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Single-chain insulin

Inactive Publication Date: 2007-06-07
NOVO NORDISK AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0088] In a further embodiment the present invention is related to a method of reducing the blood glucose level in mammalians by administrating a therapeutically active dose of a single-chain insulin according to the invention to a patient in need of such treatment.

Problems solved by technology

The two chain structure of insulin allows insulin to undertake multiple conformations, and several findings have indicated that insulin has the propensity to considerable conformational change and that restrictions in the potential for such change considerably decrease the affinity of the insulin receptor for ligands.
Blocking of the amino acid residue A1 in insulin also results in poor receptor binding, consistent with the dogma that a free N-terminal of the A-chain and free C-terminal of the B-chain of insulin are important for binding to the insulin receptor.
Consequently, fibrillation is especially a concern when using infusion pumps as insulin delivery system.

Method used

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Examples

Experimental program
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Effect test

example 1

[0163] A number of single-chain insulins were produced as described above and isolated from the culture medium and purified for further testing. The single-chain insulins were tested for biological insulin activity as measured by binding affinity to the human insulin receptor relative to that of human insulin as described below.

[0164] Furthermore, the affinity to the IGF-1 was tested as described below. The results are shown Table 1 where “IR” means human insulin receptor binding relative to that of human insulin.

TABLE 1Human IGF-1 receptorSingle-chainbinding relative toinsulin (PAK)Connecting peptideAmino acid substitutionsIRhuman insulin1606RSFDGK41%(SEQ ID NO: 34)1663TVGSSRGK46%(SEQ ID NO: 35)1664TGSSRGK43%(SEQ ID NO: 36)1735VGRSSGK[A21G]143%(SEQ ID NO: 31)1754AGRGSGP53%(SEQ ID NO: 18)1767AGRGSGP[A18Q_A21G]28%(SEQ ID NO: 18)1801AGRGSGK129%(SEQ ID NO: 15)1817AGRGSGK[A21G]63%(SEQ ID NO: 15)1800AGRGSGK[A18Q_A21G]175%(SEQ ID NO: 15)1805AGRGSGK[B3Q_A18Q_A21G]83%(SEQ ID NO: 15)1808A...

example 2

[0166] B(1-29)-B3Q-B29R-TGLGK((eps)myristoyl)GQ-A(1-21)-A18Q-A21 G Human Insulin (SEQ ID NO:133) (PAK1820)

[0167] B(1-29)-B30-B29R-TGLGKGQ-A(1-21)-A18Q-A21G human insulin (SEQ ID NO:133) (150 mg, 24 μmol) was dissolved in aqueous sodium carbonate (100 mM, 2.8 mL) and added a solution of myristic acid N-hydroxysuccinimide ester (7.7 mg, 24 μmol, may be prepared according to B. Faroux-Corlay et al., J. Med. Chem. 2001, 44, 2188-2203) in N-methylpyrrolidin-2-one (0.5 mL). The resulting mixture was added more N-methylpyrrolidin-2-one (3 mL) and aqueous sodium carbonate (100 mM, 0.8 mL), to pH 10-11. The resulting mixture was stirred at room temperature for 50 minutes. pH was adjusted to 5.5 with 1 N hydrochloric acid. The solid formed was isolated by centrifugation and decantation. The residue was purified by preparative HPLC in two runs on a Jones Kromasil RP18 5 μm, 15×225 mm column, using a flow of 8 mL / min with the following gradient:

[0168] 0.00-5.00 min: 10% CH3CN+0.1% TFA,

[0169...

example 3

[0186] B(1-29)-B3Q-B29R-TGLGK((eps)octadecandioyl)GQ-A(1-21)-A18Q-A21 G Human Insulin (SEQ ID NO:133) (PAK1820)

[0187] B(1-29)-B30-B29R-TGLGKGQ-A(1-21)-A18Q-A21G human insulin (SEQ ID NO:133) (150 mg, 24 μmol) was dissolved in aqueous sodium carbonate (100 mM, 2.8 mL) and added a solution of succinimidyl tert-butyl octadecandioate (prepared in analogy with the method described in example 4 (11 mg, 24 μmol) in acetonitrile (2 mL). The resulting mixture was added more acetonitrile (2 mL). pH of the mixture was 10-11. The resulting mixture was stirred at room temperature for 1 hour. pH was adjusted to 5.86 with 1N hydrochloric acid. The solid formed was isolated by centrifugation and decantation. The residue was purified by preparative HPLC in two runs on a Jones Kromasil RP18 5 μm, 15×225 mm column, using a flow of 8 mL / min with the following gradient:

[0188] 0.00-5.00 min: 10% CH3CN,

[0189] 5.00-35.0 min: 10%-50% CH3CN,

[0190] 35.0-45.0 min: 50%-90% CH3CN.

[0191] Pure fractions were...

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Abstract

The present invention is related to single-chain insulin having insulin activity comprising a B- and an A-chain or a modified B- and A-chain connected by a connecting peptide of from 6-11 amino acids. The single-chain insulins will have biological insulin activity and an IGF-1 receptor affinity similar to or lower than that of human insulin and a high physical stability. The single-chain insulin may contain at least one basic amino acid residues in the connecting peptide. The single-chain insulins may also be acylated in one or more Lys residues.

Description

FIELD OF THE INVENTION [0001] The present invention is related to single-chain insulins which have insulin activity and can be used for the treatment of diabetes. The single-chain insulins have a high physical stability and a low tendency to fibrillation and will be soluble at neutral pH. The present invention is also related to a DNA sequence encoding the single-chain insulins, a method for their production and pharmaceutical compositions containing the single-chain insulins. BACKGROUND OF THE INVENTION [0002] Insulin is a polypeptide hormone secreted by β-cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge. [0003] The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A,...

Claims

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Application Information

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IPC IPC(8): A61K38/28C07K14/62A61K38/00
CPCC07K14/62A61K38/00A61P3/10A61P43/00
Inventor KJELDSEN, THOMAS BORGLUMANDERSEN, ASSER SLOTHSCHLEIN, MORTENSORENSEN, ANDERS ROBERTMADSEN, PETER
Owner NOVO NORDISK AS
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