Cys ^B7 and between Cys ^A20 and Cys ^B19 , as in human insulin, and the A chain contains the internal disulphide bridge between Cys ^A6 and Cys ^A11 . The termB(l-29) designates a shortened B chain of human insulin from Phe ^B1 to _L y _S B2 ⁹ _and A(1-21) designates the A chain of human insulin. As an example, herein position A13 is the 13 position of the A chain of insulin. Unless otherwise indicated, the species of insulin herein is human.

Detailed description

Genes encoding the precursors of the insulin analogue can be prepared by modification of genes encoding the corresponding human insulin precursors by site specific muta- genesis to insert or substitute with codons encoding the de¬ sired mutation. A DNA-sequence encoding the precursor of the insulin analogue may also be made by enzymatic synthesis from oligonucleotides corresponding in whole or part to the insulin analogue precursor gene. DNA-sequences containing a gene with the desired mutation are then combined with a suitable promoter sequence, for example, fragments coding for the TPI promoter (TPIp) (T. Alber and G. Kawasaki, . Nucleotide Sequence of the triose Phosphate Isomerase Gene of Saccharomyces cerevisiae. J.Mol.Applied Genet. 1 (1982), 419 - 434), a suitable leader sequence and possible transcription termination sequence, for example, from TPI of S . cerevisiae (TPI _T ) . These fragments provide sequences to ensure a high rate of transcription of the precursor encoding gene and also provide a presequence which can effect the localization of the precursor into the secretory pathway and its eventual excretion into the growth medium. The expression units are furthermore provided with a yeast origin of replication, for instance the 2μ origin, and a selectable marker, for instance LEU 2. The selected plasmid is then transformed into a

suitable yeast strain by conventional technique, for example, as described in European patent application having publication No. 214,826 and transformants are grown on YPD medium (1% yeast extract, 2% peptone, and 2% glucose) . The insulin analogue precursor is isolated from the culture medium and reacted with Thr-Met,HOAC dissolved in a DMF/water mixture in the presence of trypsin as described in European patent application having publication No. 214,826 and converted into the human insulin analogue by acidic or basic hydrolysis, see European patent application having publication No. 214,826.

EXAMPLE 1

The yield obtained when preparing some compounds according to this invention appears from table I, below. All constructions used were of the type B(l-29)-Ala-Ala-Lys-A(l-21) , wherein the pertinent amino acid residues in the A and B chains were exchanged with the correct amino acid residues in a manner known per se. The transpeptidation was made using threonine ^*630 methyl ester and the hydrolysis was made using sodium hydroxide, vide Example 9 in European patent application having publication No. 214,826.

EXAMPLE 2

The association rate constant of some compounds of this in¬ vention appears from table III, below, where the constants are compared with that of human insulin which has been set to 100%.

Compound tested Association rate constant,

X69 31

X74 15

Y2 19