Insulin and its derivatives with enhanced ability to bind zinc

 

(57) Abstract:

In the present invention are described derivatives of insulin with an enhanced ability to bind zinc General formula I, where Z means the residue of histidine, or a peptide with 2-35 genetically encoded amino acid residues, containing 1-5 residue his-tag, suitable for the production of pharmaceutical compositions for the treatment of diabetes. The insulin of the formula I to form a zinc complexes containing hexamer insulin and approximately 5-9 moles of zinc on hexamer. 4 C. and 12 C.p. f-crystals, 6 PL.

The invention relates to insulin and its derivatives with enhanced ability to bind zinc.

Farmakokinetik introduced subcutaneously insulin depends on the associative behavior. Insulin forms a neutral aqueous solution hexamers. If insulin wants to penetrate tissue into the blood stream and get to the place where it has an impact, the insulin must first pass of capillary vessels. Assume that this is only possible for Monomeric and dimeric insulin, and cannot or only slightly possible for review of insulin and high-molecular associates (Brange et al., Diabetes Care: 13 (1990), pages 923-954; Capz subcutaneous tissue into the blood stream.

Associative and aggregate behavior of insulin is influenced by zinc++that leads to stabilization hexamer and at pH values around neutral point to the formation of macromolecular aggregates until precipitation. Zinc++as an additive to nezabyvaemomu solution of human insulin (pH 4) has only a minor influence on the profile of action. Although this solution after injection into the subcutaneous layer of tissue quickly neutralized and formed insulinotherapy complexes, natural ability to bind zinc, which is found in the human insulin, not enough to stabilize hexamers and higher aggregates. Therefore, by adding zinc++the release of human insulin slows down slightly and not achieved a strong effect of the prolonged action. Known insulin hexamers show content of about 2 mol of zinc++on mol hexamer insulin (Blundell et al., Adv. Protein Chem.: 26 (1972), pages 323-328). Two zinc ions on hexamer insulin firmly connected with hexameron insulin and cannot be removed using conventional dialysis. Were described by the so-called 4-zinc-insulin crystals, however, these crystaltravel 7093-7097).

The present invention is to find the derivative of insulin, which have increased ability to bind zinc, form a stable complex containing hexamer insulin and zinc++and have a slow mode of action after subcutaneous injection compared with human insulin.

So, were found insulin formula 1

< / BR>
and/or physiologically compatible salts of insulin of the formula I, which fulfill the above criteria and are distinguished by the fact that

R1is phenylalaninol residue or a hydrogen atom,

R3is genetically coded amino acid residue,

Y represents a genetically coded amino acid residue,

Z means (a) the remainder of the amino acids His, or b) a peptide with 2-35 genetically capable of encoding amino acid residues, containing 1-5 residue his-tag,

and residues A2-A20 correspond to the amino acid sequence of the a-chain of human insulin, animal insulin or derivative of insulin,

and residues B2-B29 correspond to the amino acid sequence of the B-chain of human insulin, animal insulin or derivative of insulin.

In particular, preferred is the necessaty from the group Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gln,

Y means the remainder of the amino acids from the group Ala, Thr, Ser and His,

Z means (a) the remainder of the amino acids His, or b) a peptide with 4-7 amino acid residues containing 1 or 2 histidine residue.

Further, it is preferable insulin of the formula I, where

R1means phenylalanine residue,

R3means the rest of the amino acids from the group of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gln,

Y means the remainder of the amino acids from the group Ala, Thr, Ser and His,

Z means (a) the remainder of the amino acids His, or b) a peptide with 4-7 amino acid residues containing 1 or 2 histidine residue.

Especially preferred is insulin 1, where

Z is a peptide of 1 to 5 amino acid residues, containing 1-2 histidine residue.

In particular, preferred is the insulin of the formula I, where

R1means phenylalanine residue,

R3means the rest of the amino acids from the group of Asn and Gly,

Y means the remainder of the amino acids from the group of Thr and His,

a Z means a peptide with 1-5 amino acid residues containing 1 or 2 histidine residue.

Further, it is preferable insulin of the formula I, where

R1means the residue of phenylalanine, R3is the 1 or 2 of the histidine residue.

Quite especially preferred is the insulin of the formula I, where

Z means a peptide with the sequence His His, His His Arg Ala His His Ala His His Arg Ala Ala His His Arg or Ala Ala His His.

The amino acid sequence of the peptides and the proteins from the indicated starting from the N-terminal end of the amino acid chain. The data given in the formula I in parentheses, e.g. A1, A6, A7, A11, A20, B1, B7, B19 or B30, correspond to the positions of amino acid residues in chain A or B of the insulin.

The term "genetically coded amino acid residue" represents amino acid residues Cly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp, Pro and selenocysteine.

Under the concepts of "residues A2-A20 and residues B2-B29" animal insulin understand, for example, amino acid sequence of insulin from cattle, pigs and chickens.

The concept of the residues A2-A20 and B2-B29 derived insulin is the corresponding amino acid sequence of human insulin, which are formed by substitution of other amino acids, genetically encoded amino acids.

A chain of human insulin has the following sequence (Seq Id No. 1):

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

Chain B of insulin to the forehead, Glu, Arg, Gly, Phe, Phe, Tyr, Thr, Pro, Lys, Thr.

Derived insulin of the formula I can be formed using a variety of genetically engineered constructs in microorganisms (EP 0489780, EP 0347781, EP 0368187, EP 0453969). Genetically engineered constructs expriments in such organisms as Escherichia coli or Streptomyceten during fermentation. Educated proteins are deposited inside of microorganisms (EP 0489780) or stand out in a fermentation solution.

Examples of insulin of the formula I are:

Gly(A21)-Humaninsulin-His(B31)-His(B32)-OH

Gly(A21)-Humaninsulin-His(B31)-His(B32)-Arg(B33)-OH

Gly(A21)-Humaninsulin-Ala(B31)-His(B32)-His(B33)-OH

Gly(A21)-Humaninsulin-Ala(B31) -His(B32)-His(B33) -Arg(B34)-OH

Gly(A21)-Humaninsulin-Ala(B31) -Ala(B32)-His(B33) -His(B34)-OH

Gly(A21)-Humaninsulin-Ala(B31) -Ala(B32)-His(B33) -His(B34)-Arg(B35)-OH

Deriving insulin 1 is mainly engineered by site-directed mutagenesis according to standard methods. This is constructed of gene structure, carrying out the coding for the desired derivative of formula I and is placed in the host cell is the preferred way in bacteria such as E. coli or yeast, in particular Saccharomyces cerevisiae for expression and, if the structure of the gene encodes for a fused protein, a derivative of insulin forms the WG patent P 3821159.

Cleavage fractions fused protein is carried out after opening the cell or by chemical means using halogenation - see EP-A-0180920 or enzymatically by using lysostaphin - see DE-A-3739347.

Then the precursor of insulin is exposed to oxidizing sulfatase in accordance with the methods described, for example, in R. C. Marshall, and A. S. Inglis in "Practical Protein Chemistry - A Handbook" (published by A. Darbre) 1986, pages 49-53, and then in the presence of thiol in the formation of correct disulfide bonds sanatoriums, for example, in accordance with the method described by G. H. Dixon and A. C. Wardlow in Nature (1960), pages 721-724.

The precursors of insulin can, however, also directly be (EP-A-0600372; EP-A-0668292).

Peptide With U - if - predpolozhytelno (R2in the formula (II) is removed using tripticase off - for example, in accordance with the methodology Kemmler and other J. B. C. (1971), pages 6786-6791 and derived insulin of the formula I using conventional techniques, such as chromatography - EP-A-0305760 and crystallization.

Further, the invention relates to complexes containing hexamer insulin and approximately 5-9 moles of zinc on hexamer insulin, the preferred way of 5-7 moles of zinc++++on mol hexamer insulin could not be deleted due to 40 hours of regular dialysis, for example using aqueous 10 mm Tris/HCl-buffer, pH 7,4.

The insulin of the formula I shown in the form of a subcutaneous injection, mainly in the composition not containing zinc (pH 4), very small slow steps compared with human insulin. After adding approximately 20 mg Zink2+on/ml of the composition after subcutaneous injection occurs later onset of action. Slow action is observed, the preferred way when 40 g Zink2+/ml. a higher concentration of zinc amplify this effect.

Further, the invention concerns preproinsulin formula II,

R2R1B2-B29-Y-Z-Cly-A2-A20-R3< / BR>
and R3and Y, defined in the formula I according to one or more paragraphs 1-6 of claims and

R1is phenylalaninol balance or covalent bond, and

R2represents (a) a genetically encoded amino acid residue or b) a peptide with 2-45 amino acid residues, and the residues A2-A20 and B2-B29 correspond to the amino acid sequence in chains a and b of human insulin, animal insulin or derivative of insulin and at the same time
Proinsulin of the formula II are suitable as intermediate compounds in the manufacture of insulin of the formula I.

Preferred are proinsulin formula II,

R2represents a peptide with residues 2-25 amino acids.

In particular, preferred are proinsulin formula II, and

R2means a peptide with 2 to 15 amino acid residues, in which at the end of carboxyl is the balance of amino acids from the group of Met, Lys and Arg.

Derivatives of insulin of the formula I according to the invention and/or complexes containing hexamer insulin and 5-9 mol zinc++on hexamer and/or their physiologically compatible salts (e.g. alkali metal salts or ammonium salts) are mainly used as biologically active substance is a pharmaceutical preparation for the treatment of diabetes, in particular diabetes mellitus.

The pharmaceutical preparation is preferred, the solution or suspension for purposes of injection; it is characterized by a content of at least one derivative of insulin of the formula I and/or complex according to the invention and/or at least one of their physiologically compatible salts in dissolved, amorphous and/or crystalline - preferred education is compulsory from 2.5 to 8.5, in particular, approximately from 4.0 to 8.5, contains an appropriate isotonic means corresponding to the storage means and, if necessary, the appropriate buffer, and the preferred way is also a certain concentration of zinc ions, all this, of course, in sterile aqueous solution. The set of components of the drug in addition to the active biological substance forms a solution is a carrier of the drug.

Drugs, containing solutions of insulin of the formula I, have a pH value of from 2.5 to 4.5, in particular from 3.5 to 4.5, the preferred way of 4.0.

Drugs, containing suspensions of insulin of the formula I, are set to a pH of 6.5 to 8.5, in particular 7,0-8,0, the preferred way of 7.4.

Suitable isotonic means are, for example, glycerol, glucose, mannitol, NaCl, calcium and magnesium, such as CaCl2or MgCl2.

By selecting isotonic means and/or preservatives have an effect on the solubility of the derivative of insulin and its physiologically compatible salts with weakly acidic pH values.

Appropriate preservative means are, for example, phenol, m-cresol, benzyl alcohol and/or slotmachine pH between about 4.0 and 8.5 are eligible, for example, to use sodium acetate, sodium citrate or sodium phosphate. Otherwise, to establish a pH value suitable physiologically acceptable diluted acid (a typical case of HCl) or alkaline solutions (typical case of NaOH).

If the product contains zinc, the content is from 1 μg to 2 mg of zinc++/ml, in particular from 5 to 200 μg zinc/ml

With the aim of getting the profile of action of the preparation according to the invention can be added also premeditatively insulin, the preferred way insulin cattle, pigs or humans, in particular human insulin or modified insulin, for example Monomeric insulin, fast-acting insulin or a human insulin Gly(A21)-Arg(B31)-Arg(B32).

The preferred concentration of the active substance correspond approximately 1-1500, then the preferred way approximately 40-400 international units/ml.

EXAMPLE 1

Getting Gly(A21) human insulin-His(B31)- His(B32)-HE

Obtaining expression was mainly in accordance with U.S. patent 5358857. There also describes vectors p1NT 90d and p1NT 91d (see example 17) and PCR Primer TIR Insull. These four components of SGE.

First of all the mini-proinsulin-encoding sequence is inserted codon for Gly (A21). To do this, use p1NT 91d as a model and reaction PCR is performed with primers TIR and Insu31

Insu31 (Seq Id No. 10):

5'TTT TTT GTC GAC HUNDRED TTA GCA GTA GTT CTC CAG CTG 3'

The PCR cycle is performed as follows: 1) min 94oC, 2) min 55oC, 3) min 72oC. This cycle was repeated 25 times, then 7 minutes is incubation at 72oC, and then incubated overnight at 4oC.

The resulting PCR fragment is allocated for cleaning in ethanol, dried, and then in restriction buffer in accordance with these manufacturers digested with restriction enzymes Nc01 Sail. Then the reaction mixture is separated by gelelectrophoresis, and the fragment Nc01-Prae-proinsulin-Sall isolate. DNA plasmids plNT 90d also split with Nc01 and Sall, and the fragment of proinsulin monkeys in the same way free from residual plasmid p1NT. Both fragments separated using gel electrophoresis, and residual plasmid DNA isolated. This DNA is introduced into the reaction ligase with a fragment Nc01-Sall-PCR. So, get plasmid plNT 150d, which after transformation in E. coli reproduces there, and then isolated.

DNA plasmids plNT 150d serve to the>

To construct this plasmid go by, is described in U.S. patent 5358857 (see example 6). To do this, hold two independent from each PCR reaction, for which the quality of the model as DNA plasmids plNT 150d. One reaction is carried out using pairs of primers TIR and plNT B5 (Seq Id No. 11):

5'GAT GCC GCG GGT CTT GGG TGT GTAG 3'

and the other reaction using a pair of primers Insu11 and plNT B6 (Seq Id No. 12):

5' A CCC AAG ACC GGC CGC TC GTG GAG 3'

The resulting PCR fragments are partially complementary, so that they are in the third reaction PRC lead to the fragment, which carries out the coding for microinsurance Gly (A21), elongated at position B31 and B32. This fragment were cleaved using Nc01 and Sall, and then injected into the reaction ligase with DNA described residual plasmid plNT90d obtaining plasmids plNT 302. Transformed by this plasmid E. coli K12 W3110 then fermented, as shown in example 4 of U.S. patent US 5227293, and processed.

Obtained as an intermediate product derived preproinsulin before cleavage of the trypsin has the following amino acid sequence:

Preproinsulin I (Seq Id No. 3):

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg

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

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

His His Arg

R2means a peptide of 11 amino acid residues,

R1Phe (B1),

Y Thr (B30),

Z His His Arg (B31-B33),

R3Gly (A21) and

A2-A20 is a sequence of chain a of human insulin (amino acid residues 2-20), and B2-B29 are a sequence chain of human insulin (amino acid residues 2-29).

Split preproinsulin 1 as described in U.S. patent US 5227293 according to example 4, by using trypsin. Then the obtained product is introduced into reaction with carboxypeptidase In accordance with example 11 to give the insulin 1. Insulin 1 corresponds to the formula I, with

R1means Phe (B1),

Y means Thr (B30),

Z His His Arg (B31-B32),

R3- Gly (A21) and

A2-A20 represent the amino acid sequence of chain And human insulin (amino acid residues 2-20), and B2-B29 are a sequence of chain B of human insulin (amino acid residues 2-29).

Insulin 1 has the following amino acid sequence:

Insulin 1 (Seq Id No. 4):

Chain: 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 His His

Circuit a: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly

(Disulfide bridges, as shown in the formula (I).

PR accordance with example 1.

Plasmid plNT 150d serves as a model for two independent from each other reactions PCR with pairs of primers TIR and plNT B7 (Seq Id No. 13):

5' GAT GCC GCG GGT CTT GGG TGT GTAG 3'

or Insull and plNT B8 (Seq Ind N 14):

5' ACCC AAG ACC GGC CGC ATC GTG GAG 3'

The PCR fragments, the occurrence of which are both responses are partially complementary, and give the third reaction PRC complete sequence that produces the encoding for the desired option. The fragment reaction is treated enzymes using Nc01 und Sall, and then legasuite in residual plasmid, open Nc01/Sall, plNT90d DNA. Occurs plasmid plNT303, which after transformation of E. coli K12 W3110 Skagit the basis for the expression of the desired pre-miniproinsulin. Fermentation and processing occurs as described in example 1, the reaction In carboxypeptidase disappears.

Derived derived preproinsulin has the following amino acid sequence:

Preproinsulin 2 (Seq Id No. 5):

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg

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

Ala His His Arg

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

Leu Tyr Gin Leu Glu Asn Tyr Cys Gly

Preproinsulin 2 corresponds to the formula II,

R1means Phe (B1),
Gly (A21) and

A2-A20 are the amino acid sequence of chain a of human insulin (amino acid residues 2-20), and B2-B29 are the amino acid sequence of the chain of human insulin (amino acid residues 2-29).

Preproinsulin then reacts interaction of trypsin with the formation of insulin 2. Insulin 2 corresponds to the formula II,

R1means Phe (Bl),

Y means Thr (B30),

Z - Ala His His Arg (B31-B34),

R3Gly(A21) and

A2-A20 are the amino acid sequence of chain a of human insulin (amino acid residues 2-20), and B2-B29 are the amino acid sequence of the chain of human insulin (amino acid residues 2-29).

Insulin 2 has the following amino acid sequence:

Insulin 2 (Seq Ind N 6):

Chain: 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 Tight Thr Pro Lys Thr Ala His His Arg

Circuit a: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly

(Disulfide bridges, as shown in the formula (I).

EXAMPLE 3

Getting Gly(A21) human insulin-Ala(B31)- Ala(B32)-His(B33)-His(B34)-OH

The expression vector is constructed in accordance with example 1.

Plasmid p1NT l50d serves as a model for two independent drugno Insu11 and p1NT 316b (Seq Ind N 16):

5' A CCC AAG ACC GCG GCG CAT CAT CGC GGC ATC GTG GAG 3'

The PCR fragments, the occurrence of which are both responses are partially complementary, and give the third reaction PRC complete sequence that produces the encoding for the desired option. The fragment reaction is treated enzymes using Nc01 und Sall, and then legasuite in residual plasmid, open Nc01/Sall, plNT 90d DNA. Occurs plasmid plNT 316, which after transformation of E. coli K12 W3110 serves as the basis for the expression of the desired pre-miniproinsulin. Fermentation and processing occurs as described in example 1, the reaction In carboxypeptidase disappears.

The resulting preproinsulin 3 has the following amino acid sequence:

Preproinsulin 3 (Seq Id No. 7):

Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg

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

Ala Ala His His Arg

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

Leu Tyr Gln Leu Glu Asn Tyr Cys Gly

Preproinsulin 3 corresponds to the formula II,

R1Phe (B1),

R2means a peptide of 11 amino acid residues,

Y - Thr (B30),

Z - Ala Ala His His Arg (B31-B35),

R3Gly (A21) and

A2-A20 are the amino acid sequence of chain a of human insulin (residues amino is.

Preproinsulin 3 then interacts with trypsin and carboxypeptidase B, as in example 11, with the formation of insulin 3. Insulin 3 corresponds to the formula I, with

R1means Phe (B1),

Y means Thr (B30),

Z - Ala Ala His His (B31-B34),

R3Gly(A21) and

A2-A20 are the amino acid sequence of chain a of human insulin (amino acid residues 2-20), and B2-B29 are the amino acid sequence of the chain of human insulin (amino acid residues 2-29).

Insulin 3 has the following amino acid sequence:

Insulin 3 (Seq Ind N 8):

Chain: 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 Ala Ala His His

Circuit a: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly

(Disulfide bridges, as shown in the formula (I).

EXAMPLE 4

Insulin 2 obtained in example 2, interacts with carboxypeptidase In accordance with example 11 to give the insulin 4.

Insulin 4 corresponds to the formula I, with

R1means Phe (B1),

Y means Thr (B30),

Z means Ala His His (B31-B33),

R3means Gly (A21) and

A2-A20 are the amino acid sequence of chain a of human insulin (residues aminocyclo">

Insulin 4 has the following amino acid sequence:

Insulin 4 (Seq Ind N 9):

Chain: 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 Ala His His

Circuit a: Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Gly

(Disulfide bridges, as shown in the formula (I).

EXAMPLE 5

The zinc-binding derivatives of insulin

Preparation of insulin (0,243 mM human insulin, with 0.13 M NaCl, 0.1% phenol, 80 µg/ml (1.22 mm) zinc++, 10 mm Tris/HCl, pH 7,4) is subjected to dialysis at the 15oC against 10 mM Tris/HCl, pH 7,4, a total of 40 hours (change buffer occurs after 16 hours and 24 hours). After that, the dialysates is acidified, the concentration of insulin was determined by HPLC and zinc by the spectrograph acompulsory. Values correct zinc using zinc content control original mix containing insulin (see table. 1).

EXAMPLE 6

Zinc dependence of the profile of action of human insulin in experiments on dog

Introduction: subcutaneous.

Dosage: 0,3 IE (betw.ed.)/kg: the pH of the composition is 4.0.

The number of dogs (n) the experience is 6.

Table. 2 shows the blood glucose in % from the original values.

EXAMPLE 7

Profile actions Gly (A21) Ala (B31), His (B32) is used in the following formulation preparation:

Glycerin 20 mg/ml m-cresol 2.7 mg/ml, insulin 2 40 IE (international units)/ml

IE is an international unit and corresponds to approximately 6 mmol of insulin, such as human insulin or insulin of the formula I. the pH Value is set by using NaOH or HCl.

Introduction: subcutaneous dose: 0,3 IE/kg;

the number of tested dogs is 6; the pH of the drug - 4,0.

Table. 3 shows the blood glucose in % of initial value.

EXAMPLE 8

Profile actions Gly(A21) Ala(B31) His(B32), His(B33), Arg(B34)-human insulin in the experiments on the dog (insulin 4)

Insulin 4, made according to example 4, is formulated and used as in example 7.

Introduction: subcutaneous dose: 0,3 IE/kg;

n = 6; pH of the drug - 4,0.

Table. 4 shows the blood glucose in % of initial value.

EXAMPLE 9

Profile actions Gly(A21) His(B31), His(B32)-human insulin in the experiments on the dog (insulin 1)

Insulin 1 obtained in example 1, is formulated and used as in example 7.

Introduction: subcutaneous dose: 0,3 S/kg;

n = 6; pH of the composition is 4.0.

Table. 5 shows the blood glucose in % of initial value.

EXAMPLE 10

Profile actions Gly(A21) Ala(B31) Ala(B32) His(B33), His(VLE as in example 7.

Introduction: subcutaneous dose: 0,3 IE/kg;

n = 6; pH of the composition is 4.0.

Table. 6 shows the blood glucose in % of initial value.

EXAMPLE 11

Getting insulin 1 from preproinsulin 1

200 mg of insulin with Arg at the Carboxy-end of the chain obtained in example 1, are dissolved in 95 ml of 10 mm HCl. After adding 5 ml of 1 M Tris/HCl (Tris(hydroxymethyl)aminomethan) - pH 8, set the value of pH using HCl or NaOH at around 8.

Add 0.1 mg carboxypeptidase C. After 90 min is fully cleavage of arginine. The pH of the initial mixture by adding HCl install 3.5 and is pumped to the column Reserved Phase (PLRP-S RP 300 10 μm 2,5x30 cm, Polymer Laboratories, Amherst, MA, USA). Mobile phase A: water with 0.1% triperoxonane acid. The phase consists of acetonitrile with 0,09% triperoxonane acid. Column works with the flow, amounting to 5 ml/min After application, the column is washed with 150 ml of phase A. Fractionated leaching is performed by applying a linear gradient in size from 22.5 to 40% for 400 minutes. Fractions analyzed separately using analytical Reverved Phase HPCl, and those that contain Des-Arg-insulin sufficient purity are pooled. The pH value is set using NaOH equal to 3.5, and areaaverage of 6.5. The precipitate attentivegreat, washed twice each time with 50 ml water, and then dried by freezing.

The output is 60-80% of insulin 1.

1. The insulin of the formula I,

< / BR>
and/or physiologically compatible salts of insulin of the formula I,

where R1the phenylalanine residue or a hydrogen atom;

R3- genetically coded amino acid residue;

Y - genetically coded amino acid residue;

Z means (a) the residue of histidine His or b) a peptide with 2-35 genetically encoded amino acid residues, containing 1-5 residue his-tag;

the residues A2-A20 correspond to the amino acid sequence of the a-chain of human insulin, animal insulin or derivative of insulin,

residues B2-V correspond to the amino acid sequence of the b-chain of human insulin, animal insulin or derivative of insulin.

2. The insulin of the formula I on p. 1, where R1means the residue of phenylalanine, R3means the rest of the amino acids from the group of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gln, Y means the remainder of the amino acids from the group Ala, Thr, Ser, His, Z means (a) the residue of histidine b) a peptide with 4-7 amino acid residues containing 1 or 2 histidine residue.

3. The insulin of the formula I, p. Asp, Glu and Gin, Y means the remainder of the amino acids from the group Ala, Thr, Ser, His, Z means (a) a histidine residue or b) a peptide with 2 to 7 amino acid residues containing 1 or 2 histidine residue.

4. The insulin of the formula I on p. 3, where Z is a peptide of 1 to 5 amino acid residues, containing 1-2 histidine residue.

5. The insulin of the formula I according to one or more paragraphs.1-4, where R1means the residue of phenylalanine, R3means the rest of the amino acids from the group of Asn and Gly, Y means the remainder of the amino acids from the group of Thr and His, Z means a peptide with 1-5 amino acid residues containing 1 or 2 histidine residue.

6. The insulin of the formula I on p. 1, where R1means the residue of phenylalanine, R3represents a glycine residue, Y represents a residue of threonine, Z means (a) the residue of histidine His or b) a peptide with 2-35 genetically encoded amino acid residues, containing 1-5 residue his-tag.

7. The insulin of the formula I on p. 6, where Z denotes the peptide sequence His His, His His Arg Ala His His Ala His His Arg Ala Ala His His Arg or Ala Ala His His.

8. Complexes containing hexamer insulin and 5-9 moles of zinc on hexamer insulin, in particular 5-7 moles of zinc on hexamer insulin, and hexamer insulin consists of six multidirectional activity characterized in that it contains an effective amount of at least insulin of the formula I and/or at least one physiologically acceptable salt of insulin of the formula I according to one or more paragraphs.1-8, in dissolved, amorphous and/or crystalline form.

10. The pharmaceutical composition according to p. 9, different additional content from 1 μg to 2 mg, preferably from 5 μg to 200 μg zinc/ml

11. The pharmaceutical composition under item 9 or 10, characterized by a pH value of from 2.5 to 8.5.

12. The pharmaceutical composition according to one or more paragraphs.9-11, characterized by a pH value of from 2.5 to 4.5.

13. The pharmaceutical composition according to one or more paragraphs. 9-12, different additional content of unmodified insulin, preferably unmodified human insulin or modified insulin, preferably Gly(A21)-Arg(B31)-Arg(B32)-human insulin.

14. Proinsulin of the formula II

R2R1B2-B29-Y-Z-Gly-A2-A20-R3(II)

where R3and Y have the meanings indicated in the formula I according to one or more paragraphs.1-7,

R1the residue is phenylalanine or a covalent bond;

R2and genetically encoded residual linakis is inability circuits a and b human insulin, animal insulin or derivative of insulin;

Z means a peptide with 2-40 genetically encoded amino acid residues 1-5 residues histidine.

15. Proinsulin of the formula II according to p. 14, where R2is a peptide with residues 2-25 amino acids.

16. Proinsulin of the formula II under item 14 or 15, where R2means a peptide with 2 to 15 amino acid residues, in which at the end of carboxyl is the rest of the amino acids from the group of Met, Lys and Arg.

 

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