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WO1992000322A1 - Analogues insuliniques susceptibles de cibler des organes specifiques - Google Patents

Analogues insuliniques susceptibles de cibler des organes specifiques Download PDF

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Publication number
WO1992000322A1
WO1992000322A1 PCT/DK1991/000185 DK9100185W WO9200322A1 WO 1992000322 A1 WO1992000322 A1 WO 1992000322A1 DK 9100185 W DK9100185 W DK 9100185W WO 9200322 A1 WO9200322 A1 WO 9200322A1
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WO
WIPO (PCT)
Prior art keywords
insulin
human insulin
amino acid
acid residue
analogue
Prior art date
Application number
PCT/DK1991/000185
Other languages
English (en)
Inventor
Lauge SCHÄFFER
Svend Havelund
Kirsten Drejer
Original Assignee
Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Publication of WO1992000322A1 publication Critical patent/WO1992000322A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to insulin analogues capable of being targeted to special organs after subcutaneous administration, pharmaceutical preparations containing such insulin analogues, and methods for making the insulin analogues.
  • Insulin is a hormone which regulates the blood glucose level by decreasing glucose outflow from the liver and increasing glucose uptake in peripheral tissues, for example, muscles and adipose tissues. It exerts these effects by interacting with insulin receptors present on most cells.
  • the insulin receptors on the hepatocytes bind most of the insulin to regulate the metabolism and synthesis of glucose in the liver cells.
  • Insulin reaches the insulin receptors in the peripheral tissues after transendothelial transport, which is fully or partly receptormediated. After having reached the target organs in the periphery, insulin acts among other things by facilitating glucose uptake. Thus, the total reduction of blood glucose by insulin is due to such effects both in the liver and peripheral tissues.
  • pancreatic insulin is secreted directly into the hepatic portal vein, thereby insulinizing the liver and avoiding persistent peripheral hyperinsulinaemia.
  • Targeting of insulin to speficic organs may be possible by altering the insulin receptor binding kinetics .
  • the present invention is based on the surprising fact that certain insulin analogues have remarkably low association rate constants in the insulin receptor binding process.
  • Insulin analogues stated to be hepatospecific are described in International Patent Application having publication No. WO 90/12814.
  • the amino acid residues in the positions A13, A14, A15, A19 and B16 have been exchanged with a hydrophobic group.
  • the present invention is based on the surprising recognition that a certain, selected group of insulin analogues of the type described in EP application No. 214,826A are not targeted to the liver.
  • the forementioned EP application discloses insulin analogues wherein one or more of the amino acid residues present in positions A8, A9, A10, A13, A21, B1, B2, B5, B9, B10, B12, B14 , B16, B17, B18 , B20, B26, B27 and B28 may be substituted with another naturally occuring amino acid residue.
  • the insulin analogues of this invention have in the A13 position and/or in the B17 position in the insulin molecule a naturally occurring amino acid residue different from leucine and/or have in the B18 position in the insulin molecule a naturally occurring amino acid residue different from valine.
  • the insulin analogues of this invention have interesting properties. Examples of such interesting properties are their ability of being targeted to special organs after subcutaneous administration, for example, to the muscles.
  • the insulin analogues of this invention can be used for the treatment of diabetes analogously with the treatment of diabetic patients with similar insulin compounds.
  • the insulin analogues of this invention are of potential value in the treatment of insulin resistent diabetics.
  • the insulin analogues of this invention are of potential value in the treatment of muscular dystrophy.
  • the insulin analogues of this invention are a selected, novel group of insulin analogues having additional advantageous characteristics over the known insulin analogues.
  • a specific subclass of the present insulin analogues are such wherein leucine in position A13 or leucine in both position A13 and position B17 in the human insulin molecule has been replaced by another, naturally occurring amino acid residue.
  • a polar amino acid in the A13, B17 and/or B18 position for example, glutamic acid and aspartic acid.
  • Other suitable amino acids to be inserted in these positions are serine, threonine, asparagine, glutamine, alanine and glycine.
  • Examples of such exchanges are substitution of the amino acid residue in the A17 position with glutamine, substitution of asparagine in the A21 position in the insulin molecule with another naturally occurring amino acid residue, deletion of the amino acid residue in the B1 position, substitution of the amino acid residue in the B10 position with glutamic acid, substitution of the amino acid residue in the B14 position with glutamine, substitution of the amino acid residue in the B27 position with glutamic acid, substitution of the amino acid residue in B28 with glutamic or aspartic acid and deletion of the amino acid residue in the B30 position.
  • the present invention is also related to novel pharmaceutical preparations containing the novel insulin analogues according to the present invention in a solution with a conventional additive, adjuvant, carrier and diluent used for known insulin preparations.
  • the present insulin analogues may be prepared by chemical synthesis by methods analogue to the method described by Marki et al. (Hoppe-Seyler's Z. Physiol.Chem., 360 (1979), 1619 - 1632). They may also be formed from separately .in vitro prepared A and B chains containing the appropriate amino acid residue substitutions, whereupon the modified A and B chains are linked together by establishing disulphide bridges according to known methods (for example, Chance et al., In: Rick, D.H., Gross, E. (editors) Peptides: Synthesis - Structure - Function. Proceedings of the seventh American peptide symposium, Illinois, pp. 721 - 728).
  • the insulin analogues may furthermore be prepared by a method analogue to the method described in European patent application having publication No. 195,691, the disclosure of which is incorporated by reference hereinto.
  • a method analogue precursor of human insulin wherein Lys B29 is connected to Gly A1 by means of either a peptide bond or a peptide chain of varying length is expressed and secreted by yeast and then converted into human insulin by the so-called transpeptidation reaction.
  • the present insulin analogues may be prepared by inserting a DNA-sequence encoding a precursor of the insulin analogue in question into a suitable yeast expression vehicle which when transferred to yeast is capable of expressing and secreting the precursor of the insulin analogue in which Lys B29 is connected to Gly A1 by a peptide bond or a peptide chain with the formula I
  • R is a peptide chain with n amino acid residues, n is an integer from 0 to 33, and R' is Lys or Arg when the transformed yeast strain is cultured in a suitable nutrient medium.
  • the precursor is then recovered from the culture broth and reacted with an amino acid derivative with the general formula II
  • Q-OR (II) wherein Q is the amino acid residue which is to be inserted in the B30 position, preferably threonine, and R" is a carboxy protecting group (for example, methyl or tert.butyl), using trypsin or trypsin-like enzyme as a catalyst in a mixture of water and organic solvents analogously as described in US patent specification No. 4,343,898 (the disclosure of which is incorporated by reference hereinto) whereupon the carboxy protecting group is removed and the insulin analogue is isolated from the reaction mixture.
  • R is a carboxy protecting group (for example, methyl or tert.butyl)
  • the insulin analogues may also be prepared by a method analogue to the method described in European patent application having publication No. 195,691 the disclosure of which is incorporated by reference hereinto.
  • insulin analogue precursors of the type having a bridge between the A and B chain consisting of a single pair of basic amino acid (Lys, Arg) are produced in yeast and then converted into the insulin analogue by an enzymatic conversion.
  • the present insulin analogues may be used for the preparation of novel insulin preparations.
  • Such novel insulin preparations may contain the insulin analogues according to the present invention or a pharmaceutically acceptable salt thereof in aqueous solution or suspension, preferably at neutral pH.
  • the aqueous medium is made isotonic, for example with sodium chloride, sodium acetate or glycerol.
  • the aqueous medium may contain zinc ions, buffers such as acetate and citrate and preservatives such as m-cresol, methylparaben or phenol.
  • the pH value of the preparation is adjusted to the desired value.
  • the insulin preparation is made sterile by sterile filtration.
  • insulin analogues are Ala B17 human insulin (herein designated X69), Ala B18 human insulin, Asn A13 human insulin, Asn A13 ,Ala B17 human insulin, Asn A13 ,Asp B17 human insulin, Asn A13 ,Glu B17 human insulin, Asn B18 human insulin, Asp A13 human insulin, Asp A13 ,Ala B17 human insulin, Asp A13 ,Asp B17 human insulin, Asp A13 ,Glu B17 human insulin, Asp B18 human insulin, Gln A13 human insulin, Gln A13 ,Ala B17 human insulin, Gln A13 ,Asp B17 human insulin, Gln B18 human insulin, Glu A13 human insulin (herein designated X115) , Glu A13 ,Ala B17 human insulin, Glu A13 ,Asp B17 human insulin, Glu A13 ,Glu B17 human insulin, Glu B18 human insulin, Gly A13 human insulin, G
  • amino acids are those stated in J.Biol.Chem. 243 (1968), 3558.
  • the amino acids are in the L configuration. Unless otherwise indicated, the species of insulins stated herein is human.
  • Glu A13 human insulin herein is a human insulin analogue wherein the amino acid in the 13 position in the A chain of human insulin (leucine) has been replaced by glutamic acid.
  • Glu A13 ,B(l-29)-Ala-Ala-Lys-A(1-21) human insulin is a precursor for the insulin analogue wherein the amino acid in the 13 position in the A chain (leucine) has been replaced by glutamic acid, and wherein the A chain (A1 - A21) and the partial B chain (B1 - B29) are connected by the peptide sequence Ala-Ala-Lys.
  • the partial B chain (B1 -B29) and the A chain are connected by two disulphide bridges, namely between Cys A7 and Cys B7 and between Cys A20 and Cys B19 , as in human insulin, and the A chain contains the internal disulphide bridge between Cys A6 and Cys A11 .
  • the termB(l-29) designates a shortened B chain of human insulin from Phe B1 to LyS B29 and A(1-21) designates the A chain of human insulin.
  • position A13 is the 13 position of the A chain of insulin.
  • the species of insulin herein is human.
  • Genes encoding the precursors of the insulin analogue can be prepared by modification of genes encoding the corresponding human insulin precursors by site specific mutagenesis to insert or substitute with codons encoding the desired mutation.
  • a DNA-sequence encoding the precursor of the insulin analogue may also be made by enzymatic synthesis from oligonucleotides corresponding in whole or part to the insulin analogue precursor gene.
  • DNA-sequences containing a gene with the desired mutation are then combined with a suitable promoter sequence, for example, fragments coding for the TPI promoter (TPIp) (T. Alber and G. Kawasaki,. Nucleotide Sequence of the triose Phosphate Isomerase Gene of Saccharomyces cerevisiae. J.Mol.Applied Genet. 1 (1982), 419 - 434), a suitable leader sequence and possible transcription termination sequence, for example, from TPI of S . cerevisiae (TPI T ).
  • TPIp fragments coding for the TPI promoter
  • TPI T TPI promoter
  • the expression units are furthermore provided with a yeast origin of replication, for instance the 2 ⁇ origin, and a selectable marker, for instance LEU 2.
  • the selected plasmid is then transformed into a suitable yeast strain by conventional technique, for example, as described in European patent application having publication No. 214,826 and transformants are grown on YPD medium (1% yeast extract, 2% peptone, and 2% glucose).
  • YPD medium 1% yeast extract, 2% peptone, and 2% glucose.
  • the insulin analogue precursor is isolated from the culture medium and reacted with Thr-Met,HOAC dissolved in a DMF/water mixture in the presence of trypsin as described in European patent application having publication No. 214,826 and converted into the human insulin analogue by acidic or basic hydrolysis, see European patent application having publication No. 214,826.
  • association rate constant of some compounds of this invention appears from table III, below, where the constants are compared with that of human insulin which has been set to 100%.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Diabetes (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

On décrit des analogues insuliniques susceptibles de cibler des organes spécifiques, dans lesquels la leucine en position A13 ou la leucine en position A13 aussi bien qu'en position B17 est remplacée par un autre reste d'acide aminé se produisant naturellement. On décrit également des préparations pharmaceutiques se rapportant à de tels analogues insuliniques.
PCT/DK1991/000185 1990-07-02 1991-07-02 Analogues insuliniques susceptibles de cibler des organes specifiques WO1992000322A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK158390A DK158390D0 (da) 1990-07-02 1990-07-02 Nye peptider
DK1583/90 1990-07-02

Publications (1)

Publication Number Publication Date
WO1992000322A1 true WO1992000322A1 (fr) 1992-01-09

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AU (1) AU8008091A (fr)
DK (1) DK158390D0 (fr)
WO (1) WO1992000322A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015611A1 (fr) * 1991-02-27 1992-09-17 Novo Nordisk A/S Nouveaux derives d'insuline
US5854208A (en) * 1993-08-13 1998-12-29 Deutsches Wollforschungsinstitut Hepatoselective pharmaceutical actives
US6342225B1 (en) 1993-08-13 2002-01-29 Deutshces Wollforschungsinstitut Pharmaceutical active conjugates
EP2033662A1 (fr) 2004-01-21 2009-03-11 Novo Nordisk Health Care AG Conjugaison au moyen de transglutaminase de peptides
AU2007298919C1 (en) * 2006-09-22 2014-02-06 Novo Nordisk A/S Protease resistant insulin analogues
WO2014145593A2 (fr) 2013-03-15 2014-09-18 Case Western Reserve University Analogues d'insuline de site 2
US9260502B2 (en) 2008-03-14 2016-02-16 Novo Nordisk A/S Protease-stabilized insulin analogues
US9896496B2 (en) 2013-10-07 2018-02-20 Novo Nordisk A/S Derivative of an insulin analogue
US10259856B2 (en) 2008-03-18 2019-04-16 Novo Nordisk A/S Protease stabilized acylated insulin analogues
US10265385B2 (en) 2016-12-16 2019-04-23 Novo Nordisk A/S Insulin containing pharmaceutical compositions
US10392429B2 (en) 2014-10-06 2019-08-27 Case Western Reserve University Biphasic single-chain insulin analogues

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0214826A2 (fr) * 1985-08-30 1987-03-18 Novo Nordisk A/S Analogues d'insuline et leur méthode de préparation
WO1990012814A1 (fr) * 1989-04-20 1990-11-01 Mount Sinai School Of Medicine Of The City University Of New York Analogues d'insuline hepatospecifiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0214826A2 (fr) * 1985-08-30 1987-03-18 Novo Nordisk A/S Analogues d'insuline et leur méthode de préparation
WO1990012814A1 (fr) * 1989-04-20 1990-11-01 Mount Sinai School Of Medicine Of The City University Of New York Analogues d'insuline hepatospecifiques

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992015611A1 (fr) * 1991-02-27 1992-09-17 Novo Nordisk A/S Nouveaux derives d'insuline
US5854208A (en) * 1993-08-13 1998-12-29 Deutsches Wollforschungsinstitut Hepatoselective pharmaceutical actives
US6063761A (en) * 1993-08-13 2000-05-16 Kings College London Hepatoselective pharmaceutical actives
US6342225B1 (en) 1993-08-13 2002-01-29 Deutshces Wollforschungsinstitut Pharmaceutical active conjugates
USRE39055E1 (en) * 1993-08-13 2006-04-04 Btg International Limited Hepatoselective pharmaceutical actives
EP2033662A1 (fr) 2004-01-21 2009-03-11 Novo Nordisk Health Care AG Conjugaison au moyen de transglutaminase de peptides
EP2368579A1 (fr) 2004-01-21 2011-09-28 Novo Nordisk Health Care AG Conjugaison au moyen de transglutaminase de peptides
US9018161B2 (en) 2006-09-22 2015-04-28 Novo Nordisk A/S Protease resistant insulin analogues
AU2007298919C1 (en) * 2006-09-22 2014-02-06 Novo Nordisk A/S Protease resistant insulin analogues
US9260502B2 (en) 2008-03-14 2016-02-16 Novo Nordisk A/S Protease-stabilized insulin analogues
US10259856B2 (en) 2008-03-18 2019-04-16 Novo Nordisk A/S Protease stabilized acylated insulin analogues
WO2014145593A2 (fr) 2013-03-15 2014-09-18 Case Western Reserve University Analogues d'insuline de site 2
WO2014145593A3 (fr) * 2013-03-15 2015-01-08 Case Western Reserve University Analogues d'insuline de site 2
JP2016516728A (ja) * 2013-03-15 2016-06-09 ケース ウェスタン リザーブ ユニバーシティＣａｓｅ Ｗｅｓｔｅｒｎ Ｒｅｓｅｒｖｅ Ｕｎｉｖｅｒｓｉｔｙ 第２部位インスリン類似体
EP2968473A4 (fr) * 2013-03-15 2016-11-23 Univ Case Western Reserve Analogues d'insuline de site 2
US9896496B2 (en) 2013-10-07 2018-02-20 Novo Nordisk A/S Derivative of an insulin analogue
US10392429B2 (en) 2014-10-06 2019-08-27 Case Western Reserve University Biphasic single-chain insulin analogues
US11142560B2 (en) 2014-10-06 2021-10-12 Case Western Reserve University Biphasic single-chain insulin analogues
US10265385B2 (en) 2016-12-16 2019-04-23 Novo Nordisk A/S Insulin containing pharmaceutical compositions
US10596231B2 (en) 2016-12-16 2020-03-24 Novo Nordisk A/S Insulin containing pharmaceutical compositions

Also Published As

Publication number Publication date
AU8008091A (en) 1992-01-23
DK158390D0 (da) 1990-07-02

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