HK1036808B - Novel insulin analogs with enhanced zinc binding - Google Patents

Description

novel insulin analogs with enhanced zinc binding

The present invention relates to insulin analogues with enhanced zinc binding capacity and stable zinc complexes thereof, which have delayed action compared to human insulin, and to processes for their preparation and use, especially as pharmaceutical formulations, for the treatment of type I and type II diabetes.

About 1 million to 2 million people worldwide have diabetes, of which about 1 million to 2 million are type I diabetes, for which replacement of the missing endocrine insulin secretion is the only currently available therapy. Patients need to inject insulin, usually several times a day for a lifetime. Unlike type I diabetes, type II diabetes is essentially devoid of insulin, but treatment with insulin in a large number of cases, especially in advanced cases, and appropriate combination with an oral antidiabetic agent is considered to be the optimal treatment regimen.

In healthy people, insulin release by the pancreas is closely related to blood glucose concentration. An increase in blood glucose levels, such as after a meal, is quickly compensated by a corresponding increase in the amount of insulin secreted. Fasting, plasma insulin levels fall back to basal values, sufficient to ensure a continuous glucose supply to insulin-sensitive tissues and organs and to maintain low nocturnal hepatic glucose production. The quality of physiological regulation of blood glucose, as described above, is hardly achieved by replacing endogenous insulin secretion with exogenous insulin, usually by subcutaneous administration. Often, control of blood glucose up or down is lost, which in very severe cases may be life threatening. In addition, the rise in blood glucose levels over the years without the original symptoms is also a considerable risk to health. In the United states, a number of DCCT studies (diabetes control and complications test research group (1993), New England journal of medicine 329, 977-. Late-stage damage in diabetes is damage to the tiny and large blood vessels, which can manifest as retinopathy, nephropathy, or neuropathy under certain circumstances, leading to blindness, renal failure, and loss of extremities, often with a high risk of cardiovascular disease. Therefore, a sophisticated diabetes treatment regimen must first aim to keep blood glucose levels as close to physiological ranges as possible. This is achieved by daily large injections of fast and slow acting insulin preparations, according to the concept of intensive insulin therapy. Fast acting preparations are administered at meals to balance the postprandial rise in blood glucose, while slow acting basal insulin should ensure a basic supply of insulin, especially at night, without causing hypoglycemia.

The currently available basal insulins are not sufficient to meet the above requirements, especially the commonly used NPH insulins, which have a maximum effect that is too strong and a short global effect. When administered at night, there is a risk of nocturnal hypoglycemia and early morning hyperglycemia.

EP 0821006 discloses insulin analogues with enhanced zinc binding, which after binding to zinc has a delayed-action form compared to human insulin. Basically these analogues differ from human insulin by amino acid variation at position A21 of the A chain and by the addition of a His residue or a peptide of 2-35 amino acid residues in length containing 1-5 His residues at position B30 of the B chain.

It is an object of the present invention to further provide insulin analogues (analogues of human or animal insulin) which have an enhanced zinc binding capacity, are capable of forming a stable complex comprising insulin analogue hexamers and zinc, and which are used in suitable formulations to make it possible to improve the treatment of type I and type II diabetes by subcutaneous injection due to their delayed action profile compared to human insulin.

Insulin analogs are derived from naturally occurring insulins, i.e., human insulin (see SEQ ID NO: 1: human insulin A chain and SEQ ID NO: 2: human insulin B chain) or animal insulins, obtained by substituting or deleting at least one natural amino acid residue and/or adding at least one amino acid residue in the A chain and/or the B chain of natural insulin.

The object of the present invention is achieved by

1. An insulin analogue of formula I or a physiologically tolerable salt thereof

Wherein

(A1-A5) is human insulin (see SEQ ID NO: 1) or the amino acid residues at the A1-A5 positions of an animal insulin A chain,

(A15-A19) is human insulin (see SEQ ID NO: 1) or the amino acid residues at the A15-A19 positions of an animal insulin A chain,

(B8-B18) is human insulin (see SEQ ID NO: 2) or the amino acid residues at positions B8-B18 of the B chain of animal insulin,

(B20-B29) is human insulin (see SEQ ID NO: 2) or the amino acid residues at positions B20-B29 of the B chain of animal insulin,

r8 is Thr or Ala,

r9 is Ser or Gly,

r10 is Ile or Val,

r14 is Tyr, His, Asp or Glu,

r21 is Asn, Asp, Gly, Ser, Thr, Ala, Glu or Gln,

r1 is any desirable genetically encodable amino acid residue, deletion, or hydrogen atom,

r2 is Val, Ala or Gly,

r3 is Asn, His, Glu or Asp,

r4 is Ala, Ser, Thr, Asn, Asp, Gln, Gly or Glu,

r30 is any desirable genetically encodable amino acid residue or-OH,

z is a hydrogen atom or a peptide containing 1 to 4 genetically encodable amino acid residues, including 1 to 4 His residues,

with the proviso that when Z is a hydrogen atom, R1 or R3 is His, Glu or Asp, R3 is His if R1 is a neutral or negatively charged amino acid residue, or with the proviso that if wherein Z is a hydrogen atom, R14 is His, Asp or Glu, furthermore with the proviso that the insulin analogue of formula I or a physiologically tolerable salt thereof differs from human insulin not only in the position R3, or the positions R3 and R21, or the positions R3 and R4 in the formula I in respect of amino acid residues (cf. SEQ ID NO: 1 and SEQ ID NO: 2).

Preferably, the insulin analogue or a physiologically tolerable salt thereof is one in which

R8 is Thr, R9 is Ser and R10 is Ile,

r1 is Phe, His, Asn, Asp or Gly,

r30 is Thr, Ala or Ser, or

5. Wherein R21 is Asn and R1 is Phe.

6. A preferred embodiment of the invention is an insulin analogue of formula I wherein R2 is Val, R3 is Asn and R4 is gin, or a physiologically tolerable salt thereof.

An insulin analogue of the formula I or a physiologically tolerable salt thereof, with the difference that R14 is

7.Tyr，

8.His，

Asp or

10.Glu。

An insulin analogue of the formula I or a physiologically tolerable salt thereof, with the difference that R30 is

11.Thr，

12.Ala，

13, Ser or

14.-OH。

An insulin analogue of the formula I or a physiologically tolerable salt thereof, with the difference that Z is

15.His，

His-Ala-or

17.His-Ala-Ala-。

An example of an insulin analogue according to the invention is

18. An insulin analogue of formula I or a physiologically tolerable salt thereof, differing in that the B-chain has a sequence

His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

(SEQ ID NO: 3), such as His (B0), des (B30) human insulin,

19. an insulin analogue of formula I or a physiologically tolerable salt thereof, differing in that the B-chain has a sequence

His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

(SEQ ID NO: 4), such as His (B0) -human insulin,

20. an insulin analogue of formula I or a physiologically tolerable salt thereof, differing in that the B-chain has a sequence

His Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu

Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

(SEQ ID NO: 5), e.g. His (B-1), Ala (B0) human insulin or

21. An insulin analogue of formula I or a physiologically tolerable salt thereof, differing in that the B-chain has a sequence

His Ala Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala

Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

(SEQ ID NO: 6), such as His (B-2), Ala (B-1), Ala (B0) -human insulin.

The invention also relates to a process for the preparation of an insulin analogue according to the invention or a physiologically tolerable salt thereof, which comprises constructing a replicable expression vector which contains a DNA sequence coding for an insulin analogue precursor having the amino acid sequence II:

Met-X² _m-(Arg)_p-Z-R1-R2-R3-R4-His-Leu-Cys-(B8-B18)-Cys-(B20-B29)-R30-X¹ _n-Arg-(A1-A5)-Cys-Cys-R8-Rg-R10-Cys-Ser-Leu-R14-(A15-A19)-Cys-R21II，

wherein

X¹n is a peptide chain containing n amino acid residues, n is an integer of 0 to 34,

X²m is a peptide chain containing m amino acid residues, m is an integer of 0 to 20,

p is 0, 1 or 2,

r30 is any desired genetically encodable amino acid residue, or is deleted and

z is deleted or is a peptide having 1-4 genetically encodable amino acid residues comprising 1-4 His residues,

the other variables have the meanings indicated above for No.1, the above-mentioned provisos still apply,

expression in a host cell and release of the insulin analogue from its precursor using chemical and/or enzymatic methods.

The host cell is preferably a bacterium, particularly preferably E.coli.

The host cell is preferably a yeast, particularly preferably Saccharomyces cerevisiae.

When expressed in E.coli, the fusion protein (SEQ ID NO: 7-9) typically forms insoluble inclusion bodies, which can be centrifuged after cell disruption and reconstituted with a denaturing agent (e.g., 8M urea or 6M guanidine hydrochloride). The solubilized fusion protein can be subjected to sulfitolysis to convert the Sulfhydryl (SH) group to an S-sulfonate (e.g., R.C. Marshall and A.S. Iglis, handbook of protein chemistry, A.Darbre eds. (1986), pages 49-53) to improve the solubility of the fusion protein for easy purification, such as by anion exchange or gel permeation chromatography.

Conversion of the derivatized fusion protein to preproinsulin (preproinsulin) with a native spatial structure and correctly formed disulfide bridges (folds) is carried out in a diluted aqueous solution, with addition of an amount of a sulfhydryl reagent, such as mercaptoethanol, Cys or glutathione, followed by air oxidation. As an alternative, the undissolved, underivatized fusion protein can also be folded directly under analogous conditions (EP-A-0600372, EP-A-0668292).

The preproinsulin is then converted to biologically active insulin by limited proteolytic cleavage. For this purpose, it is possible to use trypsin to substitute the precursor sequence Met-X of formula II²m- (Arg) p is removed at X¹The peptide chain shown by n-Arg is broken, thereby separating the B chain and the A chain. Generally, the sequence X¹Starting from Arg, Arg₂Or not present (n ═ 0), so that the insulin derivative obtained after cleavage is Arg or Arg at the C-terminus of the B chain₂These amino acids can be removed by carboxypeptidase B. Trypsin cleavage can also be carried out with increasing trypsin concentration or with increasing duration of action, allowing cleavage again at Lys (B29), in which case a des (B30) insulin derivative is produced.

The insulin analogue formed during the lysis can be purified by standard chromatographic procedures (e.g. ion exchange and reverse phase chromatography) and finally isolated by precipitation, crystallization or simple lyophilization.

Preferably, the insulin analogue precursor contains a sequence

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg

His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val

Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly

Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr

Cys Asn

(SEQ ID NO: 7), e.g. His (B0) -preproinsulin sequence, or sequence

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg

His Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu

Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly

Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr

Cys Asn

(SEQ ID NO: 8), e.g. His (B-1), Ala (B0) -preproinsulin sequence, or

Sequence of

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg

His Ala Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly

Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys

Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

(SEQ ID NO: 9), such as His (B-2), Ala (B-1), Ala (B0) -preproinsulin sequence.

The invention also relates to precursors of the insulin analogues of the invention described above, in particular preproinsulin, and to DNA sequences encoding the insulin analogue precursors of the invention, expression vectors containing DNA sequences encoding the novel insulin analogue precursors of the invention, and host cells transformed with such expression vectors.

According to the invention, the invention also relates to a pharmaceutical formulation comprising at least one insulin analogue and/or at least one physiologically tolerable salt.

Preferably, the pharmaceutical formulation is distinguished in that it comprises the insulin analogue of the invention in dissolved, amorphous and/or crystalline form and/or a physiologically tolerable salt thereof.

The pharmaceutical formulation may additionally comprise a storage aid, preferably protamine sulfate, wherein the insulin analogue and/or a physiologically tolerable salt thereof is preferably present in the form of a co-crystal with protamine sulfate.

The pharmaceutical preparations according to the invention may additionally contain unmodified human insulin and/or other insulin analogs, preferably Gly (A21) -Arg (B31) -Arg (B32) -human insulin.

According to the invention, the invention also relates to an injection solution with insulin activity, in which the pharmaceutical preparation according to the invention is included in dissolved form, preferably in a quantity of 1. mu.g to 2mg zinc/ml, particularly preferably in a quantity of 5. mu.g to 200. mu.g zinc/ml.

The invention also relates to the use of an insulin analogue according to the invention and/or a physiologically tolerable salt thereof for the preparation of a pharmaceutical preparation having a delayed onset of action of the insulin activity.

The objects listed at the outset are also achieved by an insulin-zinc complex comprising an insulin hexamer and 4 to 10 zinc ions per insulin hexamer, wherein the insulin hexamer consists of insulin analogs of the six formula I

Wherein the content of the first and second substances,

(A1-A5) is the amino acid residue at A1-A5 site of human insulin or animal insulin A chain,

(A15-A19) is the amino acid residue at A15-A19 site of human insulin or animal insulin A chain,

(B8-B18) is the amino acid residue at the B8-B18 site of human insulin or animal insulin B chain,

(B20-B29) is the amino acid residue at the B20-B29 site of human insulin or animal insulin B chain,

r8 is Thr or Ala,

r9 is Ser or Gly,

r10 is Ile or Val,

r14 is Tyr, His, Asp or Glu,

r21 is Asn, Asp, Gly, Ser, Thr, Ala, Glu or Gln,

r1 is any desirable genetically encodable amino acid residue, deleted or hydrogen atom,

r2 is Val, Ala or Gly,

r3 is Asn, His, Glu or Asp,

r4 is Ala, Ser, Thr, Asn, Asp, Gln, Gly or Glu,

r30 is any desirable genetically encodable amino acid residue or-OH,

z is a hydrogen atom or a peptide containing 1 to 4 genetically encodable amino acid residues, including 1 to 4 His residues,

preferably, the insulin-zinc complex comprises 5-8 zinc ions per insulin hexamer.

Preferably, the insulin-zinc complex contains an insulin hexamer consisting of 6 molecules of the insulin analogue of formula I above of the present invention.

According to the invention, preferably, the insulin-zinc complex can also be one in which the insulin analogue B-chain of formula I contains a sequence

Phe Val His Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys

Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

(SEQ ID NO: 10), e.g. His (B3) -human insulin, or wherein the B-chain of an insulin analogue of formula I contains the sequence

Phe Val Asp Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys

Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

(SEQ ID NO: 11), for example Asp (B3) -human insulin.

The invention also relates to a pharmaceutical formulation comprising at least one insulin-zinc complex according to the invention, and also to a pharmaceutical formulation comprising an acidic solution of at least one insulin analogue and/or a physiologically tolerable salt thereof, which zinc makes it possible to form an insulin-zinc complex according to the invention, which insulin analogue and/or a physiologically tolerable salt thereof preferably comprises an insulin analogue according to formula I described above according to the invention, or a B-chain having the numbering SEQ id no: 3, 4, 5, 10 or 11 sequence of an insulin analogue of formula I.

Preferably, the pharmaceutical formulation is a formulation comprising insulin-zinc complex in dissolved, amorphous and/or crystalline form.

The invention also relates to an injectable solution with insulin activity, containing a pharmaceutical preparation in dissolved form, preferably containing 1. mu.g to 2mg of zinc per ml, particularly preferably containing 5. mu.g to 200. mu.g of zinc per ml.

The invention also relates to the use of insulin-zinc complexes for the preparation of pharmaceutical preparations with delayed onset of insulin activity.

The insulin analogues according to the invention have biological activity,dogs were injected subcutaneously weekly with 80 μ g Zn⁺⁺The acid clear solution of/ml (zinc/ml) showed a strong retardation. If the N-terminus of the B-chain of an insulin analogue is extended by the presence of His, for example His (B0), des (B30) -human insulin (see SEQ ID NO: 3), the mode of action is significantly dependent on the amount of added zinc ions. The zinc-free formulation had no storage effect at all (total effect 6-8 hours, example 8), the pharmacodynamics were almost indistinguishable from human insulin, and when zinc ions (80. mu.g/ml) were added, the delayed effect was found to be strong (total effect about 16 hours, example 8), and the storage effect observed was much more pronounced than that of NPH-insulin. In addition, the analogues have the advantage that the zinc concentration can be preset within a certain range to control its pharmacodynamics, which is not possible with human insulin. Formulations with a rapid onset of action can be prepared with the active substance by varying the zinc concentration, as can those with a moderate or strong delayed action. Thus, the mode of action can be adapted to the needs of the patient, respectively, or by using a preparation with a suitable predetermined zinc concentration, or by the physician or the patient himself or herself, by mixing the preparations with a high zinc concentration and a low zinc concentration.

The analogs described herein also include analogs that have enhanced affinity for zinc ions as compared to human insulin.

In neutral aqueous solution, human insulin complexes two zinc ions via His (B10) side chains to form a hexamer, and these zinc ions cannot be removed by dialysis in neutral solution. Under the same conditions, the analogs described herein can bind more than 4 zinc ions. According to the invention, in His (B0) -des (B30) -and His (B3) -insulin, there are about 7 zinc ions per hexamer, and in Asp (B3) -insulin, 4.2 zinc ions per hexamer were measured (example 9).

It is well known that zinc in neutral solution leads to the formation of relatively high molecular weight conjugates and to the precipitation of insulin. After injection of a weakly acidic zinc-containing formulation of insulin dissolved in a clear solution, the formation of insulin-zinc complexes leads to precipitation of insulin in the subcutaneous tissue due to neutralization. Insulin re-dissolves from the pellet, passes through the blood vessel, and reaches the site of action with a delay. This delay is only weak for human insulin, but the analogs described herein have enhanced zinc affinity and thus their delay of action is significantly enhanced. Thus, enhanced zinc binding is the basis for the prolongation of the above-mentioned zinc-dependent effect.

The invention therefore relates not only to the insulin analogues described, but also to the insulin-zinc complexes concerned. This complex differs from the corresponding human insulin-zinc complex in that the former contains a high concentration of stably bound zinc. It is clear that, in addition to zinc, other transition metal ions, such as cobalt or copper, can also be used to form the corresponding complexes.

Example 1: a plasmid was constructed encoding a His (B3) -preproinsulin mutant, pINT345 d.

Us patent No. 5358857 describes the plasmid pINT90d, which is used as the starting material for the construction of the plasmid pINT345 d. pINT345d is characterized by two new properties compared to pINT90 d. In one aspect, it encodes a preproinsulin analog which contains a His instead of an Asp at position 3 of the B chain. On the other hand, the recognition sequence of restriction enzyme BssH2 immediately before the start of the preproinsulin mutant coding sequence allows for easy manipulation of the N-terminal 10 amino acid sequence encoding the preproinsulin analog, if the Dra3 cleavage site in the preproinsulin sequence is considered. To construct the plasmid pINT345d, the DNA of the plasmid pINT90d was double digested at 284bp and 351bp by restriction enzymes NcoI and Dra3, resulting in two fragments. Separating the enzyme digestion mixture by gel electrophoresis, and separating the plasmid DNA large fragment.

Then the DNA fragment and the synthetic DNA fragment in the following form are subjected to T4 ligase action,

1/2Ncol BssH2 B1 B2 His B4 B5 B6

5′-C ATG GCA ACA ACA TCA ACA GGA AAT TCG GCG CGC TTT GTG CAC CAG CAC CTG

3′- CGT TGT TGT AGT TGT CCT TTA AGC CGC GCG AAA CAC GTG GTC GTG GAC

B7 B8 B9 1/2 Dra3

TGC GGC TCC CAC CTA-3′

ACG CCG AGG GTG -5′

the ligation mixture was transformed into E.coli K12 competent cells. The transformation mixture was plated on NA plates containing 20mg/ml ampicillin and incubated overnight at 37 ℃. Plasmid DNA was extracted from the emerging clones and digested with the restriction enzyme BssH2, the desired plasmid DNA was cut linearly, unlike pINT9Od plasmid DNA, which cannot be digested because it does not contain a BssH2 cleavage site.

The correct plasmid DNA was digested and called pINT345 d.

It was used to construct the starting material for the preproinsulin mutants described below.

Example 2: construction of plasmid pINT342d encoding a His (B0) -preproinsulin mutant

pINT345d plasmid DNA was digested simultaneously with enzymes BssH2 and Dra3, the large plasmid fragment was separated by gel electrophoresis, and the fragment was reacted with a synthetic DNA fragment of the form described below with T4 ligase,

His B1B2B3B4B5B6B7B8B9B10

5′-CG CGC CAC TTT GTT AAC CAG CAC CTG TGC GGC TCC CAC CTA -3′

3′- G GTG AAA CAA TTG GTC GTG GAC ACG CCG AGG GTG -5′

1/2 BssH2 Hpal 1/2 Dra3

the resulting pINT342d plasmid contained an additional HpaI cleavage site compared to the starting plasmid, which encoded the preproinsulin mutant with His at position B0.

Example 3: construction of a plasmid pINT343d encoding a His (B-1), Ala (B0) -preproinsulin mutant

The remaining plasmid DNA fragment described in example b was ligated with a DNA in the form of

His Ala B1B2B3B4B5B6B7B8B9B10B11

5′-CG CGC CAC GCT TTT GTT AAC CAG CAC CTG TGC GGC TCC CAC CTA-3′

3′- G GTG CGA AAA CAA TTG GTC GTG GAC ACG CCG AGG GTG -5′

1/2 BssH2 Hpal 1/2 Dra3

The resulting DNA fragment of (a) was subjected to ligase action of T4 to form plasmid pINT343d which, like pINT342d, also contained an additional HpaI cleavage site compared to the starting vector.

Example 4: construction of a plasmid pINT344d encoding a His (B-2), Ala (B-1), Ala (B0) -preproinsulin mutant

The remaining plasmid DNA fragment described in example b was ligated with a DNA in the form of

His Ala Ala B1B2B3B4B5B6B7B8B9B10B11

5′-CG CGC CAC GCT GCT TTT GTT AAC CAG CAC CTG TGC GGC TCC CAC CTA-3′

3′- G GTG CGA CGA AAA CAA TTG GTC GTG GAC ACG CCG AGG GTG -5′

1/2 BssH2 Hpal 1/2 Dra3

The resulting DNA fragment of (a) was subjected to ligase action of T4 to form the plasmid pINT344d, which also contained an additional HpaI cleavage site compared to the starting vector.

Example 5: expression of the constructed insulin mutants

For example, plasmids pINT342d, 343d and 344d were transformed into E.coli K12W3110, respectively. According to example 4 of U.S. Pat. No. 5227293, recombinant bacteria containing the corresponding mutant plasmids were fermented to obtain the raw materials required for the preparation of the corresponding insulin mutants.

Example 6: preparation of His (B0), des (B30) -insulin

The preproinsulin mutant was expressed in E.coli according to example 5, disrupted, centrifuged and isolated as inclusion bodies. The inclusion bodies were solubilized in urea (8mol/l) and purified by sulfitolysis, anion exchange (Q-Sepharose) and gel permeation chromatography (Sephacryl S200). The buffer for chromatography contained 4M urea and 50mM Tris/HCl (Tris/HCl) pH 8.5. The fractional elution on the anion exchanger is carried out by gradient with 0-0.5M NaCl. The pre-proinsulin-S-sulfonate is then isolated by precipitation at pH4 by reducing the urea concentration to below 1M by ultrafiltration and dilution, and finally dried.

To form the correct disulfide bridges as in native proinsulin, preproinsulin-S-sulfonate was dissolved at a concentration of 0.3g/l in a buffer containing 20mM glycine at pH10.8, stirred overnight at 4 ℃ under the action of mercaptoethanol (approximately 25-50mol/mol preproinsulin), then adjusted to pH3.5 with phosphoric acid and centrifuged. Tris (25mM) was added to the supernatant to adjust the pH to 8.2, and the preproinsulin was converted to insulin and then treated with trypsin (1.5mg/g preproinsulin). The progress of the proteolysis was monitored by reverse phase HPLC. After about 6 hours, the product contained high concentrations of His (B0), des (B30) -insulin, and the action ended after acidification to ph 3.5. Insulin analogues were purified by ion exchange chromatography (S-Hyper-D, Sepracor) and reverse phase chromatography (PLRP-S RP300, Polymer Laboratories). Ion exchange chromatography was performed in a buffer containing 30% 2-propanol and 50mM lactic acid (pH 3.5). Bound insulin was eluted with a linear gradient of 0-0.5M NaCl. Reverse phase chromatography was performed in 0.1% trifluoroacetic acid with elution increasing the amount of acetonitrile incorporated. The product was isolated by precipitation at pH5.4 and lyophilized.

Example 7: insulin analog formulations for parenteral use

The formulation contains 40 or 100IU of insulin (1IU corresponding to about 6.2nmol), 20mg of 85% glycerol, 2.7mg of m-cresol and, if appropriate, Zn + + (zinc chloride) per ml of sterile aqueous solution at pH 4.

Example 8: action forms of His (B0), des (B30) -insulin analogues in dogs

6 dogs (beagle dogs) were injected subcutaneously with 40U/ml (example 7) of the formulation with the indicated amount of zinc at a dose of 0.3 IU/kg. During subsequent experiments, blood glucose concentrations were measured at the indicated times. The measured values are normalized in percentage to the respective starting values and averaged.

Example 9: zinc binding of insulin analogs

Insulin analog formulation (0.3mM insulin analog, 0.13mM NaCl, 0.1% phenol, 100)μg/ml Zn⁺⁺(Zinc chloride), 25mM tris/HCl, pH7.4) was extensively dialyzed against neutral buffer containing no zinc (0.15M NaCl, 10mM tris/HCl pH7.4 for 3 hours at room temperature, 10mM tris/HCl pH7.4 for 72 hours at 15 ℃ and 10mM tris/HCl pH7.4 for 16 hours at 15 ℃), and the dialysate was analyzed after acidification. Insulin analogue concentration was determined using reverse phase HPLC and zinc concentration was determined by atomic absorption spectroscopy. The zinc values were corrected for the zinc content of the insulin-free control.

Zinc binding

Sequence listing

(1) General information:

(i) the applicant:

(A) name: hoechst Marion Roussel Deutschland Gmbh

(B) Street: -

(C) City: frankfurt

(D) State: -

(E) The state is as follows: germany

(F) And (3) post code: 65926

(G) Telephone: 069-305-5307

(H) Faxing: 069-357175

(I) Telegraph: -

(ii) Application name: novel insulin derivatives for the treatment of diabetes

(iii) Sequence number: 11

(iv) Computer readable format:

(A) type of medium: flexible disk

(B) A computer: IBM PC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: PatentIn Release #1.0, Version #1.25(EPO)

(2) SEQ ID NO: 1, information:

(i) sequence characteristics:

(A) length: 21 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..21

(xi) Detailed sequence: SEQ ID NO: 1:

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

(2) SEQ ID NO: 2, information:

(i) sequence characteristics:

(A) length: 30 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..30

(xi) Detailed sequence: SEQ ID NO: 2:

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

20 25 30

(2) SEQ ID NO: 3, information:

(i) sequence characteristics:

(A) length: 30 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..30

(xi) Detailed sequence: SEQ ID NO: 3:

His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu

1 5 10 15

Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25 30

(2) SEQ ID NO: 4, information:

(i) sequence characteristics:

(A) length: 31 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..31

(xi) Detailed sequence: SEQ ID NO: 4:

His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu

1 5 10 15

Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

20 25 30

(2) SEQ ID NO: 5, information:

(i) sequence characteristics:

(A) length: 32 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..32

(x i) sequence details: SEQ ID NO: 5:

His Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala

1 5 10 15

Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

20 25 30

(2) SEQ ID NO: 6, information:

(i) sequence characteristics:

(A) length: 33 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..33

(x i) sequence details: SEQ ID NO: 6:

His Ala Ala Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu

1 5 10 15

Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys

20 25 30

Thr

(2) SEQ ID NO: and 7, information:

(i) sequence characteristics:

(A) length: 98 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..98

(xi) Detailed sequence: SEQ ID NO: 7:

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg His Phe Val Asn Gln

1 5 10 15

His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly

20 25 30

Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp

35 40 45

Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser

50 55 60

Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val

65 70 75 80

Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr

85 90 95

Cys Asn

(2) SEQ ID NO: and 8, information:

(i) sequence characteristics:

(A) length: 99 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..99

(xi) Detailed sequence: SEQ ID NO: 8:

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg His Ala Phe Val Asn

1 5 10 15

Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys

20 25 30

Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu

35 40 45

Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly

50 55 60

Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile

65 70 75 80

Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn

85 90 95

Tyr Cys Asn

(2) SEQ ID NO: 9, information:

(i) sequence characteristics:

(A) length: 100 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..100

(xi) Detailed sequence: SEQ ID NO: 9:

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg His Ala Ala Phe Val

1 5 10 15

Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val

20 25 30

Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala

35 40 45

Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala

50 55 60

Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly

65 70 75 80

Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu

85 90 95

Asn Tyr Cys Asn

100

(2) SEQ ID NO: 10, information:

(i) sequence characteristics:

(A) length: 30 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..30

(xi) Detailed sequence: SEQ ID NO: 10:

Phe Val His Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

20 25 30

(2) SEQ ID NO: 11, information:

(i) sequence characteristics:

(A) length: 30 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) Molecular type: protein

(ix) Is characterized in that:

(A) name/keyword: protein

(B) Position: 1..30

(xi) Detailed sequence: SEQ ID NO: 11:

Phe Val Asp Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

20 25 30

Claims (19)

1. An insulin analogue of formula I or a physiologically tolerable salt thereof, wherein the insulin analogue is

(i) The A chain is SEQ ID NO: 1, the chain B is SEQ ID NO: 3 his (bo), des (B30) -human insulin:

His Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu AlaLeu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys；

(ii) the A chain is SEQ ID NO: 1, the chain B is SEQ ID NO: his (B3) -human insulin of 10:

phe Val His Gln His Leu Cys Gly Ser His Leu Val Glu Ala LeuTyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr; or

(iii) The A chain is SEQ ID NO: 1, the chain B is SEQ ID NO: asp (B3) -human insulin of 11:

Phe Val Asp Gln His Leu Cys Gly Ser His Leu Val Glu Ala LeuTyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr。

2. a pharmaceutical formulation comprising at least one insulin analogue and/or at least one physiologically tolerable salt thereof as claimed in claim 1.

3. The pharmaceutical formulation of claim 2, comprising the insulin analogue and/or a physiologically tolerable salt thereof in dissolved, amorphous and/or crystalline form.

4. A pharmaceutical formulation according to claim 2 or 3, additionally comprising a storage aid.

5. A pharmaceutical formulation as claimed in claim 4, wherein the storage aid is protamine sulfate and the insulin analogue and/or a physiologically tolerable salt thereof is present in a co-crystal with protamine sulfate.

6. The pharmaceutical formulation of claim 2, further comprising unmodified human insulin.

7. The pharmaceutical formulation of claim 2, additionally comprising an insulin analog.

8. An injectable solution with insulin activity comprising the pharmaceutical formulation of any one of claims 2-7 in dissolved form.

9. The injectable solution of claim 8 comprising 1 μ g-2mg zinc/ml.

10. The injectable solution of claim 9 comprising 5 μ g to 200 μ g zinc/ml.

11. Use of an insulin analogue according to claim 1 and/or a physiologically tolerable salt thereof for the preparation of a pharmaceutical preparation with delayed onset of action of the insulin activity.

12. An insulin-zinc complex comprising one insulin hexamer and 4-10 zinc ions per insulin hexamer, wherein the insulin hexamer consists of six insulin analogs of claim 1.

13. A pharmaceutical formulation comprising at least one insulin-zinc complex of claim 12.

14. A pharmaceutical formulation comprising an acidic solution of at least one insulin analogue according to claim 1 and/or a physiologically tolerable salt thereof and a suitable amount of zinc ions, which makes it possible to form an insulin-zinc complex according to claim 12.

15. A pharmaceutical formulation according to claim 13 or 14, comprising insulin-zinc complex in dissolved, amorphous and/or crystalline form.

16. An injectable solution having insulin activity comprising the pharmaceutical formulation of claim 15 in dissolved form.

17. The injectable solution of claim 16 containing 1 μ g-2mg zinc/ml.

18. The injectable solution of claim 17 comprising 5 μ g to 200 μ g zinc/ml.

19. Use of an insulin-zinc complex according to claim 12 for the preparation of a pharmaceutical preparation with delayed onset of action insulin activity.