WO1992015611A1

WO1992015611A1 - Novel insulin derivatives

Info

Publication number: WO1992015611A1
Application number: PCT/DK1992/000059
Authority: WO
Inventors: Lauge SCHÄFFER; Svend Havelund; Kirsten Årup DREJER
Original assignee: Novo Nordisk A/S
Priority date: 1991-02-27
Filing date: 1992-02-27
Publication date: 1992-09-17
Also published as: DK33591D0; AU1372992A

Abstract

Insulin derivatives with organ preferential action having Tyr in position A13 and/or having Phe, Trp or Tyr in position B17 are provided. Also provided are pharmaceutical preparations containing such insulin derivatives.

Description

Novel insulin derivatives

Field of this invention

The present invention relates to novel insulin de¬ rivatives having tyrosine in position A13 and/or having phe- nylalanine, tryptophane or tyrosine in position B17, pharma¬ ceutical preparations containing such insulin derivatives, and a process for preparing the insulin derivatives.

Background of the art

Insulin is a hormone which regulates the blood glu- cose level by decreasing glucose outflow from the liver and by increasing glucose uptake in peripheral tissues, for example muscles and adipose tissues. Insulin 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 receptor mediated. This means that insulin has to bind to a receptor on the endo- thelial cell before it can be transported across this bar¬ rier to reach the distal tissues. After having reached the target organs in the periphery, insulin acts among other things by facilitating glucose uptake. Thus, the total re¬ duction of blood glucose by insulin is due to such effects both in the liver and peripheral tissues.

In normal man, pancreatic insulin is secreted di¬ rectly into the hepatic portal vein, thereby insulinizing the liver and avoiding persistent peripheral hyperinsulin- aemia. In Type I diabetics who lack endogenous insulin secretion, exogenous insulin must be delivered parenterally, and although the most physiological route is delivery of insulin directly into the hepatic portal vein, this is ana¬ tomically difficult. Therefore, the subcutaneous route has been used in clinical practice. However, peripheral hyper- insulinaemia results from the fact that insulin is delivered subcutaneously rather than intraportally, so that the insu¬ lin delivered reaches the peripheral tissue first rather than after passage through the liver. This route of admini- stration may, therefore, result in liver hypoinsulinaemia leading to a non-physiological regulation of the diabetic patients.

Type II diabetes is characterized by an excessive endogenous insulin secretion. Due to the high concentration of secreted insulin, the number of insulin receptors on the cell surfaces are decreased. The decrease of insulin recep¬ tors will, however, result in a decrease in the diabetics¹ sensitivity to insulin because this (among other things) is a function of the number of insulin receptors on the indivi- dual cells.

One important therapy of Type II diabetes is a diet and weight reduction. Treatment with oral hypoglucaemic drugs is another well established therapy. In certain in¬ stances it might, however, be desirable to treat Type II diabetics with insulin.

In order to diminish possible long-term complica¬ tions arising from peripheral hyperinsulineamia, it would be highly desirable to develop an insulin which primarily has an influence on the glucose production in the liver. It is also desired that the insulin concentration in liver and periphery will more closely resemble the physiological route of insulin delivery.

Insulin derivatives with remarkably high associa¬ tion rate constants will be distributed to the liver to a higher degree than to the peripheral tissues and such de¬ rivatives will be of potential value both in the treatment of Type I and Type II diabetes.

Therefore, there is a need in the art for insulin derivatives which after subcutaneous injection are targeted to special organs, for instance the liver and/or muscles.

Insulin derivatives 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, B2^"7 and B28 are the amino acid residues of human insulin or the same or different amino acid residue substitutions, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, are in principle covered by formula I in a European patent application having publication No. 214,826. More specifically, it is stated in this patent applicaion that the A13 amino acid residue can be Pro, Val, Arg, His, Ala, Glu, Asp, Thr, Gly, Gin, Asn or Asp and that the B17 amino acid residue can be Ser, Thr, Asn, Gin, Glu, Asp or His. It is stated that these known insulin derivatives have a rapid onset of effect.

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. There is no mentioning of the B17 posi¬ tion therein. The only specific insulin compounds stated therein are Leu^A10,Trp^A14 human insulin and Trp^A14 sheep insulin (being Ala^A8,Gly^A9,Val^A10,Trp^A14,Ala^B30 human insu¬ lin) .

International patent application having publication No. WO 92/00322 was published after the effective filing date of the present application.

Summary of the invention

This invention is based on the surprising fact that certain insulin derivatives have remarkably fast association rate constants in the insulin receptor binding process. The insulin derivatives of this invention have tyrosine in position A13 and/or have phenylalanin, trypto- phane or tyrosine in position B17. Apart from these alterations, compared with human insulin, the insulin de¬ rivatives of this invention may in further positions differ from human insulin. Compared with human insulin, such al¬ terations comprise: a) compared with human insulin, exchanging the A21 amino acid with another amino acid which can be coded for by nu- cleotide sequences, for example by Ala, Gin, Glu, His, lie, Leu, Met, Gly, Ser, Thr, Trp, Tyr or Val, or with hSer, and/or b) compared with human insulin, exchanging one or more of the amino acids present in positions A4, A17, B13 and B21 with a neutral amino acid which can be coded for by nucleo- tide sequences, and/or c) compared with human insulin, exchanging the B27 amino acid with Arg, Lys or Thr and/or d) omitting 3, 4 or 5 of the amino acid residues in posi¬ tions B26, B27, B28, B29 and B30 and/or e) protecting the carboxy group in the amino acid^' residue in the C terminal end of the B chain with an amino group and/or f) exchanging the B25 amino acid with tyrosine and/or g) exchanging the A13 amino acid with phenylalanin or tryp- tophane and/or h) omitting the Bl amino acid and/or i) compared with human insulin, having one or more of the amino acid residues in positions A8, A9, A10, A13, A21, Bl, B2, B5, B9, BIO, B12, B14, B16, B17, B18 , B20, B26, B27 and B28 exchanged with another amino acid residue, the net func¬ tion of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin.

Preferably, only one or two of these exchanges are made.

The insulin derivatives of this invention have in- teresting pharmacological properties. For example, the insu¬ lin derivatives of this invention are capable of being tar¬ geted to special organs after subcutaneous administration.

The insulin derivatives of this invention will thus be of potential value both in the treatment of Type I and Type II diabetes.

The insulin derivatives of this invention are a selected, novel group of insulin derivatives having additio- nal advantageous characteristics over the known insulin de¬ rivatives.

Examples of amino acids which can be coded for by necleotide sequences are Lys, Arg, Gly, Val, lie, Leu, Phe, Tyr, Met, Asp, Glu, Ala, Ser and Thr.

Examples of neutral amino acids are Gly, Val, lie. Leu, Phe, Tyr, Met, Asp, Glu, Ala, Ser and Thr.

This invention is also related to novel pharmaceu¬ tical preparations containing the above insulin derivatives in a solution with conventional additives, adjuvants, car¬ riers and diluents used for known insulin preparations.

The insulin derivatives of this invention may be prepared by chemical synthesis by methods analogous 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 con¬ taining 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., and Gross, E., (editors) Peptides: Synthesis - Structure - Function. Pro¬ ceedings of the seventh American peptide symposium, Illi¬ nois, pp. 721 - 728) .

A more preferred method could be to make the insu- lin derivative biosynthetically. Thus, the insulin deriva¬ tives of this invention may, for example, be prepared by altering the proinsulin gene through replacement of codon coding for Leu in position A13 and/or in position B17, 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 deriva¬ tive. The gene encoding the desired insulin derivative 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 product expressed 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 derivatives of this invention may, furthermore, be prepared by a method derivative to the method described in European patent application having pub¬ lication No. 195,691, the disclosure of which is incorpo¬ rated by reference hereinto. By such a method, an insulin derivative precursor of human insulin wherein Lys^B29 is con¬ nected to Gl ^¹ by means of either a peptide bond or a pep- tide chain of varying length, is expressed and secreted by yeast and then converted into human insulin by the so-called transpeptidation reaction.

Accordingly, the insulin derivatives of this inven¬ tion may be prepared by inserting a DNA-sequence encoding a precursor of the insulin derivative in question into a suit¬ able yeast expression vehicle which when transferred to yeast is capable of expressing and secreting the precursor of the insulin derivative in which Lys^B29 is connected to -LyA2¹ _by _a eptide bond or a peptide chain with the general 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 R¹ is Lys or Arg, when the transformed yeast strain is cultured in a suitable nutrient medium. The precursor is then recovered from the culture broth and reacted with an amino compound 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 Thr, 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. Thereafter, the carb¬ oxy protecting group is removed and the insulin derivative is isolated from the reaction mixture. The insulin derivatives of this invention may also be prepared by a method analogous to the method described in European patent application having publication No. 195,691 the disclosure of which is incorporated by reference herein¬ to. By this method, insulin derivative precursors of the type having a bridge between the A and B chain consisting of a single pair of basic amino acid (Lys or Arg) are produced in yeast and then converted into the insulin derivative by an enzymatic conversion.

The insulin derivatives of this invention may be used for the preparation of novel insulin preparations. Such novel insulin preparations may contain the insulin deriva¬ tives of this invention or a pharmaceutically acceptable salt thereof in aqueous solution or suspension, preferably at an approximately neutral pH value. 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 derivatives of this present invention are Tyi^¹³ human insulin, Phe^B17 human insulin, _TrpB1⁷ h_uman insulin, Tyr^B17 human insulin, Tyr^A13,Phe^B17 human insulin, Tyr^^Trp³¹⁷ human insulin, τy_r ^A13 _fτyr^B17 human insulin, Phe^A13,Phe^B17 human insulin, Phe^A13,Trp^B17 human insulin, Phe^A13,Tyr^B17 human insulin, Trp^A13,Phe^B17 human insulin, Trp^A13,Trp^B17 human insulin and τrp^A13,Tyr^B17 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 indica- ted, the species of insulins stated herein is human.

The replacement(s) made in the human insulin mole¬ cule according to the practice of this invention is (are) indicated with a prefix referenced to human insulin. As an example, Glu^A13 human insulin is human insulin having Glu in position 13 of the A chain (in stead of Leu) .

Like other insulins, the insulin derivatives of this invention have a low toxicity.

The insulin derivatives of this invention are ad¬ ministered analogously to the administration of known insu- lins and insulin derivatives in a therapeutically effective amount. The dosage to be administered is normally determined by a physician. The route of administration may be intra¬ muscular, subcutaneously, intravenous, via a mucos, for example nasal, or by a pump. Any novel feature or combination of features de¬ scribed herein is considered essential to this inverntion.

Detailed description

Genes encoding the precursors of the insulin de¬ rivative can be prepared by modification of genes encoding the corresponding human insulin precursors by site specific mutagenesis to insert or substitute with codons encoding the desired mutation. A DNA-sequence encoding the precursor of the insulin derivative may also be made by enzymatic syn¬ thesis from oligonucleotides corresponding in whole or part to the insulin derivative precursor gene.

DNA-sequences containing a gene with the desired mutation are then combined with a suitable promoter se¬ quence, 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. pplied Genet. 1 (1982), 419 - 434), a suitable leader sequence and possible transcription termination sequence, for example from TPI of S. cerevisiae (TPI_T) . These frag¬ ments provide sequences to ensure a high rate of transcrip¬ tion of the precursor encoding gene and also provide a pre- sequence 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 pro¬ vided with a yeast origin of replication, for instance the 2μ origin, and a selectable marker, for instance LEU 2. The selected plas id is then transformed into a suitable yeast strain by conventional technique, for example as described in European patent application having publica¬ tion No. 214,826 and transfor ants are grown on YPD medium (1% yeast extract, 2% peptone, and 2% glucose) . The insulin derivative precursor is isolated from the culture medium and reacted with threonine methyl ester acetate dissolved in a N,N-dimethylformamide/water mixture in the presence of tryp- sin as described in European patent application having pub¬ lication No. 214,826 and converted into the human insulin derivative by acidic or basic hydrolysis, see European patent application having publication No 214,826.

Example l

The specificity of a compound according to this invention, i.e. Phe^B17 human insulin, compared with human insulin was determined by the following method which appeared in Dia¬ betes May 1991:

Insulin and insulin derivative were monoiodinated in the tyrosine in the A14 position with ¹²³I. Wistar rats weighing about 250 g received a bolus injection of 5 - 10 mU of ^{1 3}I-labelled insulin derivative through a catheter in¬ serted into the jugular vein. The liver activity of ^{1 3}ι was monitored by a gammacamera and expressed as percentage of total radioactivity. Results are shown in Table I below wherein "HI" designates human insulin. Table I

LIVER ACTIVITY

Time, minutes Phe^B17HI HI

1 40 33 2 50 40 3 54 42 4 54 44 5 50 41 6 48 40

7. 42 35 8 38 32 9 35 30 10 31 26

As appears from this table, the compound of this invention is preferentially targeted to the liver.

Claims

1. Insulin derivatives differing from human insulin in that they have Tyr in position A13 and/or having Phe, Trp or Tyr in position B17.

2. Insulin derivative according to claim 1 having

Phe, Trp or Tyr in position B17.

3. Derivative according to claims 1 or 2 having - compared with human insulin - the amino acid residue in position A21 exchanged with a residue of another naturally occurring amino acid.

4. Derivative according to any of the preceding claims having - compared with human insulin - one or more of the amino acid residues in positions A4, A17, B13 and B21 exchanged with a neutral amino acid which can be coded for by nucleotide sequences.

5. Derivative according to any of the preceding claims having - compared with human insulin - the amino acid residue in position B27 exchanged with Arg, Lys or Thr.

6. Derivative according to any of the preceding claims wherein 3, 4 or 5 of the amino acid residues in posi¬ tions B26, B27, B28, B29 and B30 are omitted.

7. Derivative according to any one of the preceding claims having the carboxy group in the amino acid residue at the C terminal end of the B chain protected with an amino group.

8. Derivative according to any one of the preceding claims wherein the amino acid residue in position B25 is Tyr.

9. Derivative according to any one of the preceding claims wherein the amino acid residue in position A13 is Phe or Trp.

10. Derivative according to any one of the preced¬ ing claims having - compared with human insulin - no Bl amino acid residue.

11. Derivative according to any one of the preced¬ ing claims having - compared with human insulin - one or more of the amino acid residues in positions A8, A9, A10, A13, A21, Bl, B2, B5, B9, BIO, B12, B14, B16, B17, B18, B20, B26, B27 and B28 exchanged with another amino acid residue, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin.

12. Derivative according to claim 1 being Ty^¹³ human insulin, Phe^B17 human insulin, Trp^B17 human insulin, Tyr^B17 human insulin, Ty^¹³,Phe^B17 human insulin, Tyr^-^Trp²¹⁷ human insulin, τyr^A13,Tyr^B17 human insulin, Phe^A13 , he^B17 human insulin, Phe^A13,Trp^B17 human insulin, Phe^A13,Tyr^B17 human insulin, Trp^A13,Phe^B17 human insulin, Trp^A13,Trp^B17 human insulin or τrp^A13,Tyr^B17 human insulin.

13. Derivative according to any one of the prece- ding claims having organ preferential action.

14. Derivative according to Claim 13, wherein the organ is the liver.

15. Pharmaceutical preparations containing an insu¬ lin derivative according to any one of the preseding claims or a pharmaceutically acceptable salt thereof and conventio¬ nal pharmaceutical additives, adjuvants, carriers, diluents and solvents.

16. Preparation according to claim 15 for use in the treatment of diabetes.

17. A method of treating diabetes in a patient in need of such treatment comprising administering to the patient a therapeutically active amount of an insulin de¬ rivative as defined in any one of the claims 1 through 14, optionally together with a pharmaceutically acceptable car- rier.

18. .Any novel feature or combination of features described herein.