Claims for Patent: 6,221,633

Summary for Patent: 6,221,633

Title:	Insulin derivatives having a rapid onset of action
Abstract:	The present invention relates to insulin derivatives which in comparison to human insulin, have an accelerated onset of action, to a process for their preparation and to their use, in particular in pharmaceutical preparations for the treatment of diabetes mellitus. In particular, the present invention relates to insulin derivatives or physiologically tolerable salts thereof in which asparagine (Asn) in position B3 of the B chain is replaced by a naturally occurring basic amino acid residue and at least one amino acid residue in the positions B27, B28 or B29 of the B chain is replaced by another naturally occurring amino acid residue, it optionally being possible for asparagine (Asn) in position 21 of the A chain to be replaced by Asp, Gly, Ser, Thr or Ala and for phenylalanine (Phe) in position B1 of the B chain and the amino acid residue in position B30 of the B chain to be absent.
Inventor(s):	Ertl; Johann (Bremthal, DE), Habermann; Paul (Eppstein, DE), Geisen; Karl (Frankfurt, DE), Seipke; Gerhard (Hofheim, DE)
Assignee:	Aventis Pharma Deutschland GmbH (Frankfurt am Main, DE)
Application Number:	09/099,307
Patent Claims:	1. An insulin derivative or a physiologically tolerable salt thereof, in which asparagine (Asn) in position B3 of the B chain is replaced by a naturally occurring basic amino acid residue and at least one amino acid residue in the positions B27, B28 or B29 of the B chain is replaced by another naturally occurring neutral or acidic amino acid residue. 2. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, of formula I ##STR2## in which (A1-A5) are the amino acid residues in the positions A1 to A5 of the A chain of human insulin or animal insulin, (A12-A19) are the amino acid residues in the positions A12 to A19 of the A chain of human insulin or animal insulin, A21 is Asn, Asp, Gly, Ser, Thr or Ala, (B8-B18) are the amino acid residues in the positions B8 to B18 of the B chain of human insulin or animal insulin, (B20-B26) are the amino acid residues in the positions B20 to B26 of the B chain of human insulin or animal insulin, A8, A9, A10 are the amino acid residues in the positions A8, A9 and A10 of the A chain of human insulin or animal insulin, B30 is --OH or the amino acid residue in position B30 of the B chain of human insulin or animal insulin, B1 is a phenylalanine residue (Phe) or a hydrogen atom, B3 is a naturally occurring basic amino acid residue, B27, B28 and B29 are the amino acid residues in the positions B27, B28 and B29 of the B chain of human insulin or animal insulin or in each case are another naturally occurring amino acid residue, where at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is replaced by another naturally occurring amino acid residue which is selected from the group consisting of the neutral or acidic amino acids. 3. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 2, wherein A8 is alanine (Ala), A9 is serine (Ser), A10 is valine (Val) and B30 is alanine (Ala). 4. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 2, wherein A8 is threonine (Thr), A9 is serine (Ser) and A10 is isoleucine (Ile). 5. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 4, wherein B30 is alanine (Ala). 6. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 4, wherein B30 is threonine (Thr). 7. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 6, wherein (A1-A5) are the amino acid residues in the positions A1 to A5 of the A chain of human insulin, (A12-A19) are the amino acid residues in the positions A12 to A19 of the A chain of human insulin, (B8-B18) are the amino acid residues in the positions B8 to B18 of the B chain of human insulin and (B20-B26) are the amino acid residues in the positions B20 to B26 of the B chain of human insulin. 8. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein the amino acid residue in position B1 of the B chain is a phenylalanine residue (Phe). 9. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein the amino acid residue in position B3 of the B chain is a histidine (His), lysine (Lys) or arginine residue (Arg). 10. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 9, wherein the amino acid residue in position B3 of the B chain is a histidine residue (His). 11. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 9, wherein the amino acid residue in position B3 of the B chain is an arginine residue (Arg). 12. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 9, wherein the amino acid residue in position B3 of the B chain is a lysine residue (Lys). 13. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is a naturally occurring amino acid residue which is selected from the group consisting of isoleucine (Ile), aspartic acid (Asp) and glutamic acid (Glu). 14. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is a naturally occurring amino acid residue which is selected from the group consisting of the acidic amino acids. 15. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 14, wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is an aspartic acid residue (Asp). 16. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 15, wherein the amino acid residue in position B27 of the B chain is an aspartic acid residue (Asp). 17. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 15, wherein the amino acid residue in position B28 of the B chain is an aspartic acid residue (Asp). 18. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 15, wherein the amino acid residue in position B29 of the B chain is an aspartic acid residue (Asp). 19. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 14, wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is a glutamic acid residue (Glu). 20. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 19, wherein the amino acid residue in position B27 of the B chain is a glutamic acid residue (Glu). 21. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 19, wherein the amino acid residue in position B28 of the B chain is a glutamic acid residue (Glu). 22. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 19, wherein the amino acid residue in position B29 of the B chain is a glutamic acid residue (Glu). 23. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 22, wherein the B chain has the sequence Phe Val Lys 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 Glu Thr (SEQ ID NO 3). 24. A process for the preparation of an insulin derivative as claimed in claim 23, comprising a) constructing a replicable expression vehicle which contains a DNA sequence which codes for a precursor of the insulin derivative, in which the amino acid residue in position A1 of the A chain is linked to the amino acid residue B30 of the B chain via a peptide chain of the formula II in which R.sup.1.sub.n is a peptide chain having n amino acid residues and n is an integer from 0 to 34, and the B chain is modified by covalent linkage of the amino acid at position B1 to a peptide chain of the formula III in which R.sup.2.sub.m is a peptide chain having m amino acid residues, m is an integer from 0 to 40 and p is 0, 1 or 2, b) expressing the DNA sequence which codes for a precursor of the insulin derivative in a host cell, and c) releasing the insulin derivative from its precursor using chemical and/or enzymatic methods, wherein the precursor of the insulin derivative has the sequence Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Lys 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 Glu Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (SEQ ID NO.: 6). 25. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein at least one of the amino acid residues in the positions B27 and B28 of the B chain is replaced by a naturally occurring amino acid residue which is selected from the group consisting of the neutral amino acids. 26. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 25, wherein at least one of the amino acid residues in the positions B27, B28 and B29 of the B chain is an isoleucine residue (Ile). 27. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 26, wherein the amino acid residue in position B28 of the B chain is an isoleucine residue (Ile). 28. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 27, wherein the B chain has the sequence Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr IIe Lys Thr (SEQ ID NO 4). 29. A process for the preparation of an insulin derivative as claimed in claim 28, comprising a) constructing a replicable expression vehicle which contains a DNA sequence which codes for a precursor of the insulin derivative, in which the amino acid residue in position A1 of the A chain is linked to the amino acid residue B30 of the B chain via a peptide chain of the formula II in which R.sup.1.sub.n is a peptide chain having n amino acid residues and n is an integer from 0 to 34, and the B chain is modified by covalent linkage of the amino acid at position B1 to a peptide chain of the formula III in which R.sup.2.sub.m is a peptide chain having m amino acid residues, m is an integer from 0 to 40 and p is 0, 1 or 2, b) expressing the DNA sequence which codes for a precursor of the insulin derivative in a host cell, and c) releasing the insulin derivative from its precursor using chemical and/or enzymatic methods, wherein the precursor of the insulin derivative has the sequence Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Ile Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (SEQ ID NO.: 7). 30. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 26, wherein the amino acid residue in position B27 of the B chain is an isoleucine residue (Ile). 31. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 30, wherein the B chain has the sequence Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr lIe Pro Lys Thr (SEQ ID NO 5). 32. A process for the preparation of an insulin derivative as claimed in claim 31, comprising a) constructing a replicable expression vehicle which contains a DNA sequence which codes for a precursor of the insulin derivative, in which the amino acid residue in position A1 of the A chain is linked to the amino acid residue B30 of the B chain via a peptide chain of the formula II in which R.sup.1.sub.n is a peptide chain having n amino acid residues and n is an integer from 0 to 34, and the B chain is modified by covalent linkage of the amino acid at position B1 to a peptide chain of the formula III in which R.sup.2.sub.m is a peptide chain having m amino acid residues, m is an integer from 0 to 40 and p is 0, 1 or 2, b) expressing the DNA sequence which codes for a precursor of the insulin derivative in a host cell, and c) releasing the insulin derivative from its precursor using chemical and/or enzymatic methods, wherein the precursor of the insulin derivative has the sequence Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Ile Pro Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Lcu Tyr Gln Leu Glu Asn Tyr Cys Asn (SEQ ID NO.: 8). 33. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein the amino acid residue in position A21 of the A chain is an asparagine residue (Asp). 34. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 33, wherein the A chain has the sequence Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asp (SEQ ID NO.: 9) and the B chain has the sequence Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Ile Lys Thr (SEQ ID NO.: 10). 35. A process for the preparation of an insulin derivative as claimed in claim 33, comprising a) constricting a replicable, expression vehicle which contains a DNA sequence which codes for a precursor of the insulin derivative, in which the amino acid residue in position A1 of the A chain is linked to the amino acid residue B30 of the B chain via a peptide chain of the formula II in which R.sup.1.sub.n is a peptide chain having n amino acid residues and n is an integer from 0 to 34, and the B chain is modified by covalent linkage of the amino acid at position B1 to a peptide chain of the formula III in which R.sup.2.sub.m is a peptide chain having m amino acid residues, m is an integer from 0 to 40 and p is 0, 1 or 2, b) expressing the DNA sequence which codes for a precursor of the insulin derivative in a host cell, and c) releasing the insulin derivative from its precursor using chemical and/or enzymatic methods, wherein the precursor of the insulin derivative has the sequence Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Ile Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asp (SEQ ID No.: 11). 36. A pharmaceutical preparation, which comprises at least one insulin derivative and/or a physiologically tolerable salt thereof as claimed in claim 1. 37. A pharmaceutical preparation as claimed in claim 36, which comprises the insulin derivative and/or the physiologically tolerable salt thereof in dissolved, amorphous and/or crystalline form. 38. A pharmaceutical preparation as claimed in claim 36, which further comprises a depot auxiliary. 39. A pharmaceutical preparation as claimed in claim 38, wherein the depot auxiliary is protamine sulfate, where the insulin derivative and/or the physiologically tolerable salt thereof is present with the protamine sulfate in a cocrystallizate. 40. A method for the treatment of diabetes mellitus comprising administering an effective amount of the pharmaceutical preparation of claim 36. 41. An injectable solution having insulin activity, comprising the pharmaceutical preparation as claimed in claim 36 in dissolved form. 42. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein asparagine (Asn) in position 21 of the A chain is replaced by Asp, Gly, Ser, Thr or Ala. 43. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein phenylalanine (Phe) in position B1 of the B chain is absent. 44. An insulin derivative or a physiologically tolerable salt thereof as claimed in claim 1, wherein the amino acid residue in position B30 of the B chain is absent. 45. A process for the preparation of an insulin derivative or of a physiologically tolerable salt thereof as claimed in claim 1, comprising a) constructing a replicable, expression vehicle which contains a DNA sequence which codes for a precursor of the insulin derivative, in which the amino acid residue in position A1 of the A chain is linked to the amino acid residue B30 of the B chain via a peptide chain of the formula II in which R.sup.1.sub.n is a peptide chain having n amino acid residues and n is an integer from 0 to 34, and the B chain is modified by covalent linkage of the amino acid at position B1 to a peptide chain of the formula III in which R.sup.2.sub.m is a peptide chain having m amino acid residues, m is an integer from 0 to 40 and p is 0, 1 or 2, b) expressing the DNA sequence which codes for a precursor of the insulin derivative in a host cell, and c) releasing the insulin derivative from its precursor using chemical and/or enzymatic methods. 46. The process as claimed in claim 45, wherein the host cell is a bacterium. 47. The process as claimed in claim 46, wherein the bacterium is E. coli. 48. The process as claimed in claim 45, wherein the host cell is a yeast. 49. The process as claimed in claim 48, wherein the yeast is Saccharomyces cerevisiae. 50. A precursor of an insulin derivative, wherein the precursor has a sequence selected from the group consisting of SEQ ID NO.: 11, SEQ ID NO.:6, SEQ ID NO.:8, and SEQ ID NO.:7. 51. The precursor of claim 50, wherein the precursor has the sequence of SEQ ID NO.:6. 52. The precursor of claim 50, wherein the precursor has the sequence of SEQ ID NO.:8. 53. The precursor of claim 50, wherein the precursor has the sequence of SEQ ID NO.:7. 54. The precursor of claim 50, wherein the precursor has the sequence of SEQ ID NO.:11. 55. An isolated or purified nucleic acid comprising a sequence which codes for a precursor of an insulin derivative having a sequence selected from the group consisting of SEQ ID NO.:11, SEQ ID NO.:6, SEQ ID NO.8, and SEQ ID NO.:7. 56. The isolated or purified nucleic acid of claim 55, having the sequence of SEQ ID NO.11. 57. An expression vehicle comprising a nucleic acid as claimed in claim 56. 58. A host cell which is transformed using an expression vehicle as claimed in claim 57. 59. The isolated or purified nucleic acid of claim 55, having the sequence of SEQ ID NO.:6. 60. An expression vehicle comprising a nucleic acid as claimed in claim 59. 61. The isolated or purified nucleic acid of claim 55, having the sequence of SEQ ID NO.:8. 62. An expression vehicle comprising a nucleic acid as claimed in claim 61. 63. The isolated or purified nucleic acid of claim 55, having the sequence of SEQ ID NO.:7. 64. An expression vehicle comprising a nucleic acid as claimed in claim 63.