Insulin derivatives with increased zinc binding

Details Insulin derivatives with increased zinc binding Insulin derivatives with increased zinc binding

1997-07-25

2001-04-24

Saoud, Christine J. (Department: 1647)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S004300, C514S866000, C530S303000, C530S304000

Reexamination Certificate

active

06221837

ABSTRACT:

BACKGROUND OF THE INVENTION
The pharmacokinetics of subcutaneously administered insulin is dependent on its association behavior. Insulin forms hexamers in neutral aqueous solution. To get from the tissue into the blood stream and to the site of action, insulin 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 such solution is rapidly neutralized in the subcutaneous tissue on injection and insulin-zinc complexes are formed, the natural zinc binding of human insulin is insufficient to stabilize hexamers and higher aggregates. Therefore, by addition of zinc
++
, the release of human insulin is not markedly delayed and a strong depot effect is not 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 firmly bound to the insulin hexamer and cannot be removed by normal dialysis. So-called 4-zinc insulin crystals have been described, but these crystals on average contain only less than three mol of zinc
++
per mole of insulin hexamer (G.D. Smith et al., Proc. Natl. Acad. Sci. USA: 81, pages 7093-7097).
SUMMARY OF THE INVENTION
The object of the present invention is to find insulin derivatives which have an increased zinc binding power, form a stable complex comprising insulin hexamer and zinc
++
and have a delayed profile of action on subcutaneous injection in comparison with human insulin.
Insulins of the formula I
and/or physiologically tolerable salts of the insulins of the formula I have now been found which fulfill the abovementioned criteria and wherein
R
1
is a phenylalanine residue or a hydrogen atom,
R
3
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, of which 1 to 5 are 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
R
1
is a phenylalanine residue,
R
3
is an amino acid residue selected from the group consisting of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gln,
Y is an amino acid residue selected from the group consisting of Ala, Thr, Ser and His,
Z is
a) the amino acid residue His or
b) a peptide having 4 to 7 amino acid residues, of which 1 or 2 are histidine residues.
An insulin of the formula I is furthermore preferred where
R
1
is a phenylalanine residue,
R
3
is an amino acid residue selected from the group consisting of Asn, Gly, Ser, Thr, Ala, Asp, Glu and Gln,
Y is an amino acid residue selected 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, of which 1 or 2 are histidine residues.
An insulin of the formula I is particularly preferred where
Z is a peptide having 1 to 5 amino acid residues, of which 1 or 2 are histidine residues.
An insulin of the formula I is particularly preferred, where
R
1
is a phenylalanine residue,
R
3
is an amino acid residue selected from the group consisting of Asn and Gly,
Y is an amino acid residue selected from the group consisting of Thr and His, and
Z is a peptide having 1 to 5 amino acid residues, of which 1 or 2 are histidine residues.
An insulin of the formula I is furthermore preferred where R
1
is a phenylalanine residue, R
3
is a glycine residue, Y is a threonine residue and Z is a peptide having 1 to 5 amino acid residues, of which 1 or 2 are 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 (Seq. ID No.17) or Ala Ala His His (Seq. ID No.18).
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 B30, correspond to the position of amino acid residues in the A or B chains of the insulin.
The expression “genetically encodable amino acid residue” represents the residues of the amino acids Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp, Pro and selenocysteine.
The expressions “residues A2-A20” and “residues B2-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 B2-B29 ” 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, Ile, Val, Glu, Gln, Cys, Cys, Thr, Ser, Ile, Cys, Ser, Leu, Tyr, Gln, Leu, Glu, Asn, Tyr, Cys, Asn.
The B chain of human insulin has the following sequence (Seq ID No. 2): Phe, Val, Asn, Gln, His, Leu, Cys, Gly, Ser, His, Leu, Val, Glu, Ala, Leu, Tyr, Leu, Val, Cys, Gly, Glu, Arg, Gly, Phe, Phe, Tyr, Thr, Pro, Lys, Thr.
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(21)-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
Sacchamomyces 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 may be cleaved chemically by means of cyanogen 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. Darbr

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