DK166730B - Human insulin analogues and their pharmaceutically tolerable salts, and pharmaceutical preparations which comprise them - Google Patents

Description

DK 166730 B

The present invention relates to novel human insulin analogues which exhibit low degree of association in solution, pharmaceutically tolerable salts thereof, and insulin preparations containing them.

THE PRIOR ART

Since the discovery of insulin in 1922, many different types of insulin have been used to treat diabetes mellitus. Initially, only io insulin solutions with fast onset and relatively fast onset insulin action were used, but later on, insulin preparations with a more extended effect profile were produced by lowering the solubility of the insulin by adding e.g. zinc salt and / or protamines. For accessibility reasons, the insulin used for this purpose has usually been extracted from pancreas from livestock, most commonly oxen, pigs and sheep, but in recent years preparations containing human insulin of biotechnological origin have also emerged.

Human insulin has the structure listed below: A chain

S-S

I 7 | H-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-25 1 2 3 4 5 6 I 8 9 10 11 12

S

B-chain S

30 H-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-1234 56789 10 11 12 20

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2 A-chain (continued) -Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn-OH 13 14 15 16 17 18 19 | 21

5 S

'^ 1

B-chain (continued) S

-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-10 13 14 15 16 17 18 19 20 21 22 23 24 B-chain (continued) -Phe-Tyr-Thr -Pro-Lys-Thr-OH 25 26 27 28 29 30

The insulins of certain domestic animals are structurally very similar to human insulin. Dog and swine insulin simply differs from human insulin by containing the Ala i-30 position in the B chain, and rabbit insulin differs from human insulin only by holding Ser in the same position. By means of semisynthetic methods it is possible to convert these insulins to human insulin by replacing the B30 amino acid residue with Thr, cf. eg. Morihara et al., Nature 280 (1979), 412-413, and Markussen (U.S. Patent No. 4,343,898).

When such insulin is dissolved at physiological pH, a concentration-dependent association equilibrium is established between monomeric, dimeric, tetrameric, hexameric and even polymeric insulin. For example, the equilibrium can be determined by ultracentrifugation, by osmometry or by gel filtration, cf. eg. R. Valdes Jr. and G.A. Ackers, "Methods in Enzymology", Volume 61 (Enzyme Structure, Part H, Publishers: 30 Hirs & Timasheff), Academic Press 1979, pages 125 - 142. In normal formulations of insulin preparations, this equilibrium is shifted in such a way that the insulin in is very high in a hexameric form.

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Substitutions can be made in the insulin molecule to improve the action profile of insulin in the treatment of diabetes. Thus, from the specification of PCT patent application WO 86/05497, it is known to substitute 5 one or more of the amino acid residues Glu of the insulin molecule with a neutral amino acid residue, thereby displacing the insulin's precipitation zone so that slow release after injection.

Further, from the specification to European patent application with publication number 214,826 there are known insulin analogues which are absorbed particularly rapidly after injection. This effect is due to the fact that, by certain substitutions, especially in the B9-B12 positions and in the B26-B28 positions of the insulin molecule, a suppression of the association ability of the insulin is achieved, so that it is largely in monomer or dimeric form. However, many of these insulin analogues exhibit a reduced biological effect.

Over the years, a large number of artificial insulin analogues have been described, most often for the purpose of elucidating the structure's influence on activity, cf. eg. Mårke et al., Hoppe-Seyler's Z. Physiol. Chem. 360 (1979), 1619 - 1632. Studies on the effect of substitutions on insulin's (B22-B26) sequence on receptor binding have been particularly interesting, as this sequence is thought to be an essential site for the binding of the insulin receptor, and since naturally occurring mutations has been found with substitutions in this area, cf. eg. S. Shoelson et al. PNAS 80 (1983), 7390-94, and M. Kobayashi et al .: Biomed. Res. 5, (3) (1984), 267-72. Very low biological activities are found for analogs in which PheB24 or PheB25 are substituted, and it is therefore concluded that the presence of these two amino acids is of crucial importance for receptor binding.

Surprisingly, it has been found that certain human insulin in analogues in which one of the amino acid residues PheB24 or PheB25 35 is not present exhibit low association tendency in solution and at the same time in vitro exhibit unchanged or even higher biological activity than human insulin. The omission of either

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PheB24 or PheB25 has the effect of changing LysB2 ^ to LysB28, leading to a positive charge at this position in the human insulin molecule.

SUMMARY OF THE INVENTION

Accordingly, in the broadest aspect the present invention relates to see human insulin analogues in which there is a positively charged amino acid residue at the B28 position, i.e. Lys or Arg, i.e. in the 8-position of the B-chain calculated from GlyB20.

Surprisingly, the present insulin analogues have an io low association rate and at the same time an increased physical stability compared to other low association degree insulin analogues. The introduction of a positive charge into the B28 position can be achieved in two ways. Either by omitting one of the amino acid residues at the B24, B25, B26, B27 or B28 position of the human insulin molecule, which yields a human insulin analog with Lys at the B28 position, or by replacing ProB28 in the human insulin molecule with Lys or Arg. If Arg is preferred in the B28 position, the deletion of one of the amino acids in the B24, B25, B26, B27 or B28 position can be further combined with the replacement of the original LysB29 with an Argrest.

The present human insulin analogues may further have one or more changes in the C-terminal end of the B chain, as compared to human insulin. The amino acid residues at positions B25 - B27 and the amino acid residues following LysB28 or ArgD may be arbitrarily selected from the naturally occurring amino acid residues, or the B29 amino acid or B30 amino acid or both may be missing.

According to another aspect of the present invention, TyrB26 may be replaced by another uncharged amino acid residue in which the second carbon atom in the side chain (C 1) is sp2 hybridized (the bonds have a (more) planar structure).

In order to stabilize the molecule against chemical degradation, Asn can be in the A21 position and / or the B3 position.

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also be replaced by another amino acid residue.

The present human insulin analogues "are characterized by the following Formula I: A chain

5 S-S

I "" r H-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-s 10 |

B-chain S

H-Phe-Val-Y2-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-A Chain (continued) (I)

-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Y1-OH

p

B-chain (continued) S

20 | -Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-B chain (continued) -X1-X2-X3-X4-X5-X6 wherein X1, X2, X3, X5, Y1 and Y2 may be any naturally occurring amino acid residue, however, X ^ is not Pro, X4 is Lys or Arg, X6 may be any naturally occurring amino acid residue bearing the C-term nal hydroxy group, or -OH, or X5 and X6 together form the C-terminal hydroxy group, or are pharmaceutically tolerable salts thereof.

In another embodiment of the invention, X5 is selected

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6 among the group consisting of any naturally occurring amino acid residue except Faith.

In one embodiment, Y1 and / or Y2 in the above formula may be selected from the group consisting of any 5 naturally occurring amino acid residues except. of Asn.

More specifically, in the above formula I, X1 may be Phe, Ala, His, Thr, Ser, Asn or Tyr, X2 may be more specifically Tyr, Thr, Glu, Asp, Ala, His, Ser or Phe, X3 may be more specifically more specifically, Pro, Glu, Asp, Ser, Thr or His, X5 may be more specifically Lys, Thr, Ser, Ala, Asp or Glu, X6 may be more specifically Thr-OH, Ser-OH, Ala-OH, Asp. OH, Glu-OH or -OH, Y1 may be Asn, Glu, Asp, His, Ser, Thr, Val, Leu, Ile, Ala, Met, Trp, Tyr, Gln or Gly, more preferably Gly, Asp, Glu or Ala, and Y 2 may be Asn, Glu, Asp, His, Ser, Thr, Val, Leu, Ile, Ala, Met, Trp, Tyr, Gln or Gly, more preferably Glu or Asp.

A group of the present human insulin analogues may be characterized as one in which one of the amino acid residues 20 at the B24 or B25 position is omitted, that the amino acid residue at the B26 position is optionally replaced by another, uncharged amino acid residue in which the carbon atom at the γ position is sp2 hybridized, optionally one or more of the amino acid residues at the A21, B3 and B3O positions is different from the amino acid residue at the corresponding position in human insulin, and that there may be no amino acid residue at the B3O position.

By a simpler definition, such analogs are human insulin analogues in which there is no TyrB2 ^, in which PheB25 is optionally replaced by another uncharged amino acid residue in which the carbon atom at the γ position is SP hybridized, in which one or more of the the amino acid residues at the A21, B3 and B30 positions may be too different from the amino acid residues in human insulin and in which there may be no amino acid residues at the B3O position.

Examples of uncharged amino acid residues in which are sp-hybridized are Tyr, Phe, His, Trp or Asn.

A group of human insulin analogues of this invention 7

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has the structure shown by Formula II below, wherein X represents Tyr, His, Phe or Asn, Y calculates Thr, Ser, Ala, Asp or Glu or is omitted and where optionally one or both of the underlined Asn is replaced with Asp at 5 replacement or deamidation, or the underlined Asn in the A chain may be Gly.

A chain

S-S

I I

H-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-s

B-chain S

15 | H-Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-A-Chain (continued) (II) -Leu-Tyr-Gln-Leu-Glu-Asn-Tyr Cys-Asn-OH 20 |

S

B-chain (continued) S

25-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-1 B Chain (continued)

-X-Thr-Pro-Lys-Y-OH

Preferred human insulin analogues of the present invention are the following: des (PheB25) -human insulin, des

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8 (TyrB26) -human insulin, des (Thr527) -human insulin, des (Pro528) -human insulin, des (Phe525) -piginsulin, des (Pro528) -vignin-insulin, des (ProB28)-rabbit insulin, des (PheB25), des (Thr530) -human insulin, des (Tyr526), des (Thr530) -human insulin, (Ser ^ 21) -5 des (Pro528) -human insulin, (GlyÅ21) -des (Pro528) -human insulin, (GlyA2 ^) - des (Phe525) -human insulin, (AspA21j-des (Phe525) -human insulin, (His525) -des (Tyr526), des (Thr530) -human insulin, (AsnB25) -des (TyrB26), des (ThrB30) -human insulin, (AspA21) ) -des (PheB25), des (Thr530) -human insulin, (Asp528) -des (PheB25) -10 human insulin, (Asp53) -des (Phe525) -human insulin, (Lys528) -human insulin, (Lys528, Thr529) -human insulin and (Arg528) -des- (Lys529) -human insulin.

The human insulin analogues of the present invention may advantageously be used in the treatment of diabetes, as the decreased association rate leads to faster uptake into blood flow than is the case with regular insulin, not only after the commonly used subcutaneous injection but also with non-parenteral submission, cf. International Patent Application No. WO '87 / 06137. Their 20 improved physical stability makes them more advantageous in the treatment of diabetes.

The insulin analogues of the present invention can be prepared by altering the proinsulin network by replacing codons at suitable sites in the natural human proinsula 25 with codons encoding the desired amino acid residues and / or by omitting codons corresponding to the desired deletions. Alternatively, one can synthesize the entire DNA sequence encoding the desired insulin analog. The gene encoding the desired insulin analog is then inserted into a suitable expression vector which, when transferred to a suitable host organism, e.g. E. coli, Bacillus or yeast, make the desired product. The expression product is then isolated from the cells or culture medium depending on whether or not the expression product is secreted from the cells.

The novel insulin analogues may also be prepared by chemical synthesis by methods analogous to the method of the invention.

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those described by Mårki et al. (Hoppe-Seyler's Z. Physiol. Chem., 360 (1979), 1619-1632). DeT' can also be formed from separate, in vitro prepared A and B chains containing the relevant amino acid residues and deletions, after which the 5 modified A and B chains are linked together by forming disulfide bridges in known manner (e.g. Chance et al., In: Rick DH, Gross E (publishers) Peptides: Synthesis - structure - Function. Proceedings of the seventh American peptide symposium, Illinois, 721 - 728).

The insulin analogues may further be prepared by a method analogous to the method described in European Patent Application Publication No. 163,529, the contents of which are incorporated herein by reference. In such methods, an insulin precursor for the human insulin analog is expressed in which the basic amino acid at the B28 or B29 position (if the final product is to have a basic amino acid at this position) is linked to GlyA1 either by a peptide bond or a peptide chain of varying length and is excreted by yeast with the disulfide bridges in the proper positions and then converted to the desired human insulin analogue using the Mori-haras method (Morihara supra) or the so-called transpeptide reaction (see U.S. Patent No. 4,343,898).

Accordingly, the present insulin analogs may be prepared by inserting a DNA sequence encoding a precursor of the insulin analog in question into a suitable yeast expression vector which, when transformed into yeast, is capable of expressing and secreting the precursor of the insulin analog. wherein LysB28, ArgB28, Lys629 or ArgB29 are attached to GlyA1 by a peptide bond or a peptide chain of formula 30 III

-Rn-R1- (III) where 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 strains are grown in a suitable nutrient medium. The precursor is then recovered from the culture medium and reacted with 10

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an amino compound of formula IV

Q-QR "(IV) wherein Q is a single amino acid residue, preferably Thr, or a dipeptide, and R" is a carboxyl protecting group (e.g.

(Methyl or tert.butyl), using trypsin or a trypsin-like enzyme as catalyst in a mixture of water and organic solvent in an analogous manner as described in U.S. Patent No. 4,343,898 (the contents of which are incorporated herein by reference), and the carboxyl protecting io group is removed and the insulin analog is isolated from the reaction mixture.

If the insulin anal oges as the C-terminal residue in the B chain contain an amino acid residue different from Lys or Arg, they may also be prepared by a method analogous to the method described in European Patent Application Publication No. 195,691 , the disclosure of which is incorporated herein by reference. By this method, insulin analog precursors of the kind having a bridge between the A and B chain consisting of a single pair of basic amino acids 20 (Lys, Arg) are prepared in yeast and then converted to. insulin analogue by enzymatic conversion.

If the C-terminal amino acid residue in the B chain is Lys or Arg, the insulin analogs can be prepared from the above biosynthetic precursors by enzymatic cleavage with 25 trypsin.

The human insulin analogues of this invention, in which there are only substitutions within the last amino acid residues closest to the C-terminal end of the B chain, can also be prepared in a manner known per se, e.g. swine insulin as described in K. Inoye et al .; JACS 101 (3), (1979), 751-752, whereby pig insulin is first cleaved with trypsin to des- (B23-30) -human insulin, after which the latter is enzymatically coupled to a synthetic peptide having the desired amino acid sequence.

The present insulin analogues may be used for preparation

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The present invention also relates to pharmaceutical compositions characterized by containing a human insulin analogue of the formula of claim 1 or to the present invention. Such compositions are present in aqueous solution or suspension, preferably at neutral pH. The aqueous medium is made isotonic, for example with sodium chloride, sodium acetate or glycerol. In addition, the aqueous medium may contain zinc ions. , buffering agents such as acetate and citrate, and preservatives such as m-cresol, methylparaben or phenol The pH of the preparation is adjusted to a desired value and the insulin preparation is sterilized by sterile filtration.

The present insulin analogues may also be mixed with other insulin analogues with retarded insulin activity to produce insulin preparations comprising a mixture of fast-acting and protracted-acting insulin.

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

TERMINOLOGY

The abbreviations used for the amino acids are those given in J. Biol.Chem. 243 (1968), 3558. The amino acids are in L configuration. The nature of the insulins disclosed herein is human unless otherwise indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated with reference to the accompanying drawings, in which

FIG. 1 shows the expression plasmid pYGABA 14-276, FIG. 2 shows the yeast vector pAB24,

FIG. Figure 3 shows the DNA sequence of the 0.4 kb EcoRI-XbaI fragment from plasmid pKFN-864, and

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FIG. 4 shows the preparation of the expression plasmid pKFN-866.

DETAILED DESCRIPTION

DNA sequences encoding modified insulin precursors are constructed based on the expression cassette contained in the BamHI restriction fragment of the expression plasmid pYGABA as shown in FIG. 1. It has a length of 1103 base pairs and contains essentially the following (mentioned in order starting from the 5 'end): The GAPDH promoter (Travis io et al., J. Biol. Chem., 260 (1985), 4384 - 4389) followed by the coding region consisting of the following: The 83 N-terminal amino acids of the MF α leader sequence encoded by the wild-type yeast DNA sequence as described by Kurjan & Herskowitz followed by the two codons AAA and AGA for Lys and Arg and followed by the coding region for insulin precursor single chain des [ThrB30] human insulin (SCI), which is a synthetically produced gene using preferred yeast codons. After two stop codons, a SalI restriction site is found and the rest of the sequence is constituted by the MFal sequence containing the terminator region. The sequence is prepared using standard techniques only.

The method used is the so-called "oligonucleotide site directed mutanegesis" described by Zoller & Smith, DNA, Vol. 3. (1984), no. 6, 479 - 488. The method is briefly described below and is described in detail in Example 1: Isolated from the expression plasmid, the insulin precursor sequence is inserted into a single-stranded, genome-circular M13 bacteriophage vector. For the single-stranded genome, a chemically synthesized complementary DNA strand is canceled. The DNA strand 30 contains the desired sequence surrounded by sequences which are completely homologous to insulin sequences in the circular DNA. Then, in vitro, the primer is extended biochemically throughout the length of the circular genome using Klenow polymerase. This strand will cause the formation of single-stranded phages which, when grown in E. coli, allow the double-strand 13 to be isolated.

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DNA of the desired sequence. From this double-stranded DNA, a restriction fragment which can be reinserted into the expression vector can be isolated.

PROCEDURE FOR CARRYING OUT THE INVENTION

The invention is further illustrated by the following examples: EXAMPLE 1

Construction of the expression plasmid that can be used to express des (PheB25] -SCI.

The expression cassette contained in the expression plasmid pYGABA (shown in Figure 1) on a BamHI restriction fragment is isolated: The expression plasmid is incubated with the restriction endonuclease BamHI. The conditions were: 20 µg of plasmid, 50 units of BamHI, 100 mM NaCl, 50 mM Tris-HCl, pH-15: 7.5, 10 mM MgCl2, and 1 mM DTT in a volume of 100 μΐ. The temperature was 37 ° C and the reaction time 2 hours. The two DNA fragments are cleaved on a 1% agarose gel and the dried fragments isolated.

Ligation to the M13 vector M13mpi8: 20 The isolated restriction fragment is also ligated to the bacteriophage vector M13mpl8 also clipped with the restriction endonuclease BamHI in the following reaction mixture: 0.2 µg fragment, 0.02 µg vector, 50 mM Tris-HCl, pH: 7.4, 10 mM MgCl2, 10 mM DTT and 1 mM ATP in a volume of 20 μΐ. 5 μΐ of 25 this mixture is transformed into E. coli strain JM101. The presence of fragment in the vector and orientation of the fragment is determined by restriction enzyme mapping on double stranded M13 DNA isolated for transformants.

Isolation of single stranded (ss) DNA (template): 30 ss DNA is isolated from the above transformant using a method described by Messing in Gene 19

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14th

(1982), 269 - 276.

5'-phosphorylation of the mutagenization primer:

The mutagenization primer of the sequence 51-TTGGAGTGTA-GAAACCTCTT-31 is phosphorylated at the 5 'end in a 30 μΐ reaction-5 mixture containing 70 mM Tris-HCl, pH: 7.0, 10 mM MgCl2 / 5 mM DTT, 1 mM ATP , 100 pmol oligonucleotide and 3.6 units of T4 polynucleotide kinase. The reaction is carried out for 30 minutes at 37 ° C. The enzyme is then inactivated by incubating the mixture for 10 minutes at 65 ° C.

io Template joining and phosphorylated mutagenization primer:

Template and primer joins are performed in a 10 μΐ volume containing 0.5 pmol template, 5 pmol primer, 20 mM Tris-HCl, pH: 7.5, 10 mM MgCl 2, 50 mM NaCl, and 1 mM DTT upon heating. for 10 minutes at 65 ° C and then cool to 0 ° C.

Extension / ligation reaction:

To the above reaction mixture is added 10 μΐ of the following mixture: 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, 0.3 mM 20 TTP, 1 mM ATP, 20 mM Tris-HCl, pH: 7 , 5, 10 mM MgCl2, 10 mM DTT, 3 units of T4 DNA ligase and 2.5 units of Klenow polymerase. Then the reaction is carried out for 16 hours at 16 ° C.

Transforming JM101:

The above reaction mixture is transformed into different dilutions into CaCl2 treated E.coli JM191 cells using standard procedures and spread on 2 x YT top agar on 2 x YT agar plates. (2 x TY = tryptone: 16 g / 1, yeast extract: 10 g / 1, NaCl 5 g / 1. 2 x YT top agar = 2 x YT added 0.4% agarose and autoclaved. 2 x YT agar plates = 2 x YT 30 added 2% agar and autoclaved). The plates are incubated overnight at 37 ° C.

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Identification of positive clones:

Plaque lifter hybridization is used, as described below: A nitrocellulose filter is applied to a plate of suitable plaque density so that the filter is wetted.

The filter is then bathed in the following solutions: 1.5 M NaCl, 0.5 M. NaOH for 30 seconds, 1.5 M NaCl, 0.5 M Tris-HCl, pH: 8.0 for 1 minute , 2 x SSC (0.3 M NaCl, 0.03 M "sodium citrate) for later use. The filter is dried on a 3 MM filter paper and heated for 2 hours at 8 ° C in a vacuum oven.

io The mutagenization primer of the sequence 5'-TTGGAGTGTA-GAAACCTCTT-31 is radiolabelled at the 5 'end in a 30 μΐ reaction volume containing 70 mM Tris-HCl, pH: 7.5, 10 mM MgCl2, 5 mM DTT, 10 pmol oligonucleotide, 20 pmol γ- · * 2Ρ-ΑΤΡ and 3.5 units of T4 polynucleotide kinase. The mixture is incubated at 37 ° C for 30 minutes and then for 5 minutes at 100 ° C.

The dry filter is prehybridized for 2 hours at 65 ° C in 6 x SSC, 0.2% bovine serum albumin, 0.2% "Ficoll" ®, 0.2% polyvinylpyrrolidone, 0.2% sodium dodecyl sulfate (SDS), and 50 µg / ml salmon sperm DNA. Then, the reaction mixture containing the labeled probe is added to 15 ml of fresh prehybridization mixture and the filter is bathed therein overnight at 28 ° C with gentle shaking. After hybridization, the filter is washed three times in 2 x SSC + 0.1% SDS for 15 min. time after which autoradiographing. After washing in the same solution, but now at 52 ° C and new autoradiographing, plaques containing DNA sequences that are complementary to the mutagenization primer are identified.

Re-screening of positive clones

Since the identified clone is a result of a heteroduplex, the plaque is plated again. The hybridization and identification is repeated.

Purification of double stranded M13 phage DNA

A rescreened clone is used to infect E. coli strain JM101. A culture containing approx. 108 phages and 5 colo- 16

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kidneys of JM101 are grown for 5 hours in a 5 ml 2 x YT medium at 37 ° C. Then, double-stranded circular DNA is purified from the pellet using the method described by Birnboim, & Doly, Nucleic Acids, Res., 2 (1979), 1513.

Isolation of a restriction fragment containing modified insulin precursor The DNA preparation (about 5 μg) purified as described above is cleaved with 10 units of the restriction endonuclease io BamHI in 60 μΐ 100 mM NaCl, 50 mM Tris-HCl, pH: 7.5, 10 mM MgCl2 and 1 mM DTT for 2 hours at 37 ° C. The DNA products are separated on an agarose gel and the fragment is purified from the gel.

Ligation to the yeast vector pAB24 (Fig. 2)

The isolated restriction fragment is ligated to the yeast vector pAB24 cut with the restriction endonuclease BamHI in the following reaction mixture: Fragment 0.2 µg, vector 0.02 µg, 50 mM Tris-HCl, pH: 7.4, 10 mM MgCl2, 10 mM

DTT, 1 mM ATP in a total volume of 20 μΐ-. 5 µl of the reaction mixture is used to transform E. coli strain 20 MC1061 in which the modified expression piamide is identified and propagated. The plasmid is identical to pYGABA except for the deleted codon.

Transformation of yeast

Transformation of the expression plasmid to the yeast strain 25 Saccharomyces cerevisiae JC482ApepALeu2cir ° (a, his4, pep4, ura3, leu2, cir °) is performed as described by Ito et al., J. Bact., Vol 153 (1983), 1, 153 - 168. The transformed cells are plated on SC-ura medium (0.7% Yeast Nitrogen Base, 2.0% glucose, 0.5% casamino acids, 2.0% agar) for selection 30 for plasmid-containing cells.

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Example 2

Construction of an expression plasmid that can be used to produce des (TyrB26) -SCI.

The procedure is similar to that described above in Example 1 except that the mutagenization primer has the sequence 5 '-ACCCTTTGGAGTGAAGAAACCTCT if the hybridization temperature is 36 ° C and that the wash temperature after the hybridization is 60 ° C. The modified plasmid has a sequence identical to pYGABA except for the deleted io codon.

Example 3

Construction of an expression plasmid that can be used to produce (HisB25), des (TyrB26) -SCI.

The procedure is similar to that described above in Example 1 except that the mutagenization primer has the sequence 5'AATACCCTTTGGAGTGTGGAAACCTCTTTCACC-3 ', the hybridization temperature is 43 ° C and the wash temperature after hybridization is 66'C. The modified plasmid has a sequence identical to pYGABA, except for the modified and deleted codons.

Example 4

Construction of an expression pyramid which can be used to produce (AsnB25), des (TyrB26) -SCI.

Much the same procedure as described above in Example 1 is used except that the mutagenization primer has the sequence 51-AATACCCTTTGGAGTGTTGAAACCTCTTTCACC-31, the hybridization temperature is 42 ° C and the wash temperature after the hybridization is 65 ° C. The modified plasmid has a sequence identical to pYGABA except 30 of the modified and deleted codons.

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Example 5

Expression of precursor and isolation thereof from the culture medium.

Yeasts transformed as described in Examples 1-4 are propagated on Petri dishes containing minimal medium 5 udeniracil for 48 hours at 30 ° C. 100 ml shake flasks containing minimal medium without uracil + 5 g / l casamino acids + 10 g / l succinic acid + 30 g / l glucose at pH 5.0 are inoculated with a single colony from Petri plate. The flasks are then shaken at 30 ° C in an incubator for 72 hours.

10 After centrifugation, 1 liter of collected supernatant is sterilized by filtration and adjusting to pH 4-4.5 and a conductivity below 10 mS by addition of 5 M hydrochloric acid and water. At a flow rate of 120 ml / hour, the supernatant is then placed on a 1.6 x 6 cm column of S / "Se-15 pharose" ® FF, which is already equilibrated with 50 mM acetic acid, 50% (volume) ethanol, adjusted at pH 4.0 with NaOH. The column is then washed with 60 ml of buffer, after which the precursor is eluted with a linear gradient of NaCl from 0 to 0.35 M in 360 ml of buffer at a flow rate of 10 20 ml / hour. The eluate is divided into 4 ml fractions and detected for UV absorbance. Fractions containing precursor are identified by RP-HPLC analysis and the fractions are pooled. After desalting on a column of "Sephadex" ® G25 in 1 M acetic acid, the precursor is isolated by freeze drying.

Example 6

Preparation of des (PheB2S), des (ThrB30) human insulin.

400 mg of des (PheB25) -SCI, prepared using the procedures described in Examples 1 and 5, are dissolved in 40 ml of 50 mM tris (hydroxymethyl) aminomethane, 20% (volume) of ethanol 30 adjusted to pH 9 with HCl, and 40 ml (sedimented volume) of "Sepharose" ® containing 32 mg of immobilized trypsin in the same buffer is added. The suspension is left for 24 hours at 8 - 10 ° C with gentle stirring and then filtered. The gel is washed with 40 ml of buffer and the collected filtrates are placed on a 2.6 x 7.5 cm column of Q- "Sepharose" ® FF, previously equilibrated with 50 mM tris * (hydroxymethyl) -aminomethane, 50% (by volume) ethanol, adjusted to pH 8.0 with HCl. Then, the column is eluted with a linear 5 gradient of NaCl from 0 to 0.15 M in the same buffer over -6 hours at a flow rate of 225 ml / hour. The eluate is detected for UV absorbance and fractions containing the protein head peak are pooled. The protein is precipitated at pH 5.4 after removal of ethanol in vacuo.

io 250 mg of des (PheB25), des (ThrB3 °) -human insulin is isolated by freeze-drying.

The identity of the product is confirmed by amino acid analysis, by plasma sorption mass spectrometry and by sequential Ed-Man degradation of the separated vinyl pyridylated A and B chains.

Example 7

Preparation of des (PheB25) human insulin.

200 mg of des (PheB25), des (ThrB3 °) -human insulin prepared by the procedure described in Example 6 are dissolved in a mixture containing 400 mg of threonine methyl ester, 2.0 ml of ethanol and 0.8 ml of water. Adjust the pH to 6.3 with acetic acid and add 4 ml (sedimented volume) of "Sepharose" ® containing 3.2 mg of immobilized trypsin. After standing for 2 hours at 20 ° C with slight stirring, the gel is filtered off and the protein precipitated by the addition of 10 volumes of 2-propanol. The air-dried precipitate is redissolved in 20 mM tris (hydroxymethyl) aminomethane / HCl, 60% (by volume) ethanol, pH 8.25, and put on a 2.6 x 20 cm Q-SepharoseM® FF column which is equilibrated with the same buffer and eluting with a linear NaCl gradient in the same buffer increasing from 0 to 0.1 M over 15 hours at a flow rate of 125 ml / hour. The fractions containing des (PheB25) human insulin (B30 methyl ester), are combined and the ethanol is removed in vacuo, the protein is precipitated at pH 6.1. The suspension is centrifuged and the precipitate is freeze-dried.

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is then hydrolyzed for 10 minutes in cold 0.1 M NaOH at a protein concentration of 10 mg / ml. The reaction is quenched by adjusting the pH to 8.5 and adding 2 volumes of 20 mM tris (hydroxymethyl) aminomethane / HCl, pH 8.5. The solution 5 is then put on a 2.6 x 20 cm Q "Sepharosen® FF column and eluted as described above. The protein is precipitated at pH 5.5 after removal of the ethanol in vacuo.

80 mg of des (PheB25) human insulin is obtained after lyophilization.

The identity of the product is confirmed by amino acid analysis, plasma sorption mass spectrometry and sequential Edman degradation of the separate vinyl pyridylated A and B chains.

Example 8 Preparation of des (TyrB26), des (ThrB30) -human insulin.

250 mg of des (TyrB2®) sci prepared using the procedures described in Examples 2 and 5 are dissolved in 25 ml of 50 mM tris (hydroxymethyl) aminomethane, 20% (by volume) ethanol adjusted to pH 9 with HCl and 20 ml (sedimented volume) of "Sepharose" ® containing 20 mg of immobilized trypsin in the same buffer are added. The suspension is left for 24 hours at 8 '- 10 ° C with slight stirring and then filtered. The gel is washed with 25 ml of buffer and the collected filtrates are placed on a 2.6 x 7.5 cm column of Q- "Se-25 pharose" ® FF, previously equilibrated with 50 mM tris (hydroxymethyl) aminomethane, 50% (by volume) ethanol, adjusted to pH 8.0 with HCl. The column is then eluted with a linear NaCl gradient from 0 to 0.15 M in the same buffer over 6 hours at a flow rate of 225 ml / hour. The eluate 30 is detected for UV absorbance and fractions containing the protein head are pooled. The protein is precipitated at pH 5.4 after removal of the ethanol in vacuo.

Freeze-drying gives 130 mg of des (TyrB26), des (ThrB30) -human insulin.

The identity of the product is confirmed by amino acid analysis and 21 sequential Edman degradation of the separate vinyl pyridylated A and B chains.

Example 9

Preparation of (His®25), des (TyrB26), des (ThrB30) -human insulin.

450 mg (His®25), des (Tyr®25) -sci, prepared by the procedures described in Examples 3 and 5, are dissolved in 45 ml of 50 mM tris (hydroxymethyl) aminomethane, 20% (by volume) of ethanol adjusted to pH 9 with HCl and 45 ml (sedimented volume) of "Sepharose" ® containing 36 mg of immobilized trypsin in the same buffer are added. The suspension is left for 24 hours at 8 - 10 ° C with slight stirring and then filtered. The gel is washed with 40 ml of buffer and the combined filtrates are placed on a 2.6 x 7.5 cm column of Q- "Sepha-rose" ® FF, which is pre-calibrated with 50 mM tris (hydroxymethyl) aminomethane, 50 % (by volume) of ethanol adjusted to pH 8.0 with HCl. Then, the column is eluted with a linear NaCl gradient from 0 to 0.15 M in the same buffer over 6 hours at a flow rate of 225 ml / hour. The eluate is detected for 20 UV absorbance and fractions containing. the protein head-top is joined. The protein is precipitated at pH 5.4 after removal of the ethanol in vacuo.

Freeze-drying isolates 200 mg (His®25), des- (TyrB26), des (ThrB3 °) -human insulin.

The identity of the product is confirmed by amino acid analysis and sequential Edman degradation of the separate vinyl pyridylated A and B chains.

Example 10

Preparation of (AsnB25), des (Tyr®26), des (Thr®30) -human insulin 30 mg (AsnB25), des (Thr®26) -SCI, prepared using the procedures described in Examples 4 and 5, dissolved in 15 ml of 50 mM tris (hydroxymethyl) aminomethane, 20% (by volume) ethanol adjusted to pH 9 with HCl, and 22

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Add 15 ml (sedimented volume) of "Sepharose" ® containing 12 mg of immobilized trypsin in the same "buffer. The suspension is left for 24 hours at 8-10 ° C with light stirring, then filtered. The gel is washed with 40 ml of buffer and the Five mixed filtrates are loaded onto a 1.6 x 10 cm column of Q- "Sepharose" ® FF, previously equilibrated with 50 mM tris- (hydroxymethyl) aminomethane, 50% (by volume) ethanol, adjusted to pH 8.0 Thereafter, the column is eluted with a linear NaCl gradient from 0 to 0.15 in the same buffer over 6 10 hours at a flow rate of 90 ml / hour. The eluate is detected for UV absorption and fractions containing the protein head peak The protein is precipitated at pH 5.4 after removal of the ethanol in vacuo.

Freeze drying gives 80 mg (AsnB25), des (TyrB26) -15 des (ThrOJU) human insulin.

The identity of the product is confirmed by amino acid analysis and sequential Edman degradation of the separate vinyl pyridylated A and B chains.

Example 11 20 Preparation of (AspA21), des (PheB25), des (ThrB30) -human insulin 50 mg of des (PheB2 5), des (ThrB30) -human insulin prepared by the procedures described in Example 6 is dissolved in 10 ml water by adjusting the pH of 2 with 1 M HCl. The solution is left for 16 days at 30 ° C. After cooling 25 (to 20 ° C), 7.5 g of urea is added and the pH is adjusted to 8.1 with 1 M NaOH. The solution is then added to a 1.6 x 20 cm Q "SepharoseM® FF column which is already equilibrated with 20 mM tris (hydroxymethyl) aminomethane / HCl, 7 M urea, pH 8.1, at 4 ° C. and eluting with a linear NaCl gradient in the same buffer increasing from 0 to 0.05 M over 24 hours at a flow rate of 40 ml / hr. The mixed fractions containing the protein from the last elution peak are desalted on a column of "Sephadex" ® G25 in 1 M acetic acid and freeze-dried.

30 mg (AspA21), des (PheB25), des (ThrB30) human insulin are obtained.

23

The identity of the product is confirmed by amino acid analysis, 5-step Edman degradation and c-terminal analysis using carboxypeptidase A.

Example 12 Preparation of (SerA21), des (ProB28) -human insulin

Construction of an expression plasmid that can be used to prepare (SerA21), des (Pro328) -human insulin and produce (Ser3 · 2 - * -), des (Pro328) -human insulin.

A plasmid derived from pUC-19, pKFN-864, encoding 10 for this analog, is produced by cleaved duplex mutagenesis (Y. Morinaga et al., Biotechnology 2 (1984), 636-639) of plasmid pKFN-734 using two the mutagenic primers, NOR-648 CTAGAGCCTGCGGGCTGCGTCTAGCTGCAGTA and NOR-745 ATTGTTC-GACAATACCCTTAGCAGCCTTGGTGTAGAAGAAACCTCTTTCACC. Plasmid 15 pKFN-734 is prepared by ligating the 0.4 kb Eco RI-XbaI fragment encoding a synthetic yeast signal leader fused internally to a synthetic insulin precursor gene, B (1-29) -Ala-Ala. Lys-A (1-21), from the plasmid pLaC212spx3 to the 2.7 kb EcoRI-Xbl fragment from pUC-2019 (c. Yannisch-Perron et al., Gene 33 (1985), 103-111) .

The plasmid pLaC212spx3 is described in Example 3 and in FIG. 6 and 13 of PCT Application No. WO 89/02463.

DNA sequence of the 0.4 kb EcoRI-XbaI fragment of pKFN-864 encoding signal leader insulin, B (1-29, des-25 Pro28) -Ala-Ala-Lys-A (1-21, Ser21 ) is shown in FIG. Third

pKFN-864 is cut with EcoRI and Xbal, and the 0.5 kb fragment is joined to the 9.5 kb NcoI-Xbal fragment from pMT636 and the 1.4 kb NcoI-EcoRI fragment from pMT636 to form the plasmid pKFN -866, cf. Figure 4. The plasmid pMT636 30 is produced from pMT608 after removal of the LEU-2 gene and from pMT479, cf. 4. pMT608 is described in EP 195,691. pMT479 is described in EP 163,529. pMT479 contains the Schizosaccharomyces pombe TPI gene (POT), the S. cerevisiae triose phosphatisomerase promoter and terminator, TPIp and TPIT 35 (Alber, T. and Kawasaki, G.J. Mol. Appl. Gen. 1 (1982), 419 -

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24 434) The plasmid pKFN-866 contains the following sequence: TPIp signal leader insulin B (1-29, des-Pro28) -Ala-Ala-Lys-A (1-21, Ser21) -TPIT.

S. cerevisiae strain MT663 (E2-7B XE11-36 a / α, AtpiA-5 tpi, pep 4-3 / pep 4-3) is grown on YPGal (1% Bacto yeast extract, 2% galactose, 1% lactate) to a OD of 0.6 at 600 nm.

100 ml of the resulting culture is harvested by centrifugation, washed with 10 ml of water, centrifuged and suspended in 10 ml of a solution containing 1.2 M sorbitol, 25 mM 10 Na2 EDTA, pH = 8.0, and 6.7 mg / ml dithiothreitol. The suspension is incubated at 30 ° C for 15 minutes and centrifuged, and the cells are suspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na 2 EDTA, 0.1 M sodium citrate, pH = 5.8, and 2 mg Novozym® 234. The suspension incubated at 30 ° C for 30 minutes and the cells collected by centrifugation, washed in 10 ml 1.2 M sorbitol and 10 ml CAS (1.2 M sorbitol, 10 mM CaCl 2, 10 mM Tris HCl) (Tris = Tris (hydroxymethyl) (aminomethane) pH = 7.5) and suspended in 2 ml of CAS. For transformation, 0.1 ml of CAS resuspended cells are mixed with ca. 1 µg of plasmid pKFN-866 and 20 are left at room temperature for approx. 15 minutes. L ml (20% polyethylene glycol 4000, 10 mM CaCl2 / io mM Tris HCl, pH = 7.5) is added and the mixture is allowed to stand for an additional 30 minutes at room temperature. The mixture is centrifuged and the pellet is suspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v / v YPD, 6.7 mM 25 CaCl 2, 14 μg / ml leucine) and incubated at 30 ° C for 2 hours. The suspension is then centrifuged, the pellet suspended in 0.5 ml of 1.2 M sorbitol. 6 ml of top agar (SC medium according to Shennan et al. (Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) containing 1.2 M sorbitol plus 2.5% agar) are added at 52 ° C and the suspension is poured on top of plates containing the same agar-dried medium containing sorbitol. Transformant colonies are collected after 3 days at 30 ° C, isolated and used to start liquid cultures. Such a transformant KFN-883 is selected for further characterization.

The yeast strain KFN-883 is grown on YPD medium (1% yeast extract, 2% peptone (from Difco Laboratories) and "2% glucose). 10 ml culture of the strain is shaken at 30 ° C to an OD of 20 at 600 nm. centrifugation, the supernatant is analyzed by HPLC as described (L. Snel et al., Chromatographia 24 (1987.), 329-332). A yield of about 0.14 mg / l insulin B (1-29, des-Pro28) is obtained. ) -Ala-Ala-Lys-A (1-21, Ser2 ^ ·).

The single-stranded insulin precursor is isolated from the fermentation supernatant by adsorption to an ion-exchange column at 10 low pH, desorption at high pH, and precipitation of the collected with zinc ions. Transpeptidation of the precursor to (SerA21), des (ProB2S), (ThrB30-OMe) human insulin is as follows: 10 mmol (2.35 g) of threonine methyl ester and ice-cold acetic acid are dissolved in DMF to 5 ml, 2.5 ml 76, Add 5% v / v DMF in water and dissolve 0.5 g of precursor in the mixture, which is kept at 12 ° C. Then 50 mg of trypsin is added in 1.25 ml of 0.05 M calcium acetate and after 24 hours at 12 ° C the reaction mixture is added to 100 ml of acetone to precipitate the 20 peptides which are swirled and dried in vacuo.

The isolated insulin analog ester is purified on a preparative HPLC column using a silica C18 matrix at acidic pH. The purified ester is hydrolyzed in an aqueous medium at a pH of 10 and 25 ° C for 24 hours. The resulting 25 (SerA21) des (ProB28) human insulin is precipitated at neutral pH with zinc ions. The precipitate is purified by anion exchange chromatography and then desalted by gel filtration. A freeze-dried (Ser ^ 21) des (ProB28) human insulin yield of 102 mg is obtained.

Example 13 Preparation of Des (ThrB27) Human Insulin 1 g of zinc-free porcine insulin is dissolved in 40 ml of water by adjusting the pH to 9 and a solution of 50 mg of pig trypsin in 10 ml of 0.25 M ammonium hydrogen carbonate adjusted to a pH of 9 with ammonium solution is added.

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26

The solution is then left at 48 ° C and after 48 hours a 65% yield is obtained by HPLC analysis. The reaction mixture is gel-filtered at 4 ° C on a 5 x 90 cm column of Sephadex® G50 superfine in 0.05 M ammonium hydrogen carbonate at a flow 5 of 90 ml per minute. hour. Fractions containing the protein head are pooled and lyophilized. A yield of 520 mg of des (B23-B30) -human insulin is obtained.

A peptide of the sequence Gly-Phe-Phe-Tyr-Pro-Lys-Thr is synthesized on a PAM resin using protective, symmetric amino acid anhydrides by a peptide synthesizer from Applied Biosystems. Then, the peptide is cleaved from the resin by anhydrous hydrogen fluoride at 0 ° C and the remaining protecting groups are similarly removed.

15 200 mg of des (B23-B30) human insulin and 400 mg of peptide are dissolved in a mixture of 2.40 ml of dimethylformamide and 1.20 ml of water and the pH of the mixture is adjusted to 6.5 with triethylamine. Then 10 mg of pig trypsin is added in 0.20 ml of water and the reaction mixture is left at 20 ° C for 4 hours. The reaction 20 is then stopped by the addition of 25 ml of 2-propanol and the precipitated proteins are isolated by centrifugation. The drained precipitate is dissolved in 10 ml of 1 M acetic acid and placed on a 2.6 x 20 cm column of Lichroprep® RP-18 (25 - 40 μιιι) previously equilibrated with 0.5 mM hydrochloric acid, 0.1 M sodium chloride in 30% (by volume) ethanol. The column is then eluted at 20 ° C at a flow rate of 20 ml. hour with the same buffer, but with a linear increase in ethanol content to 50% over 24 hours. The eluate is monitored for UV absorption and fractions containing the protein head are collected. The protein was precipitated by dilution with the same volume as water and adjusting the pH to 5.5 with sodium hydroxide solution, and after standing at 4 ° C for 1 hour, the precipitate was isolated by centrifugation and freeze-drying.

A yield of 80 mg of des (ThrB27) -human insulin is obtained, which is identified by sequential Edman degradation of the separate vinyl pyridylated A and B chains.

27

Example 14

Formulation of injection solution 60 μιηοΐ of a human insulin analogue of the invention is dissolved in 4 ml of 0.1 M HCl and 20 ml of 1.5% m-5 cresol is added. The solution is then mixed with 40 ml of 4% glycerol and 20 ml of 65 mM disodium hydrogen phosphate and the pH is adjusted to 7.3. Finally, adjust the solution to 100 ml with water and then sterilize it.

Example 15 io Assessment of association degree

A 2.6 x 88 cm column of "Sephadex" ® G-75 is equilibrated with 13 mM sodium phosphate buffer pH 7.3 at a flow rate of 22 ml / hour. Using des (octapeptide B23-30) human insulin, cytochrome C, ribonuclease, and mono- and dimer myoglobin as 15 molecular weight markers, a curve is recorded indicating the molecular weight as a function of the elution volume.

Using 1 ml of solution containing 0.6 mM zing-free human insulin or 0.6 mM insulin analog prepared as described in Example 12, it is found that zinc-free human insulin is eluted as a so-called "tailing" peak with an apparent molecular weight of approx. . 14 kD and that the analogs prepared as described in Examples 6-10 are all eluted as a symmetrical peak with an apparent molecular weight of approx. 5 kD.

These results show that the human insulin analogues of the invention are largely monomeric in solution at pH 7.3, whereas the normal human insulin under the same conditions is extensively present as a mixture of dimers and higher oligomers.

Example 16 30 Assessment of biological effect

The in vitro biological effect is determined by measuring the binding affinity of the insulin receptors on isolated ones

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28 rat adipocytes and rat hepatocytes largely as described in J. Gliemann, S. Gammeltoft: Diabetologia ITT (1974), 105 - 113.

The insulin analogues are compared to semi-synthetic human insulin, whose potency is set to 100% and the results are given in the table below:

Table

Adipocytes Hepatocytes des (PheB25), des (ThrB3 0) - io human insulin 223% 201% des (PheB25) -human insulin 225% 249% (AspA21), des (PheB25) des (ThrB3 °) -human insulin 250% 242%

Claims (19)

1. Human insulin analogues, characterized in that they have the following formula: -A-chain S --- S 5 I 7 'Τ' H-Gly-Ile-Val-Glu-Gln-Cys-Cys ~ Thr-Ser-Ile-Cys-Ser-1 2 3 4 5 6 I 8 9 10 11 12 S 10 B-chain S H-Phe-Val-Y2-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val ~ 123456789 10 11 12 A-chain (continued) »15 13 14 15 16 17 18 19 20 21 -Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Y1-OH s 20 I B-chain (continued) S -Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-13 14 15 16 17 18 19 20 21 22 23 24 B-chain (continued) -X1-X2-X3-X4-X5-X6 25 26 27 28 29 30 wherein X1, X2, X3, X5, Y1 and Y2 are any naturally occurring amino acid residues, with the exception of X5 is Pro, X4 is Lys or Arg, and Xs is any naturally occurring amino acid residue bearing the c-terminal hydroxy group, or -OH, or X1 and X2 together form the C-terminal hydroxy group, or a pharmaceutically tolerable salt thereof. 52. · - Human insulin analogues according to claim 1, characterized by, 1 Λ - ·> -. that Y and / or Y is selected from the group consisting of any naturally occurring amino acid residue except Asn. Human insulin analogues according to claim 1, characterized in that X1 is selected from the group consisting of Phe, Ala, His, Thr, Ser, Asn and Tyr, X2 is selected from the group consisting of Tyr, Thr, Glu, Asp, Ala, His, Ser and Phe, X3 are selected from the group consisting of Pro, Glu, Asp, Ser, Thr and His; X1 is selected from the group consisting of Lys, Thr, Ser, Ala, Asp and Glu, X2 are selected from the group consisting of Thr-OH, Ser-OH, Ala-OH, Asp-OH, Glu-OH and -OH, or X1 and X2 together form the C-terminal hydroxy group, Y1 is selected from the group consisting of Asn, Asp, Gly, Ser, Glu and Ala, and Y2 is selected from the group consisting of Asn, Gln, Glu and Asp. Human insulin analogues according to claim 1, characterized in that η is that X is selected from the group consisting of Phe, Ala, His, Thr, Ser, Asn and Tyr, X2 is selected from the group consisting of Tyr, Thr, Glu, Asp,. Ala, His, Ser and Phe, X3 are selected from the group consisting of Pro, Glu, Asp, Ser, Thr and His, X1 is selected from the group consisting of Lys, Thr, Ser, Ala, Asp and Glu, X2 is -OH, Y1 is selected from the group consisting of Asn, Asp, Gly, Ser, Glu and Ala, and Y2 is selected from the group consisting of Asn, Gin, Glu and Asp. Human insulin analogues according to claim 1, characterized in that X1 is selected from the group consisting of Phe, Ala, His, Thr, Ser, Asn and Tyr, X2 is selected from the group consisting of Tyr, Thr, Glu, Asp, Ala, His , Ser and Phe, X3 are selected from the group consisting of Pro, Glu, Asp, Ser, Thr and His, X5 and X6 together form the C-terminal hydroxy group, Y1 is selected from the group consisting of Asn, Asp, Gly, Glu, Ser and Ala, and Y2 is selected from the group consisting of Asn, 5 Gln, Glu and Asp. Human insulin analogues according to claim 1, characterized in that X 1 is Phe, X 2 is Tyr, X 3 is Thr, X 2 is Lys, X 5 is Thr-OH, Y 1 is selected from the group consisting of Asn, Asp, Ser and Gly, and Y2 is selected from the group consisting of Asn, Gin, ίο Asp and Glu. Human insulin analogues according to claim 1, characterized in that X1 is Tyr, X2 is Thr, X3 is Pro, X5 is Thr, X6 is -OH, Y1 is selected from the group consisting of Asn, Asp, Ser and Gly, and Y2 is selected from the group consisting of Asn, Gin, Asp and Glu. Human insulin analogues according to claim 1, characterized in that X 1 is Phe, X 2 is Tyr, X 3 is Thr, X 3 is Lys, X 6 is Thr-OH, Y 2 is selected from the group consisting of Asn, Asp, Ser and Gly. and Y2 is selected from the group consisting of Asn, Gin, Asp and Glu. Human insulin analogues according to claim 1, characterized in that X1 is Tyr, X2 is Thr, X3 is Pro, X5 is Thr, X6 is -OH, Y1 is selected from the group consisting of Asn, Asp, Ser and Gly, and Y2 is selected from the group consisting of Asn, Gin, Asp and 25 GlU. Pharmaceutical composition, characterized by containing a human insulin analogue of the following formula: 16 chain S-S I 7 - | H-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-1 2 3 4 5 6 I 8 9 10 11 12 5 S ...... In B-chain S H-Phe-Val-Y2-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-10 1 2 3 4 5 6 7 8 9 10 11 12 A-chain (continued) 13 14 15 16 17 18 19 20 21 -Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Y1-OH 15 | S B-chain (continued) S 20 -Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- 13 14 15 16 17 18 19 20 21 22. 23 24 B-chain (continued) -X1-X2- X3-X4-X5-X6 25 26 27 28 29 30 25 wherein X1, X2, X3, x5, Y1 and Y2 are any naturally occurring amino acid residues, however X5 is not Pro, X4 is Lys or Arg, and X6 is any naturally occurring amino acid residue carrying the C-terminal hydroxy group, or -OH, or X5 and X6 together form the C-30 terminal hydroxy group, or a pharmaceutically tolerable salt thereof and a pharmaceutically tolerable carrier.