Title of Invention	AN INSULIN OF THE FORMULA I WITH AN INCREASED ZINC BINDING POWER
Abstract	Abstract Insulin derivatives with increased zinc binding where Z is a histidine residue or a peptide having 2 to 35 genetically encodable amino acid residues, containing 1 to 5 histidine residues, are suitable for the production of pharmaceutical preparations for the treatment of diabetes. Insulins of the formula I form complexes with zinc, containing an insulin hexamer and approximately 5 to 9 mol of zinc per hexamer.

Title of Invention

AN INSULIN OF THE FORMULA I WITH AN INCREASED ZINC BINDING POWER

Abstract

Abstract Insulin derivatives with increased zinc binding where Z is a histidine residue or a peptide having 2 to 35 genetically encodable amino acid residues, containing 1 to 5 histidine residues, are suitable for the production of pharmaceutical preparations for the treatment of diabetes. Insulins of the formula I form complexes with zinc, containing an insulin hexamer and approximately 5 to 9 mol of zinc per hexamer.

Full Text	The pharmacokinetics of subcutaneously administered insulin is dependent on its association behavior. Insulin forms hexamers in neutral aqueous solution. If the insulin is to get from the tissue into the blood stream and to the site of action, it must first pass through the walls of the capillaries. It is assumed that this is only possible for monomeric and for dimeric insulin - but not possible or only slightly possible for hexameric insulins or relatively high molecular weight associates (Brange et al., Diabetes Care: 13 (1990), pages 923-954; Kang et al. Diabetes Care: 14 (1991), pages 942-948). The dissociation of the hexamer is therefore a prerequisite for rapid passage from the subcutaneous tissue into the blood stream. The association and aggregation behavior of insulin is affected by zinc , which leads to a stabilization of the hexamer and at pHs around the neutral point to the formation of relatively high molecular weight aggregates until precipitation occurs. Zinc** as an additive to an unbuffered human insulin solution (pH 4), however, only slightly affects the profile of action. Although the solution in the tissue is rapidly neutralized and insulin-zinc complexes are formed, the natural zinc binding of the insulin is insufficient to stabilize hexamers and higher aggregates if the release of the insulin is markedly delayed and a strong depot effect is achieved. Known insulin hexamers have a content of approximately 2 mol of zinc per mole of insulin hexamer (Blundell et al., Adv. Protein Chem.: 26 (1972), pages 323-328). Two zinc molecules per insulin hexamer are so firmly bound to the insulin hexamer that they cannot be removed by normal dialysis. So-called 4-zinc insulin crystals have admittedly been described, but these crystals on average contain only less than three mol of zinc per mole of insulin hexamer (G.D. Smith etal., Proc. Natl. Acad. Sci. USA: 81, pages 7093-7097). The object of the present invention is to find insulin derivatives which have an increased zinc binding power, form a stable complex containing insulin hexamer and zinc and have a delayed profile of action on subcutaneous injection in companson with human insulin. and/or physiologically tolerable salts of the insulins of the formula I have now been found which fulfil the abovementioned criteria and wherein R is a phenylalanine residue or a hydrogen atom, R1 is a genetically encodable amino acid residue, Y is a genetically encodable amino acid residue, Z is a) the amino acid residue His or b) a peptide having 2 to 35 genetically encodable amino acid residues, containing 1 to 5 histidine residues, and the residues A2-A20 correspond to the amino acid sequence of the A chain of human insulin, animal insulin or an insulin derivative and the residues B2-B29 correspond to the amino acid sequence of the B chain of human insulin, animal insulin or an insulin derivative. An insulin of the formula I is particularly preferred where R1 is a phenylalanine residue, R is an amino acid residue from the group consisting of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gin, - Y is an amino acid residue from the group consisting of Ala, Thr, Ser and His, 2. is a) the amino acid residue His or b) a peptide having 4 to 7 amino acid residues, containing 1 or 2 histidine residues. An insulin of the formula I is furthermore preferred where R is a phenylalanine residue, R is an amino acid residue from the group consisting of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gin, Y is an amino acid residue from the group consisting of Ala, Thr, Ser and His, Z is a) the amino acid residue His or b) a peptide having 2 to 7 amino acid residues, containing 1 or 2 histidine residues. An insulin of the formula I is particularly preferred where Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues. An insulin of the formula I is particularly preferred, where R is a phenylalanine residue, R is an amino acid residue from the group consisting of Asn and Gly, Y is an amino acid residue from the group consisting of Thr and His, and Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues. An insulin of the formula I is furthermore preferred where R is a phenylalanine residue, R is a glycine residue, Y is a threonine residue and Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues. An insulin of the formula I is very particularly preferred where Z is a peptide having 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 peptides and proteins is indicated from the N-terminal end of the amino acid chain onward. The details given in brackets in formula I, e.g. A1, A6, A7, A11, A20, B1, B7, B19 or 830, correspond to the position of amino acid residues in the A or 8 chains of the insulin. The expression "genetically encodable amino acid residue" represents the residues of the amino acids Gly, Ala, Ser, Thr, Val, Leu, lie. Asp, Asn, Glu, Gin, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp, Pro and selenocysteine. The expressions "residues A2 - A20" and "residues 82 - B 29" of animal insulin are understood, for example, as meaning the amino acid sequences of insulin from cattle, pigs or chickens. The expression residues A2 - A20 and 82 - 829 of insulin derivatives represents the corresponding amino acid sequences of human insulin which are formed by the replacement of amino acids by other genetically encodable amino acids. The A chain of human insulin has the following sequence (Seq Id No. 1): Gly, lie, Val, Glu, Gin, Cys, Cys, Thr, Ser, lie, Cys, Ser, Leu, Tyr, Gin, Leu, Glu, Asn, Tyr, Cys, Asn. The B chain of human insulin has the following sequence (Seq Id No. 2 ): Phe, Val, Asn, Gin, 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. The insulin derivative of the formula I can be formed in microorganisms with the aid of a multiplicity of genetic engineering constructs (EP 0 489 780, EP 0 347 781, EP 0 368 187, EP 0 453 969). The genetic engineering constructs are expressed in microorganisms such as Escherichia coli or Streptomycetes during fermentation. - The proteins formed are stored in the interior of the microorganisms (EP 0 489 780) or secreted into the fermentation solution. Exemplary insulins of the formula I are: Gly(A21 )-human insulin-His(B31 )-His(B32)-OH Gly(A21)-human insulin-His(B31)-His(B32)-Arg (B33)-OH Gly(A21 )-human insulin-Ala(B31 )-His(B32)-His(B33)-OH Gly(A21 )-human insulin-Ala(B31 )-His(B32)-His(B33)-Arg(B34)-OH Gly(A21 )-human insulin-Ala(B31 )-Ala(B32)-His(B33)-His(B34)-OH Gly(A21 )-human insulin-Ala(B31 )-Ala(B32)-His(B33)-His(B34)-Arg(B35)-OH The insulin derivatives of the formula I are mainly prepared by genetic engineering by means of site-directed mutagenesis according to standard methods. For this purpose, a gene structure coding for the desired insulin derivative of the formula I is constructed and expressed in a host cell - preferably in a bacterium such as E. coli or a yeast, in particular Saccharomyces cerevisiae - and - if the gene structure codes for a fusion protein - the insulin derivative of the formula I is released from the fusion protein; analogous methods are described, for example, in EP-A-0 211 299, EP-A-0 227 938, EP-A-0 229 998, EP-A-0 286 956 and the DE Patent Application P 38 21 159. After cell disruption, the fusion protein portion is cleaved either chemically by means of cyangen halide - see EP-A-0 180 920 - or enzymatically by means of lysostaphin or trypsin - see DE-A-37 39 347. The insulin precursor is then subjected to oxidated sulfitolysis according to the method described, for example, by R.C. Marshall and A.S. Inglis in "Practical Protein Chemistry - A Handbook" (Editor A. Darbre) 1986, pages 49 - 53 and then renatured in the presence of a thiol with formation of the correct disulfide bridges, e.g. according to the method described by G.H. Dixon and A.C. Wardlow in Nature (1960), pages 721-724. The insulin precursors, however, can also be directly folded (EP-A-0 600 372 ; EP-A-0 668 292). - The C peptide is removed by means of tryptic cleavage - e.g. according to the method of Kemmieretal., J.B.C. (1971), pages 6786 - 6791 and the insulin " derivative of the formula I is purified by means of known techniques such as chromatography - e.g. EP-A-0 305 760 - and crystallization. The invention furthermore relates to complexes containing an insulin hexamer and approximately 5 to 9 mol zinc per insulin hexamer preferably 5 to 7 mol of zinc per insuin hexamer, the insulin hexamer consisting of six insulin molecules of the formula I. The zinc binding to the insulin hexamer is so firm that the 5 to 9 mol of zinc per mole of insulin hexamer cannot be removed by 40 hours of normal dialysis, for example with an aqueous 10 mM tris/HCI buffer, pH 7.4. After subcutaneous administration, insulins of the formula I, as in the essentially zinc-free preparation (pH 4), show a small delay in action in comparison with human insulin. After addition of approximately 20 pg of zinc'/ml of preparation a later onset of action is observed even after subcutaneous administration. The delay in action is preferably observed at 40 pg of zinc'/ml. Higher zinc concentrations potentiate this effect. The invention furthermore relates to preproinsulin of the formula II R2-Rl-B2-B29-Y-Z-Gly-A2-A20-R (II) where R, Y and Z as in formula I are defined as set forth in one or more of claims 1 to 6, and R' is a phenylalanine residue or a covalent bond, and R2 is a) a genetically encodable amino acid residue or b) a peptide having 2 to 45 amino acid residues, and the residues A2 - A20 and 82 - 829 correspond to the amino acid sequences of the A and B chains of human insulin, animal insulin or an insulin derivative. - The proinsulin of the formula II is suitable as an intermediate in the preparation of the insulins of the formula I. Preferred proinsulins of the formula 11 are those where R is a peptide having 2 to 25 amino acid residues. Particularly preferred proinsulins of the formula II are those where R is a peptide having 2 to 15 amino acid residues, in which an amino acid residue from the group consisting of Met, Lys and Arg is at the carboxyl end. The insulin derivatives of the formula I and/or the complexes according to the invention, containing an insulin hexamer and 5 to 9 mol of zinc per hexamer and/or their physiologically tolerable salts (e.g. the alkali metal or ammonium salts), are mainly used as active compounds for a pharmaceutical preparation for the treatment of diabetes, in particular of diabetes mellitus. The pharmaceutical preparation is preferably a solution or suspension for injection purposes; it comprises at least one insulin derivative of the formula I and/or the complex according to the invention and/or at least one of its physiologically tolerable salts in dissolved, amorphous and/or crystalline - preferably in dissolved - form. The preparation preferably has a pH from approximately 2.5 to 8.5, in particular from approximately 4.0 to 8.5, contains a suitable isotonicizing agent, a suitable preservative and, if appropriate, a suitable buffer, and preferably also a specific zinc ion concentration, all, of course, in sterile aqueous solution. The whole of the preparation constituents apart from the active compound forms the preparation excipient. Preparations containing solutions of the insulin of the formula I have a pH from 2.5 to 4.5, in particular from 3.5 to 4.5, preferably 4.0. Preparations containing suspensions of the insulin of the formula I have a pH from 6.5 to 8.5, in particular from 7.0 to 8.0, preferably 7.4. - Suitable isotonicizing agents are, for example, glycerol, glucose, mannitol, NaCI, calcium compounds or magnesium compounds, such as CaCl2 or MgCl2. As a result of the choice of the isotonicizing agent and/or preservative, the solubility of the insulin derivative or of its physiologically tolerable salts Is affected at the weakly acidic pHs. Suitable preservatives are, for example, phenol, m-cresol, benzyl alcohol and/or p-hydroxybenzoic acid esters. Buffer substances which can be used, in particular for adjusting a pH of between approximately 4.0 and 8.5, are, for example sodium acetate, sodium citrate or sodium phosphate. Otherwise, physiologically acceptable dilute acids (typically HCI) or alkalis (typically NaOH) are also suitable for adjusting the pH. If the preparation has a content of zinc, one of from 1 pg to 2 mg of zinc/ml, in particular from 5 \|jg to 200 pg of zinc/ml, is preferred. To vary the profile of action of the preparation according to the invention, unmodified insulin, preferably bovine, porcine or human insulin, in particular human insulin, or modified insulins, for example monomeric insulins, rapid-acting insulins or Gly(A21)-Arg(B31)-Arg(B32)-human insulin, can also be admixed. Preferred active compound concentrations are those corresponding to approximately 1 -1500, furthermore preferably approximately 5 -1000 and in particular approximately 40 - 400, international units/ml. - Example 1 " Preparation of Gly(A21)-human insulin-His(B31)-His(B32)-OH The preparation of the expression system was carried out essentially as in US 5,358,857. The vectors pINT 90d and pINT 91 d (see Example 17) and the PCR primers TIR and Insul 1 are also described there. These four components are used, inter alia, as starting materials for the vector constructs described in the following. First, the codon for Gly (A21) is inserted in the sequence coding for the mini-proinsulin. To do this, pINT 91 d is used as a template and a PCR reaction is carried out with the primers TIR and Insu31 Insu31 (SeqIdNo. 10): 5'TTT TTT GTC GAC CTA TTA GCC GCA GTA GTT CTC CAG CTG 3' The PCR cycle is carried out as follows. 1st minute 94°C, 2nd minute 55'C, 3rd minute 72°C. This cycle is repeated 25 times then the mixture is incubated at 72"'C for 7 minutes and subsequently at 40 overnight. The resulting PCR fragment is precipitated in ethanol to purify it, dried and then digested in restriction buffer using the restriction enzymes NcOl and Sail according to the details of the manufacturers. The reaction mixture is then separated by gel electrophoresis and the NcOI-pre-proinsulin-Sall fragment is isolated. DNA of the plasmid plNT90d is likewise cleaved using NcOl and Sail and the monkey proinsulin fragment is in this manner released from the pINT residual plasmid. Both fragments are separated by gel electrophoresis and the residual plasmid DNA is isolated. This DNA is reacted with the NcOl-Sail PCR fragment in a ligase reaction. The plasmid plNT150d is thus obtained, which after transformation by E.coli is replicated and then reisolated. DNA of the plasmid plNT150d is used as a starting material for the plasmid plNT302, which allows the preparation of the desired insulin variant. For the construction of this plasmid, the route described in US 5,358,857 (see Example 6) is taken. Two PCR reactions which are independent of one another are carried out to this end, for which DNA of the plasmid plNT150d is used as a template. One reaction is carried out using the primer pair TIR and pINT B5 (SeqIdNo. 11): 5' GAT GCC GCG GIG GIG GGT CTT GGG TGT GTAG 3' and the other reaction using the primer pair Insu11 and pINT B6 (SeqIdNo. 12): 5' A CCC AAG ACC CAC CAC CGC GGC ATC GTG GAG 3'. The PCR fragments which result are partially complementary, so that in a third PCR reaction they lead to a fragment which codes for a Gly (A21) miniproinsulin lengthened by the position B31 and 632. This fragment is cleaved using NcOl and Sail and then reacted with the DNA of the described plNT90d residual plasmid in a ligase reaction to give the plasmid plNT302. An E. coli K12 W3110 transformed with this plasmid is then fermented and worked up as in Example 4 of US 5,227,293. The preproinsulin derivative obtained as intermediate (before trypsin cleavage) has the following amino acid sequence: Preproinsulin 1(Seq Id No. 3): Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin 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 Arg Gly lie Val Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly Preproinsulin 1 corresponds to the formula ILin-ttiiscase R is a peptide having 11 amino acid residues, R is Phe (81), Y is Thr (B30), Z isHisHisArg(B31-B33), R isGly(A21)and A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and 82-829 is the amino acid sequence of the 8 chain of human insulin (amino acid residues 2 to 29). The preproinsulin 1 is cleaved with trypsin as in US 5,227,293 according to Example 4. The product obtained is then reacted with carboxypeptidase 8 according to Example 11 to give insulin 1. Insulin 1 corresponds to the formula I, in this case R isPhe(81), Y is Thr (830), Z is His His Arg (831-833), R isGly(A21)and A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and 82-829 is the amino acid sequence of the 8 chain of human insulin (amino acid residues 2 to 29). Insulin 1 has the following amino acid sequence: Insulin 1 (Seq Id No. 4): Phe Val Asn Gin 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 Gly He Val Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly Example 2 Preparation of Gly(A21 )-human insulin-Ala(831)-His(832)-His(B33)- Arg(834)-OH The expression vector is constructed according to Example 1. Plasmid plNT150d is used as the template for two PCR reactions, which are independent of one another, with the primer pairs TIR and pINT 87 (Seq Id No. 13): 5' GAT GCC GCG GTG GTG CGC GGT CTT GGG TGT GTAG 3' or Insu11 and pINT B8 (Seq Id No. 14): 5' ACCC AAG ACC GCG CAC CAC CGC GGC ATC GTG GAG 3'. The PCR fragments to which both reactions lead are partially complementary and in a third PCR reaction afford the complete sequence which codes for the desired insulin variant. The fragment of the reaction is treated with the enzymes Nc01 and Sail and then ligated into the Nc01/Sal1-opened residual plasmid of the plNT90d DNA. Plasmid plNT303 results, which after transformation by E.coli K12 W3110 is used as a basis for the expression of the desired pre-miniproinsulin. Fermentation and working up is carried out as in Example 1, the carboxypeptidase B reaction being dispensed with. The preproinsulin derivative obtained has the following amino acid seqence: Preproinsulin 2 (Seq Id No. 5): Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin 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 He Val Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly Preproinsulin 2 corresponds to the formula II, in this case R is Phe (81), R is a peptide having 11 amino acid residues, Y is Thr (830), Z is Ala His His Arg (831-834), R isGly(A21)and A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and 82-829 is the amino acid sequence of the 8 chain of human insulin (amino acid residues 2 to 29). - The preproinsulin 2 is then reacted with trypsin to give insulin 2. Insulin 2 corresponds to the formula II, in this case ■ R isPhe(B1), Y is Thr (B30), Z is Ala His His Arg (B31-B34), R isGly(A21)and A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and B2-B29 is the amino acid sequence of the B chain of human insulin (amino acid residues 2 to 29). Insulin 2 has the following amino acid sequence: Insulin 2 (Seq Id No. 6): Phe Val Asn Gin 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 lie Val Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly Example 3 Preparation of Gly(A21)-human insulin-Aia(B31)-Ala (B32)-His(B33)-His(B34)- Arg(B35)-OH The expression vector is constructed according to Example 1. Plasmid plNT150d is used as the template for two PCR reactions, which are independent of one another, with the primer pairs TIR and pINT 316a (Seq Id No. 15): 5' GAT GCC GCG ATG ATG CGC CGC GGT CTT GGG TGT GTA G 3' or Insul 1 and pINT 316b (Seq Id No. 16): 5' A CCC AAG ACC GCG GCG CAT CAT CGC GGC ATC GTG GAG 3'. The PCR fragments to which both reactions lead are partially complementary and in a third PCR reaction afford the complete sequence which codes for the desired insulin variant. The fragment of the reaction is treated with the enzymes Nc01 and Sail and then ligated into the Nc01/Sail-opened residual plasmid of the plNT90d DNA. Plasmid plNT316 results, which after transformation by E.coli K12 W3110 is used as a basis for the expression of the desired pre-miniproinsulin. Fermentation and working up are carried out as in Example 1, the carboxypeptidase B reaction being dispensed with. The preproinsulin 3 obtained 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 Gin 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 lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly Preproinsulin 3 corresponds to the formula II, in this case R isPhe(B1), R is a peptide having 11 amino acid residues, Y is Thr (830), Z is Ala Ala His His Arg (831 -835), F? isGly(A21)and A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and B2-B29 is the amino acid sequence of the B chain of human insulin (amino acid residues 2 to 29). The preproinsulin 3 is then reacted with trypsin to give insulin 3. Insulin 3 corresponds to the formula I, in this case R is Phe (81), Y is Thr (830), Z is Ala Ala His His (831-B34), R2 isGly(A21)and A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and B2-B29 is the amino acid sequence of the B chain of human insulin (amino acid residues 2 to 29). Insulin 3 has the following amino acid sequence: Insulin 3 (Seq Id No. 8): Phe Val Asn Gin 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 Gly lie Val Glu Gin Cys Cys Thr Ser He Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly Example 4 Insulin 2 prepared according to Example 2 is reacted with carboxypeptidase B according to Example 11 to give insulin 4. Insulin 4 corresponds to the formula I, in this case R isPhe(B1), Y is Thr (B30), Z isAlaHisHis(B31-B33), R isGly(A21)and A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and B2-B29 is the amino acid sequence of the B chain of human insulin (amino acid residues 2 to 29). Insulin 4 has the following amino acid sequence: Insulin 4 (Seq Id No. 9): Phe Val Asn Gin 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 Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly Example 5 Zinc binding of insulin derivatives Insulins of the formula I according to the invention: A preparation of insulin (0.243 mM human insulin, 0.13 M NaCI, 0.1 % phenol, 80 \|jg/ml (1.22 mM) of zinc, 10 mM tris/HCI, pH 7.4) is dialyzed at ISX against 10 mM tris/HCI pH 7.4 for a total of 40 hours (buffer exchange after 16 h and 24 h). The dialyzates are then acidified and the concentration of insulin is determined by HPLC and zinc by atomic absorption spectroscopy. The zinc values are corrected using the zinc content of a control batch which contains no insulin. Table 1 shows the results: Example 6 Zinc dependence of the profile of action of human insulin in the dog Administration: Subcutaneous Dose: 0.3 lU/kg; pH of the preparation 4.0 Number of dogs (n) per experiment is 6 Table 2 shows the blood glucose in % of the starting value. Table 2 Example 7 Profile of action of Gly(A21 )Ala{B31 )His(B32),His(B33),Arg(B34) human insulin in the dog (Insulin 2) The insulin 2 prepared according to Example 2 is employed in the following formulation: Glycerol 20 mg/ml, m-cresol 2.7 mg/ml, insulin 2 40 lU/ml lU represents international units and corresponds to approximately 6 nmol of insulin, e.g. human insulin or insation of the formula I. The pH is adjusted using NaOH or HCI. Administration: Subcutaneous; dose: 0.3 lU/kg; the number of dogs tested is 6; pH of the preparation 4.0 Table 3 shows the blood glucose in % of the starting value. Table 3 Example 8 Profile of action of Gly(A21)Ala(B31)His(B32),His(B33) human insulin in the dog (Insulin 4) The insulin 4 prepared according to Example 4 is formulated and employed as in Example 7. Administration: Subcutaneous; dose: 0.3 lU/kg; n = 6; pH of the preparation 4.0 Table 4 shows the blood glucose in % of the starting value. Example 9 Profile of action of Gly(A21)His(B31),His(B32) human insulin in the dog (Insulin 1) The insulin 1 prepared according to Example 1 is formulated and employed as in Example 7. Administration: Subcutaneous; dose; 0.3 lU/kg; n = 6; pH of the preparation 4.0 Table 5 shows the blood glucose in % of the starting value. Table 5 Example 10 Profile of action of GIy(A21 )Ala(B31 )Ala(B32)His(B33)Hls(B34) human insulin in the dog (Insulin 3) The insulin 3 prepared according to Example 1 is formulated and employed as in Example 7. Administration: Subcutaneous; dose: n = 6; pH of the preparation 4.0 Table 6 shows the blood glucose in % of the starting value. Table 6 Example 11 Preparation of insulin from preproinsulin 1 y 200 mg of the insulin, one Arg being on the carboxy terminus of the B chain, prepared according to Example 1 is dissolved in 95 ml of 10 mM HCl. After addition of 5 ml of 1 M tris/HCI (tris(hydroxymethyl)aminomethane) pH 8, the pH is adjusted to 8 using HCl or NaOH. 0.1 mg of carboxypeptidase B is added. After 90 minutes, the cleavage of the arginine is complete. The mixture is adjusted to pH 3.5 by addition of HCl and pumped onto a reversed phase column (PLRP-S RP 300 10 M, 2.5 x 30 cm. Polymer Laboratories Amherst, MA, USA). The mobile phase A is water with 0.1% trifluoroacetic acid. Phase B consists of acetonitrile with 0.09% trifluoroacetic acid. The column is operated at a flow rate of 5 ml/min. After application, the column is washed with 150 ml of A. The fractional elution is carried out by applying a linear gradient of 22.5 to 40% B in 400 minutes. The fractions are analyzed individually by analytical reversed phase HPLC and those which contain Des-Arg-insulin of sufficient purity are combined. The pH is adjusted to 3.5 using NaOH and the acetonitrile is removed in a rotary evaporator. The Des-Arg-insulin is then precipitated by setting a pH of 6.5. The precipitate is centrifuged off, washed twice with 50 ml of water in each case and finally freeze-dried. The yield is 60 to 80% of insulin 1. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Hoechst Aktiengesellschaft (B) STREET: - (C) CITY: Frankfurt am Main (D) STATE: - (E) COUNTRY: Bundesrepublik Deutschland (F) POSTAL CODE: 65926 (G) TELEPHONE: 069-305-6047 (H) TELEFAX: 069-35-7175 (I) TELEX: 041234-700 hod (ii) TITLE OF APPLICATION: Insulin derivatives with increased zinc binding (iii) NUMBER OF SEQUENCES: 16 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin 15 10 15 Leu Glu Asn Tyr Cys Asn 20 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: l.,30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val GLu Ala Leu Tyr 15 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 65 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: 1..65 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His 15 10 15 Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu 20 25 30 Arg Gly Phe Phe Tyr Thr Pro Lys Thr His His Arg Gly lie Val Glu 35 40 45 Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys 50 55 60 Gly 65 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: 1..53 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 15 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr His His 20 25 30 Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu 35 40 45 Glu Asn Tyr Cys Gly 50 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: 1..66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His 15 10 15 Leu Cys Gly Ser His Leu Val Glu Ala Leu Tvr Leu Val Cys Gly Glu 20 25 ' 30 Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala His His Arg Gly lie Val 35 40 45 Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr 50 55 60 Cys Gly 65 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS. Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: 1,.55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 15 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala His 20 25 30 His Arg Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr 35 40 45 Gin Leu Glu Asn Tyr Cys Gly 50 55 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 67 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: l.,67 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His 15 10 15 Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu 20 25 30 Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala Ala His His Arg Gly lie 35 40 45 Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn 50 55 60 Tyr Cys Gly 65 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: 1..55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 15 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala Ala 20 25 30 His His Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr 35 40 45 Gin Leu Glu Asn Tyr Cys Gly 50 55 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 amino acids (B) TYPE: Amino acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Escherichia coli (ix) FEATURES: (A) NAME/KEY: Protein (B) LOCATION: 1..54 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 15 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala His 20 25 30 His Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin 35 40 45 Leu Glu Asn Tyr Cys Gly 50 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURES: (A) NAWE/KEY: exon (B) LOCATION: 1..39 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TTTTTTGTCG ACCTATTAGC CGCAGTAGTT CTCCAGCTG (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURES: (A) NAME/KEY: exon (B) LOCATION: 1,.31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 GATGCCGCGG TGGTGGGTCT TGGGTGTGTA G (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURES: (A) NAME/KEY: exon (B) LOCATION: 1..31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12; ACCCAAGACC CACCACCGCG GCATCGTGGA G (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURES: (A) NAME/KEY: exon (B) LOCATION: 1..34 (xi) SEQUKNCE DESCRIPTION: SEQ ID NO: 13; GATGCCGCGG TGGTGCGCGG TCTTGGGTGT GTAG (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURES: (A) NAME/KEY: exon (B) LOCATION: 1..34 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACCCAAGACC GCGCACCACC GCGGCATCGT GGAG (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURES: (A) NAME/KEY: exon (B) LOCATION: 1..37 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GATGCCGCGA TGATGCGCCG CGGTCTTGGG TGTGTAG (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURES: (A) NAME/KEY: exon (B) LOCATION: 1..37 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: ACCCAAGACC GCGGCGCATC ATCGCGGCAT CGTGGAG WE CLAIM: 1. An insulin of the formula I or a physiologically tolerable salt of the insulin of the formula I, where R1 is a phenylalanine residue or a hydrogen atom, R is a genetically encodable amino acid residue, Y is a genetically encodable amino acid residue (position B30), Z is a) a histidine residue or b) a peptide having 2 to 35 genetically encodable amino acid residues, containing 1 to 5 histidine residues, and the residues A2-A20 correspond to the amino acid sequence of the A chain of human insulin, animal insulin or an insulin derivative and the residues B2-B29 correspond to the amino acid sequence of the B chain of human insulin, animal insulin or an insulin derivative. 2. The insulin of the formula I as claimed in claim I, where R1 is a phenylalanine residue, R3 is an amino acid residue from the group consisting of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gin, Y is an amino acid residue from the group consisting of Ala, Thr, Ser and His, Z is a) the histidine residue or b) a peptide having 4 to 7 amino acid residues, containing 1 or 2 histidine residues. 3. The insulin of the formula I as claimed in claim 1, where R1 is a phenylalanine residue, R3 is an amino acid residue from the group consisting of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gin, Y is an amino acid residue from the group consisting of Ala, Thr, Ser and His, Z is a) the histidine residue or b) a peptide having 2 to 7 amino acid residues, containing 1 or 2 histidine residues. 4. The insulin of the formula I as claimed in claim 3, where Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues. 5. The insulin of the formula I as claimed in one or more of 1 to 4, where R is a phenylalanine residue, R is an amino acid residue from the group consisting of Asn and Gly, Y is an amino acid residue from the group consisting of Thr and His, and Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues. 6. The insulin of the formula I as claimed in claim I, where R is a phenylalanine residue, R is a glycine residue, Y is a threonine residue' and Z is a peptide having 1 to 5 amino acid residues. 7. The insulin of the formula I as claimed in claim 6, where Z is a peptide having the sequence His His, His His Arg, Ala His His, Ala His His Arg, Ala Ala His His Arg or Ala Ala His His. 8. A complex containing an insulin hexamer and 5 to 9 mol of zinc per insulin hexamer, in particular 5 to 7 mol of zinc per insulin hexamer, the insulin hexamer consisting of six insulin molecules of the formula I as claimed in one or more of claims 1 to 7. 9. A pharmaceutical preparation, which contains an efficacious amount of at least one insulin of the formula I and/or at least one physiologically tolerable salt of the insulin of the formula I as claimed in one or more of claims 1 to 8 in dissolved, morphous and/or crystalline form. 10. The pharmaceutical preparation as claimed in claim 9, which has an additional content of 1 ug to 2 mg, preferably from 5 ug to 200 ug, of zinc/ml. 11. The pharmaceutical preparation as claimed in claim 9 or 10, which has a pH of 2.5 to 8.5. 12. The pharmaceutical preparation as claimed in one or more of claims 9 to 11, which has a pH of 2.5 to 4.5. 13. The pharmaceutical preparation as claimed in one or more of claims 9 to 12, which has an additional content of unmodified insulin, preferably unmodified human insulin, or modified insulin, preferably Gly(A21)-Arg(B31)-Arg(B32)-human insulin. 14. A proinsulin of the formula II where R3 and Y as in formula I are defined as set forth in one or more of claims 1 to 7, R is a phenylalanine residue or a covalent bond, R2 is a) a genetically encodable amino acid residue or b) a peptide having 2 to 45 amino acid residues, and the residues A2 -A20 and B2 - B29 correspond to the amino acid sequences of the A and B chains of human insulin, animal insulin or an insulin derivative, and Z' is a peptide having 2 to 40 genetically encodable amino acids residues, having 1 to 5 histidine residues. 15. The proinsulin of the formula II as claimed in claim 14, where R2 is a peptide having 2 to 25 amino acid residues. 16. The proinsulin of the formula II as claimed in claim 14 or 15, where R2 is a peptide having 2 to 15 amino acid residues, in which an amino acid residue from the group consisting of Met, Lys and Arg is at the carboxyl end.

Full Text

The pharmacokinetics of subcutaneously administered insulin is dependent on its association behavior. Insulin forms hexamers in neutral aqueous solution. If the insulin is to get from the tissue into the blood stream and to the site of action, it must first pass through the walls of the capillaries. It is assumed that this is only possible for monomeric and for dimeric insulin - but not possible or only slightly possible for hexameric insulins or relatively high molecular weight associates (Brange et al., Diabetes Care: 13 (1990), pages 923-954; Kang et al. Diabetes Care: 14 (1991), pages 942-948). The dissociation of the hexamer is therefore a prerequisite for rapid passage from the subcutaneous tissue into the blood stream.
The association and aggregation behavior of insulin is affected by zinc , which leads to a stabilization of the hexamer and at pHs around the neutral point to the formation of relatively high molecular weight aggregates until precipitation occurs. Zinc** as an additive to an unbuffered human insulin solution (pH 4), however, only slightly affects the profile of action. Although the solution in the tissue is rapidly neutralized and insulin-zinc complexes are formed, the natural zinc binding of the insulin is insufficient to stabilize hexamers and higher aggregates if the release of the insulin is markedly delayed and a strong depot effect is achieved. Known insulin hexamers have a content of approximately 2 mol of zinc per mole of insulin hexamer (Blundell et al., Adv. Protein Chem.: 26 (1972), pages 323-328). Two zinc molecules per insulin hexamer are so firmly bound to the insulin hexamer that they cannot be removed by normal dialysis. So-called 4-zinc insulin crystals have admittedly been described, but these crystals on average contain only less than three mol of zinc per mole of insulin hexamer (G.D. Smith etal., Proc. Natl. Acad. Sci. USA: 81, pages 7093-7097).
The object of the present invention is to find insulin derivatives which have an increased zinc binding power, form a stable complex containing insulin hexamer and

zinc and have a delayed profile of action on subcutaneous injection in companson with human insulin.

and/or physiologically tolerable salts of the insulins of the formula I have now been
found which fulfil the abovementioned criteria and wherein
R is a phenylalanine residue or a hydrogen atom,
R1 is a genetically encodable amino acid residue,
Y is a genetically encodable amino acid residue,
Z is a) the amino acid residue His or
b) a peptide having 2 to 35 genetically encodable amino acid residues, containing 1 to 5 histidine residues, and the residues A2-A20 correspond to the amino acid sequence of the A chain of human insulin, animal insulin or an insulin derivative and the residues B2-B29 correspond to the amino acid sequence of the B chain of human insulin, animal insulin or an insulin derivative.
An insulin of the formula I is particularly preferred where R1 is a phenylalanine residue,
R is an amino acid residue from the group consisting of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gin,

- Y is an amino acid residue from the group consisting of Ala, Thr, Ser and
His, 2. is a) the amino acid residue His or
b) a peptide having 4 to 7 amino acid residues, containing 1 or 2 histidine residues.
An insulin of the formula I is furthermore preferred where R is a phenylalanine residue,
R is an amino acid residue from the group consisting of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gin,
Y is an amino acid residue from the group consisting of Ala, Thr, Ser and
His, Z is a) the amino acid residue His or
b) a peptide having 2 to 7 amino acid residues, containing 1 or 2 histidine residues.
An insulin of the formula I is particularly preferred where
Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues.
An insulin of the formula I is particularly preferred, where
R is a phenylalanine residue,
R is an amino acid residue from the group consisting of Asn and Gly,
Y is an amino acid residue from the group consisting of Thr and His, and
Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues.
An insulin of the formula I is furthermore preferred where
R is a phenylalanine residue, R is a glycine residue, Y is a threonine residue and
Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine
residues.

An insulin of the formula I is very particularly preferred where
Z is a peptide having 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 peptides and proteins is indicated from the N-terminal end of the amino acid chain onward. The details given in brackets in formula I, e.g. A1, A6, A7, A11, A20, B1, B7, B19 or 830, correspond to the position of amino acid residues in the A or 8 chains of the insulin.
The expression "genetically encodable amino acid residue" represents the residues
of the amino acids Gly, Ala, Ser, Thr, Val, Leu, lie. Asp, Asn, Glu, Gin, Cys, Met,
Arg, Lys, His, Tyr, Phe, Trp, Pro and selenocysteine.
The expressions "residues A2 - A20" and "residues 82 - B 29" of animal insulin are
understood, for example, as meaning the amino acid sequences of insulin from
cattle, pigs or chickens.
The expression residues A2 - A20 and 82 - 829 of insulin derivatives represents the
corresponding amino acid sequences of human insulin which are formed by the
replacement of amino acids by other genetically encodable amino acids.
The A chain of human insulin has the following sequence (Seq Id No. 1): Gly, lie, Val, Glu, Gin, Cys, Cys, Thr, Ser, lie, Cys, Ser, Leu, Tyr, Gin, Leu, Glu, Asn, Tyr, Cys, Asn.
The B chain of human insulin has the following sequence (Seq Id No. 2 ): Phe, Val, Asn, Gin, 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.
The insulin derivative of the formula I can be formed in microorganisms with the aid of a multiplicity of genetic engineering constructs (EP 0 489 780, EP 0 347 781, EP 0 368 187, EP 0 453 969). The genetic engineering constructs are expressed in microorganisms such as Escherichia coli or Streptomycetes during fermentation.

- The proteins formed are stored in the interior of the microorganisms (EP 0 489 780) or secreted into the fermentation solution.
Exemplary insulins of the formula I are:
Gly(A21 )-human insulin-His(B31 )-His(B32)-OH
Gly(A21)-human insulin-His(B31)-His(B32)-Arg (B33)-OH
Gly(A21 )-human insulin-Ala(B31 )-His(B32)-His(B33)-OH
Gly(A21 )-human insulin-Ala(B31 )-His(B32)-His(B33)-Arg(B34)-OH
Gly(A21 )-human insulin-Ala(B31 )-Ala(B32)-His(B33)-His(B34)-OH
Gly(A21 )-human insulin-Ala(B31 )-Ala(B32)-His(B33)-His(B34)-Arg(B35)-OH
The insulin derivatives of the formula I are mainly prepared by genetic engineering by means of site-directed mutagenesis according to standard methods. For this purpose, a gene structure coding for the desired insulin derivative of the formula I is constructed and expressed in a host cell - preferably in a bacterium such as E. coli or a yeast, in particular Saccharomyces cerevisiae - and - if the gene structure codes for a fusion protein - the insulin derivative of the formula I is released from the fusion protein; analogous methods are described, for example, in EP-A-0 211 299, EP-A-0 227 938, EP-A-0 229 998, EP-A-0 286 956 and the DE Patent Application P 38 21 159.
After cell disruption, the fusion protein portion is cleaved either chemically by means of cyangen halide - see EP-A-0 180 920 - or enzymatically by means of lysostaphin or trypsin - see DE-A-37 39 347.
The insulin precursor is then subjected to oxidated sulfitolysis according to the method described, for example, by R.C. Marshall and A.S. Inglis in "Practical Protein Chemistry - A Handbook" (Editor A. Darbre) 1986, pages 49 - 53 and then renatured in the presence of a thiol with formation of the correct disulfide bridges, e.g. according to the method described by G.H. Dixon and A.C. Wardlow in Nature (1960), pages 721-724. The insulin precursors, however, can also be directly folded (EP-A-0 600 372 ; EP-A-0 668 292).

- The C peptide is removed by means of tryptic cleavage - e.g. according to the method of Kemmieretal., J.B.C. (1971), pages 6786 - 6791 and the insulin
" derivative of the formula I is purified by means of known techniques such as chromatography - e.g. EP-A-0 305 760 - and crystallization.
The invention furthermore relates to complexes containing an insulin hexamer and approximately 5 to 9 mol zinc per insulin hexamer preferably 5 to 7 mol of zinc per insuin hexamer, the insulin hexamer consisting of six insulin molecules of the formula I.
The zinc binding to the insulin hexamer is so firm that the 5 to 9 mol of zinc per mole of insulin hexamer cannot be removed by 40 hours of normal dialysis, for example with an aqueous 10 mM tris/HCI buffer, pH 7.4.
After subcutaneous administration, insulins of the formula I, as in the essentially zinc-free preparation (pH 4), show a small delay in action in comparison with human insulin. After addition of approximately 20 pg of zinc'/ml of preparation a later onset of action is observed even after subcutaneous administration. The delay in action is preferably observed at 40 pg of zinc'/ml. Higher zinc concentrations potentiate this effect.
The invention furthermore relates to preproinsulin of the formula II
R2-Rl-B2-B29-Y-Z-Gly-A2-A20-R (II)
where R, Y and Z as in formula I are defined as set forth in one or more of claims 1
to 6, and R' is a phenylalanine residue or a covalent bond, and R2 is a) a genetically encodable amino acid residue or
b) a peptide having 2 to 45 amino acid residues, and the residues A2 - A20 and 82 - 829 correspond to the amino acid sequences of the A and B chains of human insulin, animal insulin or an insulin derivative.

- The proinsulin of the formula II is suitable as an intermediate in the preparation of the insulins of the formula I.
Preferred proinsulins of the formula 11 are those where R is a peptide having 2 to 25 amino acid residues.
Particularly preferred proinsulins of the formula II are those where
R is a peptide having 2 to 15 amino acid residues, in which an amino acid
residue from the group consisting of Met, Lys and Arg is at the
carboxyl end.
The insulin derivatives of the formula I and/or the complexes according to the invention, containing an insulin hexamer and 5 to 9 mol of zinc per hexamer and/or their physiologically tolerable salts (e.g. the alkali metal or ammonium salts), are mainly used as active compounds for a pharmaceutical preparation for the treatment of diabetes, in particular of diabetes mellitus.
The pharmaceutical preparation is preferably a solution or suspension for injection purposes; it comprises at least one insulin derivative of the formula I and/or the complex according to the invention and/or at least one of its physiologically tolerable salts in dissolved, amorphous and/or crystalline - preferably in dissolved - form.
The preparation preferably has a pH from approximately 2.5 to 8.5, in particular from approximately 4.0 to 8.5, contains a suitable isotonicizing agent, a suitable preservative and, if appropriate, a suitable buffer, and preferably also a specific zinc ion concentration, all, of course, in sterile aqueous solution. The whole of the preparation constituents apart from the active compound forms the preparation excipient.
Preparations containing solutions of the insulin of the formula I have a pH from 2.5 to 4.5, in particular from 3.5 to 4.5, preferably 4.0.
Preparations containing suspensions of the insulin of the formula I have a pH from 6.5 to 8.5, in particular from 7.0 to 8.0, preferably 7.4.

- Suitable isotonicizing agents are, for example, glycerol, glucose, mannitol, NaCI, calcium compounds or magnesium compounds, such as CaCl2 or MgCl2.
As a result of the choice of the isotonicizing agent and/or preservative, the solubility of the insulin derivative or of its physiologically tolerable salts Is affected at the weakly acidic pHs.
Suitable preservatives are, for example, phenol, m-cresol, benzyl alcohol and/or p-hydroxybenzoic acid esters.
Buffer substances which can be used, in particular for adjusting a pH of between approximately 4.0 and 8.5, are, for example sodium acetate, sodium citrate or sodium phosphate. Otherwise, physiologically acceptable dilute acids (typically HCI) or alkalis (typically NaOH) are also suitable for adjusting the pH.
If the preparation has a content of zinc, one of from 1 pg to 2 mg of zinc/ml, in particular from 5 |jg to 200 pg of zinc/ml, is preferred.
To vary the profile of action of the preparation according to the invention, unmodified insulin, preferably bovine, porcine or human insulin, in particular human insulin, or modified insulins, for example monomeric insulins, rapid-acting insulins or Gly(A21)-Arg(B31)-Arg(B32)-human insulin, can also be admixed.
Preferred active compound concentrations are those corresponding to approximately 1 -1500, furthermore preferably approximately 5 -1000 and in particular approximately 40 - 400, international units/ml.

- Example 1
" Preparation of Gly(A21)-human insulin-His(B31)-His(B32)-OH
The preparation of the expression system was carried out essentially as in US 5,358,857. The vectors pINT 90d and pINT 91 d (see Example 17) and the PCR primers TIR and Insul 1 are also described there. These four components are used, inter alia, as starting materials for the vector constructs described in the following.
First, the codon for Gly (A21) is inserted in the sequence coding for the mini-proinsulin. To do this, pINT 91 d is used as a template and a PCR reaction is carried out with the primers TIR and Insu31 Insu31 (SeqIdNo. 10):
5'TTT TTT GTC GAC CTA TTA GCC GCA GTA GTT CTC CAG CTG 3'
The PCR cycle is carried out as follows. 1st minute 94°C, 2nd minute 55'C, 3rd minute 72°C. This cycle is repeated 25 times then the mixture is incubated at 72"'C for 7 minutes and subsequently at 40 overnight.
The resulting PCR fragment is precipitated in ethanol to purify it, dried and then digested in restriction buffer using the restriction enzymes NcOl and Sail according to the details of the manufacturers. The reaction mixture is then separated by gel electrophoresis and the NcOI-pre-proinsulin-Sall fragment is isolated. DNA of the plasmid plNT90d is likewise cleaved using NcOl and Sail and the monkey proinsulin fragment is in this manner released from the pINT residual plasmid. Both fragments are separated by gel electrophoresis and the residual plasmid DNA is isolated. This DNA is reacted with the NcOl-Sail PCR fragment in a ligase reaction. The plasmid plNT150d is thus obtained, which after transformation by E.coli is replicated and then reisolated.
DNA of the plasmid plNT150d is used as a starting material for the plasmid plNT302, which allows the preparation of the desired insulin variant.

For the construction of this plasmid, the route described in US 5,358,857 (see Example 6) is taken. Two PCR reactions which are independent of one another are carried out to this end, for which DNA of the plasmid plNT150d is used as a template. One reaction is carried out using the primer pair TIR and pINT B5 (SeqIdNo. 11):
5' GAT GCC GCG GIG GIG GGT CTT GGG TGT GTAG 3'
and the other reaction using the primer pair Insu11 and pINT B6 (SeqIdNo. 12):
5' A CCC AAG ACC CAC CAC CGC GGC ATC GTG GAG 3'.
The PCR fragments which result are partially complementary, so that in a third PCR reaction they lead to a fragment which codes for a Gly (A21) miniproinsulin lengthened by the position B31 and 632. This fragment is cleaved using NcOl and Sail and then reacted with the DNA of the described plNT90d residual plasmid in a ligase reaction to give the plasmid plNT302.
An E. coli K12 W3110 transformed with this plasmid is then fermented and worked up as in Example 4 of US 5,227,293. The preproinsulin derivative obtained as intermediate (before trypsin cleavage) has the following amino acid sequence:
Preproinsulin 1(Seq Id No. 3):
Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg
Phe Val Asn Gin 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 Arg
Gly lie Val Glu Gin Cys Cys Thr Ser He Cys Ser
Leu Tyr Gin Leu Glu Asn Tyr Cys Gly
Preproinsulin 1 corresponds to the formula ILin-ttiiscase R is a peptide having 11 amino acid residues, R is Phe (81),

Y is Thr (B30),
Z isHisHisArg(B31-B33),
R isGly(A21)and
A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid
residues 2 to 20) and 82-829 is the amino acid sequence of the 8 chain of human
insulin (amino acid residues 2 to 29).
The preproinsulin 1 is cleaved with trypsin as in US 5,227,293 according to Example 4. The product obtained is then reacted with carboxypeptidase 8 according to Example 11 to give insulin 1. Insulin 1 corresponds to the formula I, in this case R isPhe(81),
Y is Thr (830),
Z is His His Arg (831-833),
R isGly(A21)and
A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid
residues 2 to 20) and 82-829 is the amino acid sequence of the 8 chain of human
insulin (amino acid residues 2 to 29).
Insulin 1 has the following amino acid sequence:
Insulin 1 (Seq Id No. 4):
Phe Val Asn Gin 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
Gly He Val Glu Gin Cys Cys Thr Ser He Cys Ser
Leu Tyr Gin Leu Glu Asn Tyr Cys Gly
Example 2
Preparation of Gly(A21 )-human insulin-Ala(831)-His(832)-His(B33)-
Arg(834)-OH
The expression vector is constructed according to Example 1.
Plasmid plNT150d is used as the template for two PCR reactions, which are
independent of one another, with the primer pairs TIR and pINT 87 (Seq Id No. 13):

5' GAT GCC GCG GTG GTG CGC GGT CTT GGG TGT GTAG 3'
or Insu11 and pINT B8 (Seq Id No. 14):
5' ACCC AAG ACC GCG CAC CAC CGC GGC ATC GTG GAG 3'.
The PCR fragments to which both reactions lead are partially complementary and in
a third PCR reaction afford the complete sequence which codes for the desired
insulin variant. The fragment of the reaction is treated with the enzymes Nc01 and
Sail and then ligated into the Nc01/Sal1-opened residual plasmid of the plNT90d
DNA. Plasmid plNT303 results, which after transformation by E.coli K12 W3110 is
used as a basis for the expression of the desired pre-miniproinsulin. Fermentation
and working up is carried out as in Example 1, the carboxypeptidase B reaction
being dispensed with.
The preproinsulin derivative obtained has the following amino acid seqence:
Preproinsulin 2 (Seq Id No. 5):
Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg
Phe Val Asn Gin 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 He Val Glu Gin Cys Cys Thr Ser He Cys Ser
Leu Tyr Gin Leu Glu Asn Tyr Cys Gly
Preproinsulin 2 corresponds to the formula II, in this case
R is Phe (81),
R is a peptide having 11 amino acid residues,
Y is Thr (830),
Z is Ala His His Arg (831-834),
R isGly(A21)and
A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid
residues 2 to 20) and 82-829 is the amino acid sequence of the 8 chain of human
insulin (amino acid residues 2 to 29).

- The preproinsulin 2 is then reacted with trypsin to give insulin 2. Insulin 2
corresponds to the formula II, in this case ■ R isPhe(B1),
Y is Thr (B30),
Z is Ala His His Arg (B31-B34),
R isGly(A21)and
A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid
residues 2 to 20) and B2-B29 is the amino acid sequence of the B chain of human
insulin (amino acid residues 2 to 29).
Insulin 2 has the following amino acid sequence:
Insulin 2 (Seq Id No. 6):
Phe Val Asn Gin 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 lie Val Glu Gin Cys Cys Thr Ser He Cys Ser
Leu Tyr Gin Leu Glu Asn Tyr Cys Gly
Example 3
Preparation of Gly(A21)-human insulin-Aia(B31)-Ala (B32)-His(B33)-His(B34)-
Arg(B35)-OH
The expression vector is constructed according to Example 1.
Plasmid plNT150d is used as the template for two PCR reactions, which are
independent of one another, with the primer pairs TIR and pINT 316a (Seq Id No.
15):
5' GAT GCC GCG ATG ATG CGC CGC GGT CTT GGG TGT GTA G 3'
or Insul 1 and pINT 316b (Seq Id No. 16):
5' A CCC AAG ACC GCG GCG CAT CAT CGC GGC ATC GTG GAG 3'.

The PCR fragments to which both reactions lead are partially complementary and in
a third PCR reaction afford the complete sequence which codes for the desired
insulin variant. The fragment of the reaction is treated with the enzymes Nc01 and
Sail and then ligated into the Nc01/Sail-opened residual plasmid of the plNT90d
DNA. Plasmid plNT316 results, which after transformation by E.coli K12 W3110 is
used as a basis for the expression of the desired pre-miniproinsulin. Fermentation
and working up are carried out as in Example 1, the carboxypeptidase B reaction
being dispensed with.
The preproinsulin 3 obtained 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 Gin 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 lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser
Leu Tyr Gin Leu Glu Asn Tyr Cys Gly
Preproinsulin 3 corresponds to the formula II, in this case
R isPhe(B1),
R is a peptide having 11 amino acid residues,
Y is Thr (830),
Z is Ala Ala His His Arg (831 -835),
F? isGly(A21)and
A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid
residues 2 to 20) and B2-B29 is the amino acid sequence of the B chain of human
insulin (amino acid residues 2 to 29).
The preproinsulin 3 is then reacted with trypsin to give insulin 3. Insulin 3 corresponds to the formula I, in this case R is Phe (81),
Y is Thr (830),
Z is Ala Ala His His (831-B34), R2 isGly(A21)and

A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid residues 2 to 20) and B2-B29 is the amino acid sequence of the B chain of human insulin (amino acid residues 2 to 29).
Insulin 3 has the following amino acid sequence:
Insulin 3 (Seq Id No. 8):
Phe Val Asn Gin 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
Gly lie Val Glu Gin Cys Cys Thr Ser He Cys Ser
Leu Tyr Gin Leu Glu Asn Tyr Cys Gly
Example 4
Insulin 2 prepared according to Example 2 is reacted with carboxypeptidase B
according to Example 11 to give insulin 4.
Insulin 4 corresponds to the formula I, in this case
R isPhe(B1),
Y is Thr (B30),
Z isAlaHisHis(B31-B33),
R isGly(A21)and
A2-A20 is the amino acid sequence of the A chain of human insulin (amino acid
residues 2 to 20) and B2-B29 is the amino acid sequence of the B chain of human
insulin (amino acid residues 2 to 29).
Insulin 4 has the following amino acid sequence:
Insulin 4 (Seq Id No. 9):
Phe Val Asn Gin 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
Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser
Leu Tyr Gin Leu Glu Asn Tyr Cys Gly

Example 5
Zinc binding of insulin derivatives
Insulins of the formula I according to the invention:

A preparation of insulin (0.243 mM human insulin, 0.13 M NaCI, 0.1 % phenol, 80 |jg/ml (1.22 mM) of zinc, 10 mM tris/HCI, pH 7.4) is dialyzed at ISX against 10 mM tris/HCI pH 7.4 for a total of 40 hours (buffer exchange after 16 h and 24 h). The dialyzates are then acidified and the concentration of insulin is determined by HPLC and zinc by atomic absorption spectroscopy. The zinc values are corrected using the zinc content of a control batch which contains no insulin. Table 1 shows the results:

Example 6
Zinc dependence of the profile of action of human insulin in the dog
Administration: Subcutaneous
Dose: 0.3 lU/kg; pH of the preparation 4.0
Number of dogs (n) per experiment is 6

Table 2 shows the blood glucose in % of the starting value.
Table 2
Example 7
Profile of action of Gly(A21 )Ala{B31 )His(B32),His(B33),Arg(B34) human insulin in
the dog (Insulin 2)
The insulin 2 prepared according to Example 2 is employed in the following
formulation:
Glycerol 20 mg/ml, m-cresol 2.7 mg/ml, insulin 2 40 lU/ml
lU represents international units and corresponds to approximately 6 nmol of insulin,
e.g. human insulin or insation of the formula I. The pH is adjusted using NaOH or
HCI.
Administration: Subcutaneous; dose: 0.3 lU/kg;
the number of dogs tested is 6; pH of the preparation 4.0

Table 3 shows the blood glucose in % of the starting value.
Table 3
Example 8
Profile of action of Gly(A21)Ala(B31)His(B32),His(B33) human insulin in the dog
(Insulin 4)
The insulin 4 prepared according to Example 4 is formulated and employed as in
Example 7.
Administration: Subcutaneous; dose: 0.3 lU/kg;
n = 6; pH of the preparation 4.0
Table 4 shows the blood glucose in % of the starting value.

Example 9
Profile of action of Gly(A21)His(B31),His(B32) human insulin in the dog (Insulin 1)
The insulin 1 prepared according to Example 1 is formulated and employed as in
Example 7.
Administration: Subcutaneous; dose; 0.3 lU/kg;
n = 6; pH of the preparation 4.0
Table 5 shows the blood glucose in % of the starting value.
Table 5

Example 10
Profile of action of GIy(A21 )Ala(B31 )Ala(B32)His(B33)Hls(B34) human insulin in the
dog (Insulin 3)
The insulin 3 prepared according to Example 1 is formulated and employed as in

Example 7.
Administration: Subcutaneous; dose:
n = 6; pH of the preparation 4.0
Table 6 shows the blood glucose in % of the starting value.
Table 6

Example 11
Preparation of insulin from preproinsulin 1 y
200 mg of the insulin, one Arg being on the carboxy terminus of the B chain, prepared according to Example 1 is dissolved in 95 ml of 10 mM HCl. After addition of 5 ml of 1 M tris/HCI (tris(hydroxymethyl)aminomethane) pH 8, the pH is adjusted to 8 using HCl or NaOH.
0.1 mg of carboxypeptidase B is added. After 90 minutes, the cleavage of the arginine is complete. The mixture is adjusted to pH 3.5 by addition of HCl and pumped onto a reversed phase column (PLRP-S RP 300 10 M, 2.5 x 30 cm. Polymer

Laboratories Amherst, MA, USA). The mobile phase A is water with 0.1% trifluoroacetic acid. Phase B consists of acetonitrile with 0.09% trifluoroacetic acid. The column is operated at a flow rate of 5 ml/min. After application, the column is washed with 150 ml of A. The fractional elution is carried out by applying a linear gradient of 22.5 to 40% B in 400 minutes. The fractions are analyzed individually by analytical reversed phase HPLC and those which contain Des-Arg-insulin of sufficient purity are combined. The pH is adjusted to 3.5 using NaOH and the acetonitrile is removed in a rotary evaporator. The Des-Arg-insulin is then precipitated by setting a pH of 6.5. The precipitate is centrifuged off, washed twice with 50 ml of water in each case and finally freeze-dried. The yield is 60 to 80% of insulin 1.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Hoechst Aktiengesellschaft
(B) STREET: -
(C) CITY: Frankfurt am Main
(D) STATE: -
(E) COUNTRY: Bundesrepublik Deutschland
(F) POSTAL CODE: 65926
(G) TELEPHONE: 069-305-6047
(H) TELEFAX: 069-35-7175
(I) TELEX: 041234-700 hod
(ii) TITLE OF APPLICATION: Insulin derivatives with increased zinc binding
(iii) NUMBER OF SEQUENCES: 16
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: 1..21
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin
15 10 15
Leu Glu Asn Tyr Cys Asn 20
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli

(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: l.,30
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val GLu Ala Leu Tyr
15 10 15
Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr
20 25 30
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: 1..65
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His
15 10 15
Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu
20 25 30
Arg Gly Phe Phe Tyr Thr Pro Lys Thr His His Arg Gly lie Val Glu
35 40 45
Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys
50 55 60
Gly 65
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: 1..53

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
15 10 15
Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr His His
20 25 30
Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu
35 40 45
Glu Asn Tyr Cys Gly 50
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: 1..66
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His
15 10 15
Leu Cys Gly Ser His Leu Val Glu Ala Leu Tvr Leu Val Cys Gly Glu
20 25 ' 30
Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala His His Arg Gly lie Val
35 40 45
Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr
50 55 60
Cys Gly 65
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS. Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli

(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: 1,.55
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
15 10 15
Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala His
20 25 30
His Arg Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr
35 40 45
Gin Leu Glu Asn Tyr Cys Gly
50 55
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: l.,67
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Asn Gin His
15 10 15
Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu
20 25 30
Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala Ala His His Arg Gly lie
35 40 45
Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn
50 55 60
Tyr Cys Gly 65
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: 1..55
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
15 10 15
Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala Ala
20 25 30
His His Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr
35 40 45
Gin Leu Glu Asn Tyr Cys Gly
50 55
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 amino acids
(B) TYPE: Amino acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Escherichia coli
(ix) FEATURES:
(A) NAME/KEY: Protein
(B) LOCATION: 1..54
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
15 10 15
Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Ala His
20 25 30
His Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin
35 40 45
Leu Glu Asn Tyr Cys Gly 50
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:
(A) NAWE/KEY: exon
(B) LOCATION: 1..39
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TTTTTTGTCG ACCTATTAGC CGCAGTAGTT CTCCAGCTG
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURES:
(A) NAME/KEY: exon
(B) LOCATION: 1,.31
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 GATGCCGCGG TGGTGGGTCT TGGGTGTGTA G
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURES:
(A) NAME/KEY: exon
(B) LOCATION: 1..31
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12; ACCCAAGACC CACCACCGCG GCATCGTGGA G
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURES:
(A) NAME/KEY: exon
(B) LOCATION: 1..34

(xi) SEQUKNCE DESCRIPTION: SEQ ID NO: 13; GATGCCGCGG TGGTGCGCGG TCTTGGGTGT GTAG
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURES:
(A) NAME/KEY: exon
(B) LOCATION: 1..34
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACCCAAGACC GCGCACCACC GCGGCATCGT GGAG
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURES:
(A) NAME/KEY: exon
(B) LOCATION: 1..37
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GATGCCGCGA TGATGCGCCG CGGTCTTGGG TGTGTAG
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURES:
(A) NAME/KEY: exon
(B) LOCATION: 1..37
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: ACCCAAGACC GCGGCGCATC ATCGCGGCAT CGTGGAG

WE CLAIM:
1. An insulin of the formula I

or a physiologically tolerable salt of the insulin of the formula I, where
R1 is a phenylalanine residue or a hydrogen atom, R is a genetically encodable amino acid residue,
Y is a genetically encodable amino acid residue (position B30),
Z is a) a histidine residue or
b) a peptide having 2 to 35 genetically encodable amino acid residues, containing 1 to 5 histidine residues, and the residues A2-A20 correspond to the amino acid sequence of the A chain of human insulin, animal insulin or an insulin derivative and the residues B2-B29 correspond to the amino acid sequence of the B chain of human insulin, animal insulin or an insulin derivative.
2. The insulin of the formula I as claimed in claim I, where
R1 is a phenylalanine residue,
R3 is an amino acid residue from the group consisting of Asn, Gly, Ser, Thr,
Ala, Asp, Glu and Gin,
Y is an amino acid residue from the group consisting of Ala, Thr, Ser and His,

Z is a) the histidine residue or
b) a peptide having 4 to 7 amino acid residues, containing 1 or 2 histidine residues.
3. The insulin of the formula I as claimed in claim 1, where
R1 is a phenylalanine residue,
R3 is an amino acid residue from the group consisting of Asn, Gly, Ser, Thr,
Ala, Asp, Glu and Gin,
Y is an amino acid residue from the group consisting of Ala, Thr, Ser and
His,
Z is a) the histidine residue or
b) a peptide having 2 to 7 amino acid residues, containing 1 or 2 histidine residues.
4. The insulin of the formula I as claimed in claim 3, where
Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues.
5. The insulin of the formula I as claimed in one or more of 1 to 4, where
R is a phenylalanine residue,
R is an amino acid residue from the group consisting of Asn and Gly,
Y is an amino acid residue from the group consisting of Thr and His, and
Z is a peptide having 1 to 5 amino acid residues, containing 1 or 2 histidine residues.
6. The insulin of the formula I as claimed in claim I, where
R is a phenylalanine residue, R is a glycine residue, Y is a threonine residue' and Z is a peptide having 1 to 5 amino acid residues.

7. The insulin of the formula I as claimed in claim 6, where
Z is a peptide having the sequence His His, His His Arg, Ala His His, Ala His His Arg, Ala Ala His His Arg or Ala Ala His His.
8. A complex containing an insulin hexamer and 5 to 9 mol of zinc per insulin hexamer, in particular 5 to 7 mol of zinc per insulin hexamer, the insulin hexamer consisting of six insulin molecules of the formula I as claimed in one or more of claims 1 to 7.
9. A pharmaceutical preparation, which contains an efficacious amount of at least one insulin of the formula I and/or at least one physiologically tolerable salt of the insulin of the formula I as claimed in one or more of claims 1 to 8 in dissolved, morphous and/or crystalline form.
10. The pharmaceutical preparation as claimed in claim 9, which has an additional content of 1 ug to 2 mg, preferably from 5 ug to 200 ug, of zinc/ml.
11. The pharmaceutical preparation as claimed in claim 9 or 10, which has a pH of 2.5 to 8.5.
12. The pharmaceutical preparation as claimed in one or more of claims 9 to 11, which has a pH of 2.5 to 4.5.
13. The pharmaceutical preparation as claimed in one or more of claims 9 to 12, which has an additional content of unmodified insulin, preferably unmodified human insulin, or modified insulin, preferably Gly(A21)-Arg(B31)-Arg(B32)-human insulin.
14. A proinsulin of the formula II

where R3 and Y as in formula I are defined as set forth in one or more of claims 1 to 7,

R is a phenylalanine residue or a covalent bond,
R2 is a) a genetically encodable amino acid residue or
b) a peptide having 2 to 45 amino acid residues, and the residues A2 -A20 and B2 - B29 correspond to the amino acid sequences of the A and B chains of human insulin, animal insulin or an insulin derivative, and
Z' is a peptide having 2 to 40 genetically encodable amino acids residues, having 1 to 5 histidine residues.
15. The proinsulin of the formula II as claimed in claim 14, where
R2 is a peptide having 2 to 25 amino acid residues.
16. The proinsulin of the formula II as claimed in claim 14 or 15, where
R2 is a peptide having 2 to 15 amino acid residues, in which an amino acid residue from the group consisting of Met, Lys and Arg is at the carboxyl end.

Documents:

1565-mas-1997 abstract.jpg

1565-mas-1997 abstract.pdf

1565-mas-1997 abstractduplicate.pdf

1565-mas-1997 claims-duplicate.pdf

1565-mas-1997 claims.pdf

1565-mas-1997 correspondence-others.pdf

1565-mas-1997 correspondence-po.pdf

1565-mas-1997 description (complete)-duplicate.pdf

1565-mas-1997 description (complete).pdf

1565-mas-1997 form-19.pdf

1565-mas-1997 form-2.pdf

1565-mas-1997 form-26.pdf

1565-mas-1997 form-4.pdf

1565-mas-1997 form-6.pdf

1565-mas-1997 others.pdf

1565-mas-1997 petition.pdf

1565-mas.rtf

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Patent Number

224685

Indian Patent Application Number

1565/MAS/1997

PG Journal Number

49/2008

Publication Date

05-Dec-2008

Grant Date

21-Oct-2008

Date of Filing

11-Jul-1997

Name of Patentee

HOECHST AKTIENGESELLSCHAFT

Applicant Address

D-65926 FRANKFURT AM MAIN,

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. JOHANN ERTL	GINSTRWEG 10, 65817 BREMTHAL,
2	DR. PAUL HABERMANN	ROSSERTSTRAE 35, 65817 EPPSTEIN,
3	DR. KARL GEISEN	JAHNSTRERBE 43, 60318 FRANKFURT,
4	DR. GERHARD SEIPKE	WIESENSTRABE 44, 65719 HOFHEIM,

PCT International Classification Number

CO7K14/62

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	19630242.0	1996-07-26	Germany