WO1992015611A1 - Novel insulin derivatives - Google Patents

Novel insulin derivatives Download PDF

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Publication number
WO1992015611A1
WO1992015611A1 PCT/DK1992/000059 DK9200059W WO9215611A1 WO 1992015611 A1 WO1992015611 A1 WO 1992015611A1 DK 9200059 W DK9200059 W DK 9200059W WO 9215611 A1 WO9215611 A1 WO 9215611A1
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WO
WIPO (PCT)
Prior art keywords
human insulin
insulin
amino acid
derivative according
phe
Prior art date
Application number
PCT/DK1992/000059
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English (en)
French (fr)
Inventor
Lauge SCHÄFFER
Svend Havelund
Kirsten Årup 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 WO1992015611A1 publication Critical patent/WO1992015611A1/en

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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 novel insulin de ⁇ rivatives having tyrosine in position A13 and/or having phe- nylalanine, tryptophane or tyrosine in position B17, pharma ⁇ ceutical preparations containing such insulin derivatives, and a process for preparing the insulin derivatives.
  • Insulin is a hormone which regulates the blood glu- cose level by decreasing glucose outflow from the liver and by increasing glucose uptake in peripheral tissues, for example muscles and adipose tissues. Insulin 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 receptor mediated. This means that insulin has to bind to a receptor on the endo- thelial cell before it can be transported across this bar ⁇ rier to reach the distal tissues. After having reached the target organs in the periphery, insulin acts among other things by facilitating glucose uptake. Thus, the total re ⁇ duction of blood glucose by insulin is due to such effects both in the liver and peripheral tissues.
  • pancreatic insulin is secreted di ⁇ rectly into the hepatic portal vein, thereby insulinizing the liver and avoiding persistent peripheral hyperinsulin- aemia.
  • exogenous insulin In Type I diabetics who lack endogenous insulin secretion, exogenous insulin must be delivered parenterally, and although the most physiological route is delivery of insulin directly into the hepatic portal vein, this is ana ⁇ tomically difficult. Therefore, the subcutaneous route has been used in clinical practice.
  • peripheral hyper- insulinaemia results from the fact that insulin is delivered subcutaneously rather than intraportally, so that the insu ⁇ lin delivered reaches the peripheral tissue first rather than after passage through the liver. This route of admini- stration may, therefore, result in liver hypoinsulinaemia leading to a non-physiological regulation of the diabetic patients.
  • Type II diabetes is characterized by an excessive endogenous insulin secretion. Due to the high concentration of secreted insulin, the number of insulin receptors on the cell surfaces are decreased. The decrease of insulin recep ⁇ tors will, however, result in a decrease in the diabetics 1 sensitivity to insulin because this (among other things) is a function of the number of insulin receptors on the indivi- dual cells.
  • Type II diabetes One important therapy of Type II diabetes is a diet and weight reduction. Treatment with oral hypoglucaemic drugs is another well established therapy. In certain in ⁇ stances it might, however, be desirable to treat Type II diabetics with insulin.
  • Insulin derivatives with remarkably high associa ⁇ tion rate constants will be distributed to the liver to a higher degree than to the peripheral tissues and such de ⁇ rivatives will be of potential value both in the treatment of Type I and Type II diabetes.
  • Insulin derivatives wherein one or more of the amino acid residues present in positions A8, A9, A10, A13, A21, Bl, B2, B5, B9, BIO, B12, B14, B16, B17, B18, B20, B26, B2 " 7 and B28 are the amino acid residues of human insulin or the same or different amino acid residue substitutions, the net function of which is to impart to the molecule the same charge or a greater negative charge at neutral pH than that of human insulin, are in principle covered by formula I in a European patent application having publication No. 214,826.
  • the A13 amino acid residue can be Pro, Val, Arg, His, Ala, Glu, Asp, Thr, Gly, Gin, Asn or Asp and that the B17 amino acid residue can be Ser, Thr, Asn, Gin, Glu, Asp or His. It is stated that these known insulin derivatives have a rapid onset of effect.
  • Insulin analogues stated to be hepatospecific are described in International Patent Application having published cation No. WO 90/12814.
  • the amino acid residues in the positions A13, A14, A15, A19 and B16 have been exchanged.
  • the only specific insulin compounds stated therein are Leu A10 ,Trp A14 human insulin and Trp A14 sheep insulin (being Ala A8 ,Gly A9 ,Val A10 ,Trp A14 ,Ala B30 human insu ⁇ lin) .
  • This invention is based on the surprising fact that certain insulin derivatives have remarkably fast association rate constants in the insulin receptor binding process.
  • the insulin derivatives of this invention have tyrosine in position A13 and/or have phenylalanin, trypto- phane or tyrosine in position B17.
  • the insulin de ⁇ rivatives of this invention may in further positions differ from human insulin.
  • such al ⁇ terations comprise: a) compared with human insulin, exchanging the A21 amino acid with another amino acid which can be coded for by nu- cleotide sequences, for example by Ala, Gin, Glu, His, lie, Leu, Met, Gly, Ser, Thr, Trp, Tyr or Val, or with hSer, and/or b) compared with human insulin, exchanging one or more of the amino acids present in positions A4, A17, B13 and B21 with a neutral amino acid which can be coded for by nucleo- tide sequences, and/or c) compared with human insulin, exchanging the B27 amino acid with Arg, Lys or Thr and/or d) omitting 3, 4 or 5 of the amino acid residues in posi ⁇ tions B26, B27, B28, B29 and B30 and/or e) protecting the carboxy group in the amino acid ' residue in the C terminal end of the B chain with an amino group and/or f) exchanging
  • the insulin derivatives of this invention have in- teresting pharmacological properties.
  • the insu ⁇ lin derivatives of this invention are capable of being tar ⁇ geted to special organs after subcutaneous administration.
  • the insulin derivatives of this invention will thus be of potential value both in the treatment of Type I and Type II diabetes.
  • the insulin derivatives of this invention are a selected, novel group of insulin derivatives having additio- nal advantageous characteristics over the known insulin de ⁇ rivatives.
  • amino acids which can be coded for by necleotide sequences are Lys, Arg, Gly, Val, lie, Leu, Phe, Tyr, Met, Asp, Glu, Ala, Ser and Thr.
  • neutral amino acids are Gly, Val, lie. Leu, Phe, Tyr, Met, Asp, Glu, Ala, Ser and Thr.
  • This invention is also related to novel pharmaceu ⁇ tical preparations containing the above insulin derivatives in a solution with conventional additives, adjuvants, car ⁇ riers and diluents used for known insulin preparations.
  • the insulin derivatives of this invention may be prepared by chemical synthesis by methods analogous 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 con ⁇ taining 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., and Gross, E., (editors) Peptides: Synthesis - Structure - Function. Pro ⁇ ceedings of the seventh American peptide symposium, Illi ⁇ nois, pp. 721 - 728) .
  • the insulin deriva ⁇ tives of this invention may, for example, be prepared by altering the proinsulin gene through replacement of codon coding for Leu in position A13 and/or in position B17, in the native human proinsulin gene by codon(s) encoding the desired amino acid residue substitute(s) or by synthesizing the whole DNA-sequence encoding the desired insulin deriva ⁇ tive.
  • the gene encoding the desired insulin derivative is then inserted into a suitable expression vector which when transferred to a suitable host organism, for example E. coli., Bacillus or yeast, generates the desired product.
  • the product expressed is then isolated from the cells or the culture broth depending on whether the expressed product is secreted from the cells or not.
  • the insulin derivatives of this invention may, furthermore, be prepared by a method derivative to the method described in European patent application having pub ⁇ lication No. 195,691, the disclosure of which is incorpo ⁇ rated by reference hereinto.
  • a method derivative to the method described in European patent application having pub ⁇ lication No. 195,691 the disclosure of which is incorpo ⁇ rated by reference hereinto.
  • an insulin derivative precursor of human insulin wherein Lys B29 is con ⁇ nected to Gl ⁇ 1 by means of either a peptide bond or a pep- tide chain of varying length, is expressed and secreted by yeast and then converted into human insulin by the so-called transpeptidation reaction.
  • the insulin derivatives of this inven ⁇ tion may be prepared by inserting a DNA-sequence encoding a precursor of the insulin derivative in question into a suit ⁇ able yeast expression vehicle which when transferred to yeast is capable of expressing and secreting the precursor of the insulin derivative in which Lys B29 is connected to -LyA2 1 b y a eptide bond or a peptide chain with the general formula I
  • R is a peptide chain with n amino acid residues, n is an integer from 0 to 33, and R 1 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 compound with the general formula II
  • Q is the amino acid residue which is to be inserted in the B30 position, preferably Thr
  • 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. Thereafter, the carb ⁇ oxy protecting group is removed and the insulin derivative is isolated from the reaction mixture.
  • the insulin derivatives of this invention may also be prepared by a method analogous to the method described in European patent application having publication No. 195,691 the disclosure of which is incorporated by reference herein ⁇ to. By this method, insulin derivative precursors of the type having a bridge between the A and B chain consisting of a single pair of basic amino acid (Lys or Arg) are produced in yeast and then converted into the insulin derivative by an enzymatic conversion.
  • the insulin derivatives of this invention may be used for the preparation of novel insulin preparations.
  • Such novel insulin preparations may contain the insulin deriva ⁇ tives of this invention or a pharmaceutically acceptable salt thereof in aqueous solution or suspension, preferably at an approximately neutral pH value.
  • 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 derivatives of this present invention are Tyi ⁇ 13 human insulin, Phe B17 human insulin, Tr pB1 7 h uman insulin, Tyr B17 human insulin, Tyr A13 ,Phe B17 human insulin, Tyr ⁇ Trp 317 human insulin, ⁇ y r A13 f ⁇ yr B17 human insulin, Phe A13 ,Phe B17 human insulin, Phe A13 ,Trp B17 human insulin, Phe A13 ,Tyr B17 human insulin, Trp A13 ,Phe B17 human insulin, Trp A13 ,Trp B17 human insulin and ⁇ rp A13 ,Tyr B17 human insulin.
  • amino acids are those stated in J.Biol.Chem. 243 (1968), 3558.
  • the amino acids are in the L configuration. Unless otherwise indica- ted, the species of insulins stated herein is human.
  • the replacement(s) made in the human insulin mole ⁇ cule according to the practice of this invention is (are) indicated with a prefix referenced to human insulin.
  • Glu A13 human insulin is human insulin having Glu in position 13 of the A chain (in stead of Leu) .
  • the insulin derivatives of this invention have a low toxicity.
  • the insulin derivatives of this invention are ad ⁇ ministered analogously to the administration of known insu- lins and insulin derivatives in a therapeutically effective amount.
  • the dosage to be administered is normally determined by a physician.
  • the route of administration may be intra ⁇ muscular, subcutaneously, intravenous, via a mucos, for example nasal, or by a pump. Any novel feature or combination of features de ⁇ scribed herein is considered essential to this inverntion.
  • Genes encoding the precursors of the insulin de ⁇ rivative 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 derivative may also be made by enzymatic syn ⁇ thesis from oligonucleotides corresponding in whole or part to the insulin derivative precursor gene.
  • DNA-sequences containing a gene with the desired mutation are then combined with a suitable promoter se ⁇ quence, 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. pplied 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
  • frag ⁇ ments provide sequences to ensure a high rate of transcrip ⁇ tion of the precursor encoding gene and also provide a pre- sequence which can effect the localization of the precursor into the secretory pathway and its eventual excretion into the growth medium.
  • the expression units are furthermore pro ⁇ vided with a yeast origin of replication, for instance the 2 ⁇ origin, and a selectable marker, for instance LEU 2.
  • the selected plas id is then transformed into a suitable yeast strain by conventional technique, for example as described in European patent application having publica ⁇ tion No. 214,826 and transfor ants are grown on YPD medium (1% yeast extract, 2% peptone, and 2% glucose) .
  • the insulin derivative precursor is isolated from the culture medium and reacted with threonine methyl ester acetate dissolved in a N,N-dimethylformamide/water mixture in the presence of tryp- sin as described in European patent application having pub ⁇ lication No. 214,826 and converted into the human insulin derivative by acidic or basic hydrolysis, see European patent application having publication No 214,826.
  • Insulin and insulin derivative were monoiodinated in the tyrosine in the A14 position with 123 I.
  • Wistar rats weighing about 250 g received a bolus injection of 5 - 10 mU of 1 3 I-labelled insulin derivative through a catheter in ⁇ serted into the jugular vein.
  • the liver activity of 1 3 ⁇ was monitored by a gammacamera and expressed as percentage of total radioactivity. Results are shown in Table I below wherein "HI" designates human insulin. Table I
  • the compound of this invention is preferentially targeted to the liver.

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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)
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  • 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)
PCT/DK1992/000059 1991-02-27 1992-02-27 Novel insulin derivatives WO1992015611A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK33591A DK33591D0 (en, 2012) 1991-02-27 1991-02-27
DK335/91 1991-02-27

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WO1992015611A1 true WO1992015611A1 (en) 1992-09-17

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (en) 2004-01-21 2009-03-11 Novo Nordisk Health Care AG Transglutaminase mediated conjugation of peptides
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
WO2018094388A1 (en) 2016-11-21 2018-05-24 Case Western Reserve University Rapid-acting insulin analogues of enhanced stability
US10919949B2 (en) 2017-08-17 2021-02-16 Novo Nordisk A/S Acylated insulin analogues and uses thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986005497A1 (en) * 1985-03-15 1986-09-25 Nordisk Gentofte A/S Novel insulin derivatives and pharmaceutical preparations containing these derivatives
EP0214826A2 (en) * 1985-08-30 1987-03-18 Novo Nordisk A/S Insulin analogues and method of preparing the same
WO1990001038A1 (en) * 1988-07-20 1990-02-08 Nordisk Gentofte A/S Human insulin analogs and preparations containing them
WO1990012814A1 (en) * 1989-04-20 1990-11-01 Mount Sinai School Of Medicine Of The City University Of New York Hepatospecific insulin analogues
WO1992000322A1 (en) * 1990-07-02 1992-01-09 Novo Nordisk A/S Insulin analogues with organ preferential action

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986005497A1 (en) * 1985-03-15 1986-09-25 Nordisk Gentofte A/S Novel insulin derivatives and pharmaceutical preparations containing these derivatives
EP0214826A2 (en) * 1985-08-30 1987-03-18 Novo Nordisk A/S Insulin analogues and method of preparing the same
WO1990001038A1 (en) * 1988-07-20 1990-02-08 Nordisk Gentofte A/S Human insulin analogs and preparations containing them
WO1990012814A1 (en) * 1989-04-20 1990-11-01 Mount Sinai School Of Medicine Of The City University Of New York Hepatospecific insulin analogues
WO1992000322A1 (en) * 1990-07-02 1992-01-09 Novo Nordisk A/S Insulin analogues with organ preferential action

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (en) 2004-01-21 2009-03-11 Novo Nordisk Health Care AG Transglutaminase mediated conjugation of peptides
EP2368579A1 (en) 2004-01-21 2011-09-28 Novo Nordisk Health Care AG Transglutaminase mediated conjugation of peptides
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
WO2018094388A1 (en) 2016-11-21 2018-05-24 Case Western Reserve University Rapid-acting insulin analogues of enhanced stability
JP2019535733A (ja) * 2016-11-21 2019-12-12 ケース ウェスタン リザーブ ユニバーシティＣａｓｅ Ｗｅｓｔｅｒｎｒｅｓｅｒｖｅ Ｕｎｉｖｅｒｓｉｔｙ 安定性が増強された速効性インスリン類似体
EP3541397A4 (en) * 2016-11-21 2020-11-04 Case Western Reserve University FAST-ACTING INSULIN ANALOGUE WITH IMPROVED STABILITY
US11208453B2 (en) 2016-11-21 2021-12-28 Case Western Reserve University Rapid-acting insulin analogues of enhanced stability
JP7286160B2 (ja) 2016-11-21 2023-06-05 ケース ウェスタン リザーブ ユニバーシティ 安定性が増強された速効性インスリン類似体
US10919949B2 (en) 2017-08-17 2021-02-16 Novo Nordisk A/S Acylated insulin analogues and uses thereof

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DK33591D0 (en, 2012) 1991-02-27
AU1372992A (en) 1992-10-06

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