WO2019229242A1 - Dérivés comprenant un analogue d'apeline et leurs utilisations - Google Patents

Dérivés comprenant un analogue d'apeline et leurs utilisations Download PDF

Info

Publication number
WO2019229242A1
WO2019229242A1 PCT/EP2019/064202 EP2019064202W WO2019229242A1 WO 2019229242 A1 WO2019229242 A1 WO 2019229242A1 EP 2019064202 W EP2019064202 W EP 2019064202W WO 2019229242 A1 WO2019229242 A1 WO 2019229242A1
Authority
WO
WIPO (PCT)
Prior art keywords
chem
apelin
ethoxy
xaa
amino
Prior art date
Application number
PCT/EP2019/064202
Other languages
English (en)
Inventor
János Tibor KODRA
Jesper L. LAU
Anthony Murray
Line Marie NIELSEN
Jacob Fuglsbjerg JEPPESEN
Fangzhou WU
Fa Liu
Cecilie Mie JØRGENSEN
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 WO2019229242A1 publication Critical patent/WO2019229242A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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 derivatives comprising an apelin analogue with a methylene bridge between two sulphur-containing amino acids, and the uses thereof.
  • the human apelin receptor (also known as the human APJ receptor, abbreviated hAPJR) is a G protein-coupled receptor which is widely expressed in human tissues, e.g. in cardiovascular tissue, central nervous system and adipocytes.
  • a human endogenous ligand for the hAPJR has been identified as apelin.
  • the apelin/hAPJR system regulates cardiovascular and metabolic homeostasis
  • Apelin-36 and apelin-13 have interesting and comparable potency in cardiovascular tissues [Hypertension, 54 (2009), 598-604], but they also have some inherent disadvantages making them less than ideal as a medical treatment. These disadvantages include poor stability which in turn may result in poor shelf-life and poor systemic half-life effectively preventing pharmaceutical use.
  • WO15013167 and WO2016116842 purport to disclose hAPJR agonists modified to overcome the problem of peptide degradation in the human body.
  • WO14099984 purports to disclose hAPJR agonists with increased stability as compared to human apelin-13.
  • the present invention relates to derivatives of apelin analogues.
  • the derivatives of the invention comprise an apelin analogue, which apelin analogue comprises two amino acids that are covalently linked via a methylene bridge, which methylene bridge is attached to a sulphur atom in the side chain of each of the two covalently linked amino acids.
  • the derivatives of the invention have one or more substituents, comprising at least one functional group with pKa ⁇ 7.0.
  • the derivatives of the invention have desirable chemical stability and desirable bioactivity. Also, or alternatively the derivatives of the invention have a surprisingly high chemical stability. Also, or alternatively, the derivatives of the invention have a surprisingly high bioactivity.
  • the invention relates to the use of the derivatives as a medicament. More in particular the invention relates to the use of the derivatives in the treatment of cardiovascular disease.
  • An asterisk (*) in a chemical formula designates a point of attachment.
  • the present invention relates to a derivative comprising an apelin analogue; wherein the apelin analogue comprises a first amino acid having a sulphur atom in the side chain at a position corresponding to 28 of apelin(19-36) (SEQ ID NO: 67), and a second amino acid having a sulphur atom in the side chain at a position corresponding to 35 of apelin(19-36) (SEQ ID NO: 67), and 15-19 amino acid residues; wherein a methylene bridge of the formula: Si-G-te-Sn covalently links the first and the second amino acid having a sulphur atom in the side chain, wherein Si is the sulphur atom of the side chain of the first amino acid, and Sii is the sulphur atom of the side chain of the second amino acid; or a pharmaceutically acceptable salt, amide, or ester thereof.
  • the apelin analogue comprises a first amino acid having a sulphur atom in the side chain at
  • apelin-36 refers to human apelin-36.
  • Apelin-36 may also be referred to as "apelin(l-36)".
  • the amino acid sequence of apelin(l-36) is included in the sequence listing as SEQ ID NO: 1.
  • apelin-13 refers to human apelin-13.
  • the amino acid sequence of apelin-13 is included in the sequence listing as SEQ ID NO: 2.
  • apelin(19-36) refers to the amino acid sequence that spans from position 19 to position 36 (both included) of apelin(l-36). Apelin(19-36) is included in the sequence listing as SEQ ID NO: 67. Apelin(19-36) (SEQ ID NO:
  • 67 is essentially a truncated variant of apelin(l-36) (SEQ ID NO: 1).
  • apelin analogue refers to a peptide which has apelin activity.
  • the term "apelin analogue”, as used herein, may refer to a peptide which is an analogue of apelin(l-36) (SEQ ID NO: 1), wherein said analogue has one or more amino acid changes as compared to apelin(l-36) (SEQ ID NO: 1), wherein said amino acid changes may include amino acid additions, amino acid deletions, and/or amino acid substitutions.
  • the first amino acid residue of SEQ ID NO: 1 (Leu) i.e. the first residue of apelin(l-36)
  • any reference herein to an amino acid residue number or a position number of the apelin(l-36) sequence is to the sequence starting with Leu at position 1 and ending with Phe at position 36.
  • apelin analogue may refer to a peptide which is an analogue of apelin(19-36) (SEQ ID NO: 67), wherein said analogue has one or more amino acid changes as compared to apelin(19-36) (SEQ ID NO: 67), wherein said amino acid changes may include amino acid additions, amino acid deletions, and/or amino acid substitutions.
  • Arg the first amino acid residue of SEQ ID NO: 67 (Arg)
  • Arg residue is referred to as position 19, and subsequent amino acid residues are numbered accordingly, ending with Phe in position 36.
  • any reference herein to an amino acid residue number or a position number of the apelin(19-36) sequence is to the sequence starting with Arg at position 19 and ending with Phe at position 36.
  • the apelin analogue incorporated in the derivative of the invention is an analogue of apelin(19-36) (SEQ ID NO: 67).
  • substitution refers to one amino acid being replaced by another. Amino acids may be substituted by conservative
  • substitution denotes that one or more amino acids are replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids.
  • the apelin analogues of the derivatives of the invention may comprise substitutions by one or more non-coded amino acid.
  • Apelin analogues of the derivatives of the invention may be described by reference to i) the nature of the actual change, and to ii) the position of the amino acid residue in apelin(l-36) which is changed.
  • an amino acid change in the form of a substitution may be referred to as "aaX", wherein aa is the amino acid introduced in the substituted position, and wherein the X is a number which corresponds to the position of the amino acid residue of SEQ ID NO: 1 which is substituted.
  • An amino acid change in the form of a deletion may be referred to as "desX", wherein the X is a number which corresponds to the position of the amino acid residue of SEQ NO: l which is deleted; X may also refer an interval of positions, rather than a single position of amino acids, which is deleted.
  • Apelin(l-36) designates a sequence which, when compared to apelin(l-36), has the following amino acid changes: deletion of the amino acid residues in positions 1- 18 and position 36, as well as substitutions of Leu at position 28 with Cys, of His at position 30 with Lys, of Lys at position 31 with Arg, of Met at position 34 with Nle, and of Pro at position 35 with Cys.
  • Apelin(l-36) designates a sequence which is identical to apelin(19-36) (SEQ ID NO: 67).
  • Analogues "comprising" certain specified amino acid changes may comprise further amino acid changes, when compared to apelin(l-36) (SEQ ID NO: 1) or to apelin(19-36) (SEQ ID NO: 67).
  • the analogue "has" the specified changes.
  • a position "equivalent to” or “corresponding to” may be used to characterise the site of change in an apelin analogue by reference to apelin(l-36) (SEQ ID NO: 1) or apelin(19-36) (SEQ ID NO: 67).
  • an apelin analogue that comprises a Cys residue in a position corresponding to 28 of apelin(19-36) characterises an apelin analogue in which Leu at position 28 is substituted with Cys.
  • amino acid refers to any amino acid, naturally occurring (including the 20 standard amino acids which are encoded by the standard genetic code in humans) or not naturally occurring. Amino acid residues may be identified by their full name, their one-letter code, and/or their three-letter code as is usual in the art. These three ways are fully equivalent.
  • non-coded amino acids refers to all amino acids which aren't among the 20 standard amino acids encoded by the standard genetic code in humans. Non-coded amino acids may exist in nature or be purely synthetic. Non-limiting examples of non-coded amino acids are D-isomers of the coded amino acids, and glycine residues with a side chain attached to the nitrogen atom rather than the alpha-carbon atom.
  • D- isomers of coded amino acids may be referred to as (i) "D-" followed by the full name, followed by the one-letter code, or followed by the three-letter code of the amino acid, or as (ii) the lower case one-letter code of the amino acid. Further abbreviations for non-coded amino acids used in this application are presented in Table 1.
  • peptide as used in the context of the derivatives of the invention, may refer to the apelin analogue moiety of the derivative.
  • peptide may also refer to the amino acid sequence of human apelin-36 and human apelin 13.
  • the apelin analogue of the derivative of the invention comprises a first amino acid having a sulphur atom in the side chain at a position corresponding to 28 of apelin(19-36) (SEQ ID NO: 67), and a second amino acid having a sulphur atom in the side chain at a position corresponding to 35 of apelin(19-36) (SEQ ID NO: 67), and 15-19 amino acid residues.
  • the side chain of such an amino acid "having" a sulphur atom may have additional atoms in the side chain, e.g. as in the case of a Cys residue, and thus the term “having” may be used interchangeably with the term “comprising".
  • the first and the second amino acid having a sulphur atom in the side chain each are independently selected from a group consisting of Cys and D- Cys.
  • the maximum number of amino acid changes in the apelin analogue is ten as compared to apelin(19-36) (SEQ ID NO: 67).
  • the apelin analogue of the derivative of the invention comprises an amino acid sequence of Formula I: Xaai 8 -Xaai 9 -Xaa2o-Xaa2i-Xaa22- Xaa2 3 -Xaa24-Arg-Pro-Arg-Xaa2 8 -Ser-Xaa 30 -Xaa 3i -Gly-Pro-Xaa 34 -Xaa 35 -Xaa 36 , wherein Xaais is Arg or absent, Xaai 9 is Arg, D-Arg, Lys, or absent, Xaa2o is Lys, D-Lys, or absent, Xaa2i is Lys, Phe, or D-Phe, Xaa22 is Arg, D-Arg, Lys, or
  • the X in Formula II is H or Cl. In a more preferred embodiment, the X of Formula II is H or Cl in the para-position of the benzene ring. In a preferred embodiment, the number of amino acid changes of Formula I is 3-10 as compared to SEQ ID NO: 67.
  • the apelin analogue of the derivatives of the invention comprises 16-19 amino acids.
  • the apelin analogue of the derivatives of the invention may have an acetylated N-terminus.
  • the apelin analogue of the derivatives of the invention may have an amidated C-terminus.
  • derivative means a chemically modified apelin analogue, in which the apelin analogue has two residues, which are covalently linked via a methylene bridge.
  • the derivatives of the invention may have one or more substituents that are covalently attached to the apelin analogue.
  • the derivative may be referred to as a "compound”.
  • the derivative may be referred to as an "apelin derivative”.
  • the derivative may be referred to as an "apelin compound”.
  • the derivatives of the invention have apelin activity.
  • methylene bridge refers to a methylene moiety (i.e. -CH2-) that covalently links the side chains of two amino acids of the apelin analogue of the derivatives of the invention.
  • the methylene bridge covalently links two amino acids having a sulphur atom in the side chain.
  • the apelin analogue of the derivatives of the invention comprises a first amino acid having a sulphur atom in the side chain at a position corresponding to 28 of apelin(19-36) (SEQ ID NO:
  • a second amino acid having a sulphur atom in the side chain at a position corresponding to 35 of apelin(19-36) (SEQ ID NO: 67), wherein a methylene bridge of the formula: SrCH 2 -Sii covalently links said two amino acids having a sulphur atom in the side chain, wherein Si is the sulphur atom of the side chain of the first amino acid, and Sn is the sulphur atom of the side chain of the second amino acid residue.
  • the methylene bridge of the derivatives of the invention may be described by reference to i) the two amino acids between which the methylene bridge exists, and to ii) an indication of the existence of the methylene bridge by using the designation "(S,S'-methylenebis)".
  • a non-limiting example of methylene bridge nomenclature is "Cys28— Cys35(S,S'- methylenebis)", which indicates that a methylene bridge exists between the Cys residues at position 28 and 35.
  • Cys28— D-Cys35(S,S'-methylenebis) Another non-limiting example of methylene bridge nomenclature is "Cys28— D-Cys35(S,S'-methylenebis)", which indicates that a methylene bridge exists between the Cys residue at position 28 and the D-Cys residue at position 35.
  • substituted refers to a moiety that is covalently attached to the apelin analogue.
  • the substituent of the derivative may be covalently attached to any residue of the apelin analogue.
  • the derivative of the invention comprises one or more substituents.
  • the derivatives of the invention comprise one substituent.
  • the substituent is attached to any of the amino acid residues of the apelin analogue.
  • the substituent is attached to the epsilon position of a Lys residue of the apelin analogue, or to the N-terminal amino group of the apelin analogue.
  • the substituent is covalently attached to a Lys residue of the apelin analogue via an amide bond formed between a carboxylic acid group of the substituent and the epsilon-amino group of the Lys residue, or covalently attached to the N-terminus of the apelin analogue via an amide bond formed between a carboxylic acid group of the substituent and the N-terminal amino group of the apelin analogue.
  • the substituent is attached to the epsilon position of a Lys residue in position Xaai 9 , Xaa 2 o, Xaa 2i , Xaa 22 , Xaa 23 , Xaa 24 , Xaa 3 o, or Xaa 3i of Formula I, or to the N-terminal amino group of Formula I.
  • the substituent may be capable of forming non-covalent aggregates with albumin, thereby promoting the circulation of the derivative in the blood stream, and thus having the effect of protracting the time of action of the derivative, since the aggregate of the derivative and albumin is only slowly disintegrated to release the free form of the derivative.
  • the substituent as a whole, may also be referred to as an "albumin-binding moiety".
  • the substituent has a functional group (FG) with pKa ⁇ 7.0.
  • the pKa is the pKa of CH3-FG in water.
  • the pKa is calculated as the minus log Ka for the equilibrium of Equation 1 : CH3- FG + H2O ⁇ ??> FG + HsOT
  • the substituent is lipophilic, and/or negatively charged at physiological pH (7.4).
  • the substituent may comprise a portion which is particularly relevant for the albumin binding and thereby the protraction, which portion may be referred to as a "protracting moiety".
  • the protracting moiety may be near, preferably at, the terminal (or distal, or free) end of the substituent, relative to its point of attachment to the peptide.
  • the one or more protracting moieties comprises, or consists of, a moiety independently selected from : Chem. 1 : HOOC-(CH2)r-CO-*, wherein r is 12, 14, 16, 18, or 20 and Chem. 2: tetrazolyl-(CH2)s-CO-*, wherein s is an integer in the range of 12-20.
  • the r of Chem. 1 is 14, 16 or 18.
  • the s of Chem. 2 is 15.
  • the substituent may comprise a portion between the protracting moiety and the point of attachment to the peptide, which portion may be referred to as a "linker".
  • the linker may be optional, and hence in case no linker is present the albumin binding moiety may be identical to the protracting moiety.
  • the linker has the formula : -Z2-Z3-Z4-Z5-Z6
  • Zi is selected from :
  • Chem. 1 HOOC-(CH2)r-CO-*, wherein r is 12, 14, 16, 18, or 20,
  • Chem. 2 tetrazolyl-(CH2)s-CO-*, wherein s is an integer in the range of 12-20; wherein Z2 is selected from :
  • Chem. 5 *-NH-(CH2)2-0-(CH2)2-0-CH2-C0-*; wherein Zs and ⁇ 6 are identical and each selected from
  • the protracting moiety and the linker are interconnected via an amide bond.
  • the individual linker elements are interconnected via an amide bond.
  • the linker and the apelin analogue are interconnected via an amide bond.
  • the substituent and the apelin analogue are interconnected via an amide bond.
  • linker elements used in this application are presented in Table 2.
  • HOOC- refers to carboxy; -(CH2)s- to a number of methylene units corresponding to s; and -CO- to carbonyl.
  • Apelin-(1- 36) designates a derivative which, when compared to apelin(l-36), has the following amino acid changes: Deletion of amino acid residues 1-18, substitution of Leu at position 28 with Cys, of Met at position 34 with Nle, and of Pro at position 35 with D-Cys, and which has a methylene bridge between the Cys residue at position 28 and the D-Cys residue at position 35, and which has the [2- [2-[2-[2-[2-[2-[2-[2-[[[(4S)-4-car
  • the derivatives of the invention may exist in different stereoisomeric forms having the same molecular formula and sequence of bonded atoms but differing only in the three-dimensional orientation of their atoms in space.
  • the derivatives of the invention are selected from a group consisting of: Chem. 10, Chem. 11, Chem. 12, Chem. 13, Chem. 14, Chem. 15, Chem. 16, Chem. 17, Chem. 18, Chem. 19, Chem. 20, Chem. 21,
  • concentration of the derivatives of the invention may be analysed and determined using any suitable method, e.g. RP-HPLC with UV detection, dissociative SEC with UV detection, and HPLC-CLND.
  • the derivatives of the invention may be in the form of a pharmaceutically acceptable salt, amide, or ester.
  • Salts are e.g. formed by a chemical reaction between a base and an acid, e.g. : 2NH 3 + H2SO4 (NhU ⁇ SCM.
  • the salt may be a basic salt, an acid salt, or it may be neither nor (i.e. a neutral salt).
  • Basic salts produce hydroxide ions and acid salts hydronium ions in water.
  • the salts of the derivatives of the invention may be formed with added cations or anions between anionic or cationic groups, respectively. These groups may be situated in the apelin analogue and/or in the substituent of the derivatives of the invention.
  • Non-limiting examples of anionic groups of the derivatives of the invention include free carboxylic groups in the substituent, if any, as well as in the apelin analogue.
  • the amino acid sequence often includes a free carboxylic acid group at the C-terminus, and it may also include free carboxylic groups at internal acid amino acid residues such as Asp and Glu.
  • Non-limiting examples of cationic groups in the apelin analogue include the free amino group at the N-terminus, if present, as well as any free amino group of internal basic amino acid residues such as His, Arg, and Lys.
  • the amino group at the N-terminus of the apelin analogue of the derivatives of the invention may be free or acetylated.
  • the ester of the derivatives of the invention may, e.g., be formed by the reaction of a free carboxylic acid group with an alcohol or a phenol, which leads to replacement of at least one hydroxyl group by an alkoxy or aryloxy group
  • the ester formation may involve the free carboxylic group at the C- terminus of the apelin analogue, and/or any free carboxylic group in the substituent.
  • the amide of the derivatives of the invention may, e.g., be formed by the reaction of a free carboxylic acid group with an amine or a substituted amine, or by reaction of a free or substituted amino group with a carboxylic acid.
  • the amide formation may involve the free carboxylic group at the C- terminus of the apelin analogue, any free carboxylic group in the substituent, the free amino group at the N-terminus of the apelin analogue, and/or any free or substituted amino group in the apelin analogue and/or in the substituent.
  • the derivative is in the form of a
  • the derivative is in the form of a pharmaceutically acceptable amide. In a preferred embodiment, the derivative is in the form a pharmaceutically acceptable ester.
  • the derivatives of the invention have desirable chemical stability. Also, or alternatively, the derivatives of the invention have a surprisingly high chemical stability. In a second functional aspect, the derivatives of the invention have desirable apelin activity. Also, or alternatively, the derivatives of the invention have a surprisingly high apelin activity.
  • the derivatives of the invention have desirable chemical stability.
  • Chemical stability may be affected by the tendency of the derivatives to form aggregates.
  • chemical stability may be affected by the tendency of derivatives to undergo chemical changes in the structure leading to formation of chemical degradation.
  • the chemical stability of the derivatives may be evaluated by means of standard quantification methods for various time periods of exposure to stress in the form of heat.
  • the chemical stability may be evaluated by measuring the amount of high molecular weight product (HMWP) formation and/or purity loss at various time-points after exposure to stress in the form of heat.
  • HMWP formation may refer to the formation of covalently linked aggregates such as dimers, trimers and higher oligomeric species over time.
  • HMWP formation may be measured by size- exclusion chromatography-high performance liquid chromatography (SEC-HPLC).
  • Purity loss may refer to the quantitative loss of derivative and/or the formation of chemical degradation products. Purity loss may be measured by reverse phase- high performance liquid chromatography (RP-HPLC).
  • the HMWP formation is determined as the difference in HMWP formation, analysed by SEC-HPLC, between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C. In a preferred embodiment, the HMWP formation is determined as described in Example 65.
  • the purity loss is determined as the difference in purity loss, analysed by RP-HPLC, between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C. In a preferred embodiment, the purity loss is determined as described in Example 65.
  • the chemical stability, determined as the difference between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C, of the derivatives is higher than that of human apelin-13 (SEQ ID NO: 2).
  • the chemical stability, determined as the difference between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C, of the derivatives is higher than that of a derivative variant that contains a disulphide bridge instead of the methylene bridge.
  • the HMWP formation, determined as the difference between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C, of the derivatives is ⁇ 10%, preferably is ⁇ 5%, more preferably is ⁇ 2%, more preferably is ⁇ 1%, most preferably is ⁇ 0.5%.
  • the purity loss, determined as the relative difference between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C, of the derivatives is ⁇ 20%, preferably is ⁇ 15%, more preferably is ⁇ 10%, more preferably is ⁇ 9%, more preferably is ⁇ 5%, more preferably is ⁇ 4%, more preferably is ⁇ 3%, more preferably is ⁇ 2%, and most preferably is ⁇ 1%.
  • the HMWP formation determined as the difference between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C, is ⁇ 2%
  • the purity loss determined as the difference between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C, is ⁇ 9%.
  • the chemical stability of the derivatives of the invention is expressed by HMWP formation. In a preferred embodiment, the chemical stability of the derivatives of the invention is expressed by purity loss.
  • the chemical stability of the derivatives of the invention is expressed by both HMWP formation and purity loss.
  • the derivatives of the invention show a surprisingly high chemical stability as compared to derivatives without a methylene bridge (e.g. Chem. 43 vs. Chem. 72 in Example 65).
  • Apelin activity e.g. Chem. 43 vs. Chem. 72 in Example 65.
  • the derivatives of the invention have apelin activity.
  • Apelin activity refers to the ability to modulate hAPJR.
  • the apelin activity may also be referred to as "biological activity” and/or "bioactivity”.
  • the apelin activity may be measured as activation of hAPJR in the presence of albumin.
  • the apelin activity may be measured as activation of the hAPJR, which is encoded by the AGTRL1 gene, in the presence of albumin.
  • the hAPJR activates the inhibitory G protein alphai (Galphai). Galphai activation results in a reduced cellular level of the second messenger cyclin AMP (cAMP), and thus measurements of cAMP may be utilised to quantify apelin activity.
  • cAMP second messenger cyclin AMP
  • the hAPJR activation is determined, in the presence of albumin, using an assay measuring the ability of the derivative to inhibit the forskolin induced generation of cAMP in a cell line expressing hAPJR.
  • the level of hAPJR activation of the derivatives is expressed as a value relative to that of human apelin-13 (SEQ ID NO : 2).
  • the apelin activity is measured as human apelin receptor activation and expressed as a value relative to that of apelin-13 in the form of IC50 Fold.
  • the apelin activity of the derivatives of the invention is determined as described in Example 66.
  • the derivatives of the invention have apelin activity expressed as the IC50 Fold which is >0.010, preferably is >0.015, more preferably is >0.020, more preferably is >0.025, more preferably is >0.030, more preferably is >0.040, more preferably is >0.050, and most preferably is >0.100.
  • the derivatives of the invention have apelin activity expressed as the IC50 Fold which is ⁇ 1.000, preferably is ⁇ 2.000, and most preferably is ⁇ 5.000.
  • the derivatives of the invention show a surprisingly high bioactivity as compared to derivatives with shorter apelin analogue sequences (e.g. Chem. 43 vs. Chem. 70 in Example 66).
  • the present invention also relates to a derivative of the invention, for use as a medicament.
  • treatment refers to the medical treatment of any human subject in need thereof.
  • the timing and purpose of said treatment may vary from one individual to another, according to the status of the subject's health.
  • Said treatment may be prophylactic, palliative, symptomatic and/or curative.
  • the derivative of the invention may be used for the following :
  • cardiovascular diseases such as atherosclerosis, myocardial infarction, coronary heart disease, ischemic cardiovascular disease stroke, an early cardiac or early cardiovascular disease, left ventricular hypertrophy, cardiac fibrosis, coronary artery disease, hypertension, essential hypertension, pulmonary hypertension, acute hypertensive emergency, cardiomyopathy, heart insufficiency, exercise intolerance, acute and/or chronic heart failure, atrial fibrillation, arrhythmia, cardiac dysrhythmia, ventricular tachycardia, syncopy, angina pectoris, diastolic dysfunction, and/or systolic dysfunction, water retention and preeclampsia
  • cardiovascular diseases such as atherosclerosis, myocardial infarction, coronary heart disease, ischemic cardiovascular disease stroke, an early cardiac or early cardiovascular disease, left ventricular hypertrophy, cardiac fibrosis, coronary artery disease, hypertension, essential hypertension, pulmonary hypertension, acute hypertensive emergency, cardiomyopathy, heart insufficiency, exercise intolerance
  • hyperglycemia type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reduction of HbAlC
  • the indication is (i), (ii), and (iii). In a more preferred embodiment, the indication is (i) and (ii). In a most preferred
  • the indication is (i).
  • the apelin analogue of the derivatives of the invention may for instance be prepared by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, see, e.g. Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza Dorwald, "Organic Synthesis on solid Phase", Wiley-VCH Verlag GmbH, 2000, and "Fmoc Solid Phase Peptide
  • telomeres may be produced, in whole or in part, by recombinant methods, viz. by culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the peptide in a suitable nutrient medium under conditions permitting the expression of the peptide.
  • host cells suitable for expression of these peptides are: Escherichia coli, Saccharomyces cerevisiae, as well as mammalian BHK or CHO cell lines.
  • Those derivatives of the invention which include non-coded amino acids may e.g. be produced as described in the experimental part. Or see e.g., Hodgson et al: "The synthesis of peptides and proteins containing non-natural amino acids", Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430.
  • compositions are included in the experimental part.
  • compositions comprising a derivative of the invention or a pharmaceutically acceptable salt, amide, or ester thereof, and a pharmaceutically acceptable excipient may be prepared as is known in the art.
  • excipient broadly refers to any component other than the active therapeutic ingredient(s).
  • the excipient may be an inert substance, an inactive substance, and/or a not medicinally active substance.
  • the excipient may serve various purposes, e.g. as a carrier, vehicle, diluent, tablet aid, and/or to improve administration, and/or absorption of the active substance.
  • Non-limiting examples of excipients are: Solvents, diluents, buffers, preservatives, tonicity regulating agents, chelating agents, surfactants, and stabilisers.
  • ingredients of a pharmaceutical composition include, e.g., wetting agents, emulsifiers, antioxidants, bulking agents, metal ions, oily vehicles, proteins.
  • a pharmaceutical composition comprising the derivatives of the invention may be of several dosage forms, e.g. a solution, a suspension, a tablet, and a capsule.
  • the pharmaceutical composition comprising the derivatives of the invention may be administered to a patient in need thereof at several sites, e.g. at topical sites such as skin or mucosal sites; at sites which bypass absorption such as in an artery, in a vein, or in the heart; and at sites which involve absorption, such as in the skin, under the skin, in a muscle, orally, or in the abdomen.
  • An administered dose may contain from 0. lug/kg to lOOmg/kg of the derivatives of the invention.
  • the treatment with a derivative according to the present invention may also be combined with one or more additional pharmacologically active substances, e.g. selected from cardiovascular agents, antidiabetic agents, and/or anti-obesity agents.
  • these pharmacologically active substances are: inotropes, beta adrenergic receptor blockers, HMG-CoA reductase inhibitors, angiotensin II receptor antagonists, angiotensin converting enzyme (ACE) inhibitors, calcium channel blockers (CCB), endothelin antagonists, renin inhibitors, diuretics, aldosterone receptor blockers, endothelin receptor blockers, aldosterone synthase inhibitors, CETP inhibitor, relaxin, PCSK9 inhibitors, BNP and NEP inhibitors, GLP-1 analogues, insulin, sulphonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors,
  • the treatment with a derivative of this invention may also be combined with heart surgery.
  • the pharmaceutical composition comprising the derivatives of the invention may be used for the following :
  • cardiovascular diseases such as atherosclerosis, myocardial infarction, coronary heart disease, ischemic cardiovascular disease stroke, an early cardiac or early cardiovascular disease, left ventricular
  • hypertrophy cardiac fibrosis, coronary artery disease, hypertension, essential hypertension, pulmonary hypertension, acute hypertensive emergency, cardiomyopathy, heart insufficiency, exercise intolerance, acute and/or chronic heart failure, atrial fibrillation, arrhythmia, cardiac dysrhythmia, ventricular tachycardia, syncopy, angina pectoris, diastolic dysfunction, and/or systolic dysfunction, water retention and preeclampsia
  • hyperglycemia type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young), gestational diabetes, and/or for reduction of HbAlC
  • the indication is (i), (ii), and (iii). In a more preferred embodiment, the indication is (i) and (ii). In a most preferred embodiment the indication is (i).
  • a derivative comprising an apelin analogue
  • the apelin analogue comprises a first amino acid having a sulphur atom in the side chain at a position corresponding to 28 of apelin(19-36) (SEQ ID NO:
  • a methylene bridge of the formula: Si-G-te-Sn covalently links said two amino acids having a sulphur atom in the side chain, wherein Si is the sulphur atom of the side chain of the first amino acid, and Sn is the sulphur atom of the side chain of the second amino acid residues;
  • apelin analogue comprises an amino acid sequence of Formula I:
  • Xaa 3i -Gly-Pro-Xaa 34 -Xaa 35 -Xaa 36 ; wherein Xaais is Arg or absent, Xaai 9 is Arg, D-Arg, Lys, or absent, Xaa 2 o is Lys, D-Lys, or absent, Xaa 2i is Lys, Phe, or D-Phe, Xaa 22 is Arg, D-Arg, Lys, or N-Me-Arg, Xaa 23 is Arg, D-Arg, Lys, or N-Me-Arg, Xaa 24 is Gin or Lys, Xaa 28 is Cys or D-Cys, Xaa 3 o is Arg, His, or Lys, Xaa 3i is Arg or Lys, Xaa 34 is Leu, Nle, Phe, or Cha, Xaa 35 is Cys or D-Cys, and Xaa 36 is Leu, Phe, al
  • N- terminus may be acetylated, and wherein the C-terminus may be amidated.
  • Chem. 1 HOOC-(CH2)r-CO-*, wherein r is 12, 14, 16, 18, or 20,
  • Chem. 2 tetrazolyl-(CH2)s-CO-*, wherein s is an integer in the range of 12-20; wherein Z2 is selected from :
  • Chem. 5 *-NH-(CH2)2-0-(CH2)2-0-CH 2 -C0-*; wherein Zs and ⁇ 6 are identical and each selected from
  • Chem. 1 HOOC-(CH2)r-CO-*, wherein r is 14, 16, or 18
  • Chem. 2 tetrazolyl-(CH2)s-CO-*, wherein s is 15;
  • Chem. 49 Chem. 50, Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem. 66, Chem. 67, Chem. 68, and Chem. 69.
  • Chem. 50 Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem. 66, Chem. 67, Chem. 68, and Chem. 69.
  • Chem. 15 Chem. 16, Chem. 17, Chem. 18, Chem. 19, Chem. 20, Chem. 21, Chem. 22, Chem. 23, Chem. 24, Chem. 25, Chem. 27, Chem. 28, Chem. 29, Chem. 30, Chem. 31, Chem. 32, Chem. 33, Chem. 34, Chem. 35, Chem. 36, Chem. 37, Chem. 38, Chem. 39, Chem. 40, Chem. 41, Chem. 42, Chem. 43, Chem. 44, Chem. 45, Chem. 46, Chem. 47, Chem. 48, Chem. 49, Chem. 50, Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 65, Chem. 66, Chem. 67, and Chem. 69.
  • Chem. 16 Chem. 17, Chem. 18, Chem. 19, Chem. 20, Chem. 21, Chem. 22, Chem. 23, Chem. 24, Chem. 27, Chem. 28, Chem. 29, Chem. 30, Chem. 31, Chem. 33, Chem. 34, Chem. 35, Chem. 36, Chem. 37, Chem. 38, Chem. 39, Chem. 40, Chem. 41, Chem. 42, Chem. 43, Chem. 44, Chem. 45, Chem. 46, Chem. 47, Chem. 49, Chem. 51, Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61, Chem. 63, Chem. 65, Chem. 66, Chem. 67, and Chem. 69.
  • HMWP high molecular weight products
  • any preceding embodiment for which the purity loss, determined as the difference between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C, is ⁇ 20%, preferably is ⁇ 15%, more preferably is ⁇ 10%, more preferably is ⁇ 9%, more preferably is ⁇ 5%, more preferably is ⁇ 4%, more preferably is ⁇ 3%, more preferably is ⁇ 2%, and most preferably is ⁇ 1%.
  • apelin activity refers to the ability to inhibit forskolin induced cAMP accumulation in a cell line expressing the human apelin receptor.
  • apelin activity refers to the ability to inhibit forskolin induced cAMP accumulation in a cell line expressing the human apelin receptor encoded by the AGTRL1 gene.
  • >0.015 more preferably is >0.020, more preferably is >0.025, more preferably is >0.030, more preferably is >0.040, more preferably is >0.050, and most preferably is >0.100.
  • a derivative comprising an apelin analogue
  • apelin analogue comprises Formula I:
  • Xaa 3i -Gly-Pro-Xaa 34 -Xaa 35 -Xaa 36 ; wherein Xaais is Arg or absent, Xaai 9 is Arg, D-Arg, Lys, or absent, Xaa 2 o is Lys, D-Lys, or absent, Xaa 2i is Lys, Phe, or D-Phe, Xaa 22 is Arg, D-Arg, Lys, or N-Me-Arg, Xaa 23 is Arg, D-Arg, Lys, or N-Me-Arg, Xaa 24 is Gin or Lys, Xaa 28 is Cys or D-Cys, Xaa 3 o is Arg, His or Lys, Xaa 3i is Arg, or Lys, Xaa 34 is Leu, Nle, Phe, or Cha, Xaa 35 is Cys or D-Cys, and Xaa 36 is Leu, Phe, al
  • X is H or a halogen; wherein Xaa 28 and Xaa 35 are covalently linked through a methylene bridge of the formula: S 28 -CH2-S 35 , wherein S 28 is the sulphur atom of the side chain of Xaa 28 , and S35 is the sulphur atom of the side chain of Xaa 3 s; or a pharmaceutically acceptable salt, amide, or ester thereof.
  • a method of treating cardiovascular disease comprising administering to a patient in need thereof an effective amount of the derivative according to any preceding embodiment, optionally in combination with one or more additional therapeutically active compounds.
  • a pharmaceutical composition comprising a derivative according to any of the proceeding embodiments, and one or more excipients.
  • a pharmaceutical composition comprising a derivative according to any of the proceeding embodiments, and one or more excipients, for use as a
  • a pharmaceutical composition comprising a derivative according to any of the proceeding embodiments, and one or more excipients, for use as in treatment of all forms of cardiovascular disease.
  • a derivative of an apelin analogue wherein the derivative comprises: an apelin analogue comprising Formula I :
  • Xaa34-Xaa35-Xaa36 wherein Xaai9 is Arg, D-Arg, Lys, or absent, Xaa 2 o is Lys, D-Lys, or absent, Xaa 2i is Lys, Phe, or D-Phe, Xaa 2 2 is Arg, D-Arg, or Lys, Xaa 23 is Arg, D- Arg, or Lys, Xaa 24 is Gin or Lys, Xaa 28 is Cys or D-Cys, Xaa3o is Arg, His or Lys, Xaa3i is Arg or Lys, Xaa34 is Nle, Xaa35 is Cys or D-Cys, and Xaa36 is Leu, Phe, alpha-Me-Phe, 4-CI-Phe, Trp, Yaa, or absent; provided that if Xaa 2 o is absent then Xaai9 is absent; wherein Yaa
  • X is H or a halogen; wherein the N-terminal amino group may be acetylated wherein Xaa 28 and Xaa35 are covalently linked through a methylene bridge of the formula : S28-CH2-S35, wherein S28 is the sulphur atom of the side chain of Xaa 28 , and S35 is the sulphur atom of the side chain of Xaa3s; and one or more substituents comprising at least one functional group (FG) with pKa ⁇ 7.0; or a pharmaceutically acceptable salt, amide, or ester thereof.
  • Chem. 1 HOOC-(CH2)r-CO-*, wherein r is 12, 14, 16, 18, or 20,
  • Chem.2 tetrazolyl-(CH2)s-CO-*, wherein s is an integer in the range of 12-20; wherein Z2 is selected from:
  • Chem.4 gGlu; wherein each of Z3 and Z 4 is
  • Chem.5 *-NH-(CH2)2-0-(CH2)2-0-CH2-C0-*; wherein Zs and ZQ are identical and each selected from
  • Chem. 1 HOOC-(CH2)r-CO-*, wherein r is 12, 14, 16, 18, or 20,
  • Chem.2 tetrazolyl-(CH2)s-CO-*, wherein s is an integer in the range of 12-20; wherein Z2 is selected from:
  • Chem.4 gGlu; wherein each of Z3 and Z 4 is
  • Chem.5 *-NH-(CH2)2-0-(CH2)2-0-CH2-C0-*; wherein Z5 and ZQ are identical and each selected from
  • Z5 and ZQ are present, then ZQ is attached to the epsilon position of a Lys residue of Formula I, or to the N-terminal amino group of Formula I; provided that if Z5 and ZQ are absent, then Z 4 is attached to the epsilon position of a Lys residue of Formula I, or to the N-terminal amino group of Formula I.
  • Zi is Chem. 1 : HOOC-(CH2)r-CO-*, wherein r is 12, 14, 16, 18, or 20; wherein Z 2 is Chem. 4: gGlu.
  • HMWP high molecular weight products
  • the derivative of any of the preceding embodiments, for which the purity loss, determined as the difference between a sample stored for 2 weeks at 37°C and sample stored for 2 weeks at 5°C, is ⁇ 10.00%, preferably ⁇ 5.00%, and most preferably ⁇ 1.00%.
  • apelin activity refers to the ability to inhibit forskolin induced cAMP accumulation in a cell line expressing the human apelin receptor.
  • apelin activity refers to the ability to inhibit forskolin induced cAMP accumulation in a cell line expressing the human apelin receptor encoded by the AGTRL1 gene.
  • a method of treating cardiovascular disease comprising administering to a patient in need thereof an effective amount of the derivative according to any of the preceding embodiments, optionally in combination with one or more additional therapeutically active compounds.
  • a pharmaceutical composition comprising a derivative according to any of the proceeding embodiments, and one or more excipients.
  • DIPEA Diisopropylethylamine
  • DMAP 4-dimethylaminopyridine
  • NMP N-methyl pyrrolidone
  • TIPS triisopropylsilane
  • TIS triisopropylsilane
  • This section is divided into several subsections. First, a subsection on synthesis of protractors and linker elements are provided. Secondly, a subsection on synthesis of building blocks for introducing non-coded amino acids is provided. Thirdly, a subsection on synthesis of the derivatives of the invention is provided. Fourthly a subsection on detection and characterization of the derivatives is provided. Finally, several examples of the preparation of specific derivatives is provided. Unless otherwise stated the general methods of preparation was used in the preparation of the individual example compounds.
  • Fmoc-8-amino-3,6-dioxaoctanoic acid was commercially available from e.g. Iris Biotech.
  • Fmoc-Glu-OtBu was commercially available from e.g. Bachem and Sigma- Aldrich.
  • octadecanedioic acid mono-tert-butyl ester see patent application W02010102886 (pages 27-28).
  • the corresponding mono-tert-butyl esters of C14-, C16- and C20 diacid can be prepared accordingly.
  • Fmoc-protected Nle, alpha-Me-Phe, N-Me-Arg, N-Me-Phe, and 4Bn-Tyr were all commercially available and supplied by e.g. NovabioChem and Sigma Aldrich.
  • a 2-Chlorotrityl resin (0.6 mmol/g, 1 g) was pre-swelled in DCM (20 ml_) under stirring for 20 min. The resin was drained, and a solution of bromoacetic acid (530 mg) and DIPEA (2 ml), in DCM (20 ml) was added, and the mixture was shaken for 1 hour, dried and washed multiple times with DCM. Benzylamine (10 eq., for N-Phe) or 4-chlorobenzylamine (10 eq., for 4-CI-Nphe) dissolved in DMF was added to the mixture. Then the mixture was shaken for 1 hour, dried and washed multiple times with DCM. The preloaded resin was then used directly for peptide coupling by the general method for preparation of the peptide and introduction of the substituent as described below.
  • This section relates to methods for preparing the derivatives, including peptide preparation by SPPS, methods for introducing the substituent, methods for cleaving the compound from the resin, methods for introducing the methylene bridge as well as methods for purification and quantification.
  • the derivatives of the invention may be prepared as described in the examples herein.
  • the derivatives of the invention may be prepared as known in the art, i.e. the preparation of peptides may be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using Boc or Fmoc chemistry or other well-established techniques, see, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza Dorwald, "Organic Synthesis on solid Phase", Wiley-VCH Verlag GmbH, 2000, and "Fmoc Solid Phase Peptide Synthesis", Edited by W.C. Chan and P.D. White, Oxford University Press, 2000.
  • Fmoc-protected amino acids used in the methods were the standard recommended : Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Mmt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc- Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Fmoc)-OH Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)
  • the resin was then resuspended in HFIP/DCM (75:25)(2x20min or 2x30 min) and subsequently washed with DCM before the substituent was introduced at the epsilon-nitrogen of the Lys.
  • Introduction of the substituent at the alpha-position of the N-terminal amino acid was accomplished using a standard Fmoc-protected amino acid, i.e. Fmoc-Gly-OH serving as a first element of the substituent.
  • the moieties of the substituent were introduced in a stepwise procedure by a Prelude peptide synthesizer following the SPPS principles described for the peptide preparation above.
  • Suitable protected building blocks such as the standard Fmoc-protected amino acids, Fmoc-8-amino-3,6- dioxaoctanoic acid and Fmoc-Glu-OtBu, were used.
  • Introduction of the final element of the moiety i.e. the fatty acid group
  • the suitable building block such as but not limited to, octadecanedioic acid mono-tert-butyl- ester.
  • the coupling time was increased or the coupling step for each building block was repeated.
  • acetyl groups in the N- terminus was introduced by acylation with 1M acetic anhydride in DMF or NMP.
  • preparation of C-terminal peptide amides was carried using PAL Amide AM resin (loading e.g. 0.61 mmol/g) or H-Rink Amide-ChemMatrix resin (loading e.g. 0.52 nmol/g) or Rink Amide AM polystyrene resin (NovabioChem, loading e.g. 0.62 mmol/g).
  • the crude peptide was acidified with TFA to pH 2-3 and purified by reversed- phase preparative HPLC (Waters Deltaprep 4000 or Gilson) on a column comprising C8- or C18-silica gel. Elution was performed with an increasing gradient of MeCN in water comprising 0.1% TFA. Relevant fractions were analysed using UPLC. Fractions comprising the pure target peptide were pooled. The resulting solution was analysed (UPLC, LCMS) and the peptide derivative was quantified using a chemiluminescent nitrogen specific HPLC detector (Antek 8060 HPLC-CLND). The product was dispensed into glass vials. The vials were capped with Millipore glass fibre prefilters. Freeze-drying afforded the trifluoroacetate salt of the derivative as a white solid.
  • Apelin-13 (SEQ ID NO: 2) was used as reference material.
  • Apelin-13 was prepared according to the general method for preparation of the peptide with SPPS using Fmoc based chemistry on a Prelude Solid Phase Peptide Synthesizer as described above.
  • the LCMS027 method was used for detection and
  • Cys28 Cys35(S,S'-methylenebis), Nle34] Apelin-(l-36)
  • Cys28 Cys35(S,S'-methylenebis), Nle34, alpha-Me-Phe36] Apelin-(l-36)
  • Cys28 Cys35(S,S'-methylenebis), Lys30, Arg31, Nle34] Apelin-(l-36)
  • Cys28 Cys35(S,S'-methylenebis), Nle34] Apelin(l-36)-amide
  • SPPS was performed using Fmoc based chemistry on a SymphonyX Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 U.S.A.).
  • N-terminus amino group was trapped with Boc protection and introduction of methylene bridge by general method B.
  • the peptide was synthesized as described in the general methods. The
  • the peptide was synthesized as described in the general methods. The
  • HMWP formation refers to the formation of covalently linked aggregates such as dimers, trimers and higher oligomeric species.
  • the molecular size of such aggregates is different from that of the non-aggregated derivative, and these size differences may be utilised to analyse HMWP formation using dissociative SEC-HPLC.
  • Subjecting the derivatives to heat i.e. stressing the derivatives
  • a sample incubated for 2 weeks at 37°C was compared to a sample incubated for 2 weeks at 5°C.
  • Sample preparation the derivative was solubilized in a vehicle consisting of 10 mM His buffer (L-histidine (Alsiano), pH 6.5).
  • Sample treatment The sample solution was allocated into two UPLC vials with tight screw caps. The two UPLC vials were incubated at 37°C and 5°C,
  • the relative amount of HMWP was defined as the area percentage of peaks eluting prior to the main peak (i.e. the peak of the intact derivative) relative to the total area of all integrated peaks (not related to the method or the vehicle) in each chromatogram.
  • Sample preparation the derivative was solubilized in a vehicle consisting of 10 mM His buffer (L-histidine (Alsiano), pH 6.5).
  • Sample treatment The sample solution was allocated into two UPLC vials with tight screw caps. The UPLC vials were incubated at 37°C and 5°C, respectively, for 2 weeks, and subsequently they were analysed.
  • Purity was defined as the area percentage of the main peak (i.e. the peak of the intact derivative) relative to the total area of all integrated peaks (not related to the method or the vehicle) in each chromatogram.
  • the purpose of this method was to investigate the apelin activity (also referred to as "potency" in the following) of the derivatives of the invention in the presence of albumin.
  • the potency was determined as the in vitro hAPJR activation in a whole cell assay.
  • the hAPJR is a class G protein coupled receptor that activates the inhibitory G- protein alphai (Galphai). Galphai activation results in a reduced cellular level of the second messenger cAMP. Therefore, the potency of the derivative on the hAPJR may be measured as the ability of the derivatives to inhibit the forskolin induced generation of the second messenger cAMP in a cell line expressing hAPJR. The potency of the derivatives may be reported as the level of hAPJR activation relative to that of human apelin-13 (SEQ ID NO: 2). The ability of the derivatives to inhibit forskolin induced cAMP accumulation was performed using the CHO-K1 AGTRL1 Cell Line, part # 93-1050C2, from
  • DiscoverX i.e. a cell line expressing the hAPJR.
  • the CAMP dynamic 2 KIT (Cisbio cat. no. 62AM4PEJ) were used to detect cAMP levels.
  • This kit is specifically intended for the direct quantitative determination of cAMP.
  • the assay is based on a competition between native cAMP produced by cells and cAMP labelled with the dye d2 for binding to a cryptate labelled antibody.
  • the specific signal i.e. energy transfer signal
  • the method steps are outlined in the following :
  • CAMP dynamic 2 KIT Cisbio # 62AM4PEJ
  • the fluorescent signal from the wells was detected on a Mithras microplate multimode reader (Berthold Technologies) using the following filters: The donor signal was detected by using excitation at 320 nm and emission at 620 nm and the acceptor signal was detected by using excitation at 320 nm and emission at 665 nm.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des dérivés comprenant un analogue d'apeline ; l'analogue d'apeline comprenant deux acides aminés ayant un atome de soufre dans la chaîne latérale, lesdits deux acides aminés étant liés de manière covalente par l'intermédiaire d'un pont de méthylène. L'invention concerne également l'utilisation pharmaceutique des dérivés, de préférence dans le traitement de toutes les formes de maladie cardiovasculaire.
PCT/EP2019/064202 2018-05-31 2019-05-31 Dérivés comprenant un analogue d'apeline et leurs utilisations WO2019229242A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US201862678669P 2018-05-31 2018-05-31
US62/678,669 2018-05-31
EP18177304 2018-06-12
EP18177304.5 2018-06-12
US201862695411P 2018-07-09 2018-07-09
US62/695,411 2018-07-09

Publications (1)

Publication Number Publication Date
WO2019229242A1 true WO2019229242A1 (fr) 2019-12-05

Family

ID=66647421

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2019/064202 WO2019229242A1 (fr) 2018-05-31 2019-05-31 Dérivés comprenant un analogue d'apeline et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2019229242A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11744873B2 (en) 2021-01-20 2023-09-05 Viking Therapeutics, Inc. Compositions and methods for the treatment of metabolic and liver disorders

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006005667A2 (fr) 2004-07-08 2006-01-19 Novo Nordisk A/S Marquages de prolongation de polypeptide
WO2010102886A1 (fr) 2009-02-19 2010-09-16 Novo Nordisk A/S Modification du facteur viii
WO2014099984A1 (fr) 2012-12-20 2014-06-26 Amgen Inc. Agonistes du récepteur apj et leurs utilisations
WO2015013167A1 (fr) 2013-07-25 2015-01-29 Novartis Ag Polypeptides cyclique disulfure pour le traitement de l'insuffisance cardiaque
WO2015191781A2 (fr) * 2014-06-10 2015-12-17 Amgen Inc. Polypeptides d'apéline
WO2016116842A1 (fr) 2015-01-23 2016-07-28 Novartis Ag Conjugués d'acides gras de l'apeline synthétique présentant une demi-vie améliorée

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006005667A2 (fr) 2004-07-08 2006-01-19 Novo Nordisk A/S Marquages de prolongation de polypeptide
WO2010102886A1 (fr) 2009-02-19 2010-09-16 Novo Nordisk A/S Modification du facteur viii
WO2014099984A1 (fr) 2012-12-20 2014-06-26 Amgen Inc. Agonistes du récepteur apj et leurs utilisations
WO2015013167A1 (fr) 2013-07-25 2015-01-29 Novartis Ag Polypeptides cyclique disulfure pour le traitement de l'insuffisance cardiaque
WO2015191781A2 (fr) * 2014-06-10 2015-12-17 Amgen Inc. Polypeptides d'apéline
WO2016116842A1 (fr) 2015-01-23 2016-07-28 Novartis Ag Conjugués d'acides gras de l'apeline synthétique présentant une demi-vie améliorée

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"Optimal Alignments in Linear Space", CABIOS (COMPUTER APPLICATIONS IN THE BIOSCIENCES), vol. 4, 1988, pages 11 - 17
"Remington: The Science and Practice of Pharmacy", 1995
C. M. B. K. KOURRA ET AL: "Converting disulfide bridges in native peptides to stable methylene thioacetals", CHEMICAL SCIENCE, vol. 7, no. 12, 1 January 2016 (2016-01-01), pages 7007 - 7012, XP055507366, DOI: 10.1039/C6SC02285E *
FLORENCIO ZARAGOZA DORWALD: "Fmoc Solid Phase Peptide Synthesis", 2000, OXFORD UNIVERSITY PRESS
GREENEWUTS: "Protective Groups in Organic Synthesis", 1999, JOHN WILEY & SONS
HODGSON ET AL.: "The synthesis of peptides and proteins containing non-natural amino acids", CHEMICAL SOCIETY REVIEWS, vol. 33, no. 7, 2004, pages 422 - 430, XP008067398
HYPERTENSION, vol. 54, 2009, pages 598 - 604
INTERNATIONAL JOURNAL OF CARDIOLOGY, vol. 230, 2017, pages 164 - 170
K TRÂN ET AL: "A Systematic Exploration of Macrocyclization in Apelin-13: Impact on Binding, Signaling, Stability, and Cardiovascular Effects", JOURNAL OF MEDICINAL CHEMISTRY, vol. 61, no. 6, 20 February 2018 (2018-02-20), pages 2266 - 2277, XP055507284, DOI: 10.1021/acs.jmedchem.7b01353 *
NEEDLEMAN, S.B.WUNSCH, C.D., JOURNAL OF MOLECULAR BIOLOGY, vol. 48, 1970, pages 443 - 453

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11744873B2 (en) 2021-01-20 2023-09-05 Viking Therapeutics, Inc. Compositions and methods for the treatment of metabolic and liver disorders

Similar Documents

Publication Publication Date Title
AU2019263674B2 (en) GIP derivatives and uses thereof
CA3084004C (fr) Analogues d'incretine et leurs utilisations
KR100658961B1 (ko) 공유적으로 가교결합된 인슐린 이량체, 이를 포함하는 약제학적 조성물 및 진단 키트, 및 이의 제조방법
KR20210031533A (ko) Gip/glp1 공효능제 화합물
JP2010539132A (ja) アミリンの改良された誘導体
AU2014255608A1 (en) Stable, protracted GLP-1/glucagon receptor co-agonists for medical use
CA2939763A1 (fr) Analogues du glucagon stables et leur utilisation dans le traitement de l'hypoglycemie
KR20210071038A (ko) 단백질 타이로신-타이로신 유사체 및 그의 사용 방법
CA3184717A1 (fr) Co-agonistes au niveau des recepteurs de glp-1 et de gip appropries pour une administration par voie orale
JP2022509568A (ja) 修飾されたｇｉｐペプチド類似体
WO2023000240A1 (fr) Analogue de polypeptide glp-1 à action prolongée, son procédé de préparation et son utilisation
KR102230368B1 (ko) 아실화 옥신토모듈린 펩타이드 유사체
JP2011525895A (ja) アミリン及びサケカルシトニンの誘導体化ハイブリッドペプチド
KR20230104118A (ko) Glp-1 전구약물 및 이의 용도
WO2019229242A1 (fr) Dérivés comprenant un analogue d'apeline et leurs utilisations
US20230102339A1 (en) Incretin analogs and uses thereof
US20230346961A1 (en) Glp-1 and gip receptor co-agonists
WO2022129254A1 (fr) Polypeptides et leurs utilisations
AU2021399076B2 (en) Polypeptides and uses thereof
TW202415675A (zh) 腸促胰島素(incretin)類似物及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19726458

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19726458

Country of ref document: EP

Kind code of ref document: A1