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WO2008116913A2 - Composés peptidiques à pégylation biodégradable transitoire - Google Patents

Composés peptidiques à pégylation biodégradable transitoire Download PDF

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Publication number
WO2008116913A2
WO2008116913A2 PCT/EP2008/053658 EP2008053658W WO2008116913A2 WO 2008116913 A2 WO2008116913 A2 WO 2008116913A2 EP 2008053658 W EP2008053658 W EP 2008053658W WO 2008116913 A2 WO2008116913 A2 WO 2008116913A2
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Prior art keywords
compound
peptide
formula
alkyl
aryl
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WO2008116913A3 (fr
Inventor
Florencio Dörwald ZARAGOZA
Bernd Pesckhe
Magali Zundel
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Novo Nordisk AS
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Novo Nordisk AS
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Anticipated expiration legal-status Critical
Priority to US13/720,956 priority Critical patent/US9340544B2/en
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    • 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
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • 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
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)

Definitions

  • the present invention relates to novel peptide compounds with transient biodegradable pegylation and of the general formula V:
  • A represents a peptide-derived radical
  • n is an integer in the range of 1-20
  • -R 1 , -R 2 , -R 3 , and -R 4 independently represent hydrogen, hydroxy, C 1-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 1-6 alkoxy, C 1-6 alkylthio, Cs-scycloalkylthio, C 1-6 alkylamino, C 1-6 dialkylamino, arylamino, diarylamino, aryloxy, aryl, or arylthio; any two of -R 1 , -R 2 , -R 3 , and -R 4 taken together form a 5-7 membered ring; or any one of -R 1 , -R 2 , -R 3 , and -R 4 is covalently bound to Z, forming a 5-7 membered ring; and -Z
  • formula Vl has a solubility in water of at least 0.10 mg/ml at room temperature and a pH in the range of pH 3.0 to pH 1 1.0.
  • the invention also relates to a process for preparing such compounds, the process comprising the step of manufacturing an intermediate compound of the formula (XII); and treating a peptide or protein AH with this intermediate compound to yield a compound of formula V, as shown in the following reaction scheme:
  • the invention furthermore relates to the intermediate compound of the formula XII, wherein -X represents a leaving group selected from chloride, imidazolyl, and 2,5- dioxopyrrolidin-1-yloxy; and -Z- , -R 1 , -R 2 , -R 3 , and -R 4 are as defined above.
  • the invention relates to certain compounds of formula (V) for use as a medicament, and pharmaceutical composition comprising, as an active ingredient, at least one such compound together with one or more pharmaceutically acceptable carriers or excipients.
  • pegylation has been shown to improve their solubility, decrease their immunogenicity, and extend their halflife.
  • the conjugate may be either biologically active, show a decreased biological activity as compared to the parent protein, or be biologically inactive.
  • Pegylation can, however, lead to a significant loss of biological activity if the Peg- chain is attached close to the epitope which is responsible for activity. Furthermore, if the peptide or protein contains more than one derivatisable site (e.g. several lysines), then not only the site of pegylation but also the number of Peg-fragments introduced would be difficult to control.
  • Peg-derivative which is biodegradable, and which does not leave, after biodegradation, any molecular fragment attached to the protein ("traceless", or "transient” pegylation).
  • the invention relates to novel peptide compounds with transient biodegradable pegylation and of the general formula (V), as explained above.
  • the diradical -Z- in formula (V) is defined by its molecular weight and by the solubility in water of a corresponding phenol.
  • the molecular weight may be considered an essential feature of -Z- since the influence of this group on the biological or chemo-physical characteristics of the resulting peptide conjugate should ideally be very low, and the smaller the -Z- group, the more likely is it that its influence is indeed low.
  • solubility in water may be considered an essential feature of -Z-, since in order for the peptide conjugate to be a suitable medicament it is preferably water-soluble, and if the PEG-reagent in itself has a low water solubility, the conjugate is likely to also have a low water solubility. Furthermore, the reactions between the protein and the PEG reagent should preferably be conducted in water.
  • conjugates of formula V are biodegradable, and do only lead to the formation of carbon dioxide, the native protein and a Peg-derivative, but not to an organic small molecule byproduct:
  • conjugates of formula V may be suitable for therapy. This is also the case for conjugates of formula (I), which are described below.
  • Peg-derivatives of formula (Vl) are expected to be non-toxic, and accordingly the conjugates of the invention of formula (V) and (I) may be useful prodrugs of peptides and proteins, and be suitable for therapy.
  • Compounds of formula (V) may be prepared by single or multiple derivatisation of a peptide AH n , which must contain at least one N-terminal amino group, one unacylated lysine, and/or one alternative primary or secondary amino group, with a reagent of general formula (XII). This reagent can be prepared by treatment of a phenol of general formula (Vl) with a carbonic acid derivative (Xl).
  • Compounds of formula (I) may be prepared by single or multiple derivatisation of a peptide AH m , which must contain at least one N-terminal amino group, one unacylated lysine, or one alternative primary or secondary amino group, with a reagent of general formula (IV).
  • This reagent can be prepared by treatment of a phenol or thiophenol of general formula (II) with a carbonic acid derivative (III).
  • This invention will be particularly well suited for the pegylation of large, complex proteins, which cannot be regioselectively pegylated, or which lose biological activity upon pegylation.
  • pegylation according to the invention may extend even further the half- life of long-acting peptides such as GLP-1 analogues with protracted action such as
  • Liraglutide The duration of action of such compounds may even make them potentially suitable for once-monthly administration.
  • peptide is intended to indicate a sequence of two or more amino acids joined by peptide bonds, wherein said amino acids may be natural or unnatural.
  • the term encompasses the terms polypeptides and proteins, which may consist of two or more polypeptides held together by covalent interactions, such as for instance cysteine bridges, or non-covalent interactions. It is to be understood that the term is also intended to include peptides which have been derivatised, for instance by the attachment of lipophilic groups, PEG or prosthetic groups.
  • the peptide is composed of at least five constituent amino acids.
  • the constituent amino acids may be from the group of the amino acids encoded by the genetic code (such as proteinogenic amino acids, see below), and they may be unnatural amino acids (see below), such as natural amino acids which are not encoded by the genetic code, as well as synthetic amino acids.
  • Natural amino acids which are not encoded by the genetic code are e.g., ⁇ -carboxyglutamate, ornithine, phosphoserine, D- alanine and D-glutamine.
  • Synthetic amino acids comprise amino acids manufactured by chemical synthesis, e.g.
  • D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib ( ⁇ -aminoisobutyric acid), Abu ( ⁇ -aminobutyric acid), Tie (tert- butylglycine), ⁇ -alanine, 3-aminomethyl benzoic acid, and anthranilic acid.
  • non-proteogenic amino acid is a moiety which can be incorporated into a peptide via peptide bonds but is not a proteogenic amino acid.
  • examples are v- carboxyglutamate, ornithine, phosphoserine, the D-amino acids such as D-alanine and D- glutamine,
  • Synthetic non-proteogenic amino acids comprise amino acids manufactured by chemical synthesis, i.e.
  • D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib ( ⁇ -aminoisobutyric acid), Abu ( ⁇ -aminobutyric acid), Tie (tert- butylglycine), 3-aminomethyl benzoic acid, anthranilic acid, des-amino-Histidine, the beta analogues of amino acids such as ⁇ -alanine etc., D-histidine, desamino-histidine, 2-amino- histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ - methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylic acid, (1-amino
  • 'proteinogenic amino acid' refers to L-alanine, L-arginine, L-asparagine, L- aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L- leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L- tryptophan, L-tyrosine, or L-valine.
  • unnatural amino acid refers to any compound comprising at least one primary or secondary amino group and at least one carboxyl group, without being L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, glycine, L- histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, or L-valine.
  • analogue as used herein referring to a peptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide.
  • conjugate as a noun is intended to indicate a modified peptide, i.e. a peptide with a moiety bonded to it to modify the properties of said peptide.
  • conjugate is intended to indicate the process of bonding a moiety to a peptide to modify the properties of said peptide.
  • derivative as used herein in relation to a peptide means a chemically modified peptide or an analogue thereof, wherein at least one substituent is not present in the unmodified peptide or an analogue thereof, i.e. a peptide which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters and the like.
  • An example of a derivative of GLP-1 (7-37) is Arg 34 Lys 26 (N ⁇ -( ⁇ -Glu(N ⁇ - hexadecanoyl)))-GLP-1 (7-37).
  • spacer and “linker” are intended to mean an organic diradical with a molecular weight from 14 to 5000.
  • the spacer has a free amino group.
  • the linker has a free amino acid group.
  • radical or "diradical” is intended to indicate a molecular fragment with one or two unpaired electrons, respectively.
  • a fragment may be formally generated by removing one (e.g., a hydrogen) or two atoms or groups of atoms (e.g., a hydroxyl group) by homolytic bond cleavage, i.e. a bond cleavage, in which each of the two resulting fragments contains one of the two electrons which formed the original bond.
  • PEG poly(ethylene glycol)
  • PEO poly(ethylene oxide)
  • POE polyoxyethylene
  • PEG poly(ethylene glycol)
  • mPEG monofunctional methyl ether PEG (methoxypoly(ethylene glycol)), abbreviated mPEG.
  • PEGs are also available with different geometries, such as linear, and branched PEGs.
  • PEG has the structure HO-(CH 2 -CH 2 -O-) n -H, the molecular formula C 2n H 4n+2 O n+I , and the CAS number [25322-68-3].
  • the molar mass of course depends on n.
  • Most PEGs include molecules with a distribution of molecular weights, i.e. they are polydisperse.
  • the size distribution can be characterized statistically by its weight average molecular weight (Mw) and its number average molecular weight (Mn), the ratio of which is called the polydispersity index (Mw/Mn) (see e.g. "Polymer Synthesis and Characterization", J. A. Nairn, University of Utah, 2003). Mw and Mn can be measured by mass spectroscopy.
  • the polydispersity index is accordingly a number which is greater than or equal to one, and it may also be estimated from Gel Permeation Chromatographic data.
  • the polydispersity index is 1
  • the product is monodisperse and is thus made up of compounds with a single molecular weight.
  • the polydispersity index is greater than 1
  • the polymer is polydisperse, and the polydispersity index tells how broad the distribution of polymers with different molecular weights is.
  • the polydispersity index typically increases with the molecular weight of the PEG or mPEG.
  • PEG and “Peg” are used interchangeably and basically mean a radical or diradical comprising the structure wherein n is an integer larger than 1.
  • Peg is intended to indicate poly(ethylene glycol) as well as poly(ethylene glycol) monoalkyl ether, wherein alkyl indicates C 1-6 alkyl, such as methyl, ethyl, propyl, butyl, pentyl and hexyl. Accordingly, in a preferred embodiment, Peg for use according to the invention is represented by the following formula: in which n is an integer larger than 1 , and M and P independently designates alkyloxy, hydroxy, or is absent. As explained above, a compound of this formula in which M designates methyloxy and P is absent is also referred to as mPEG.
  • the molecular weight of the Peg for use according to the invention preferably is between approximately 100 Da and approximately 1000000 Da.
  • the molecular weight of the Peg in kDa may be indicated in parentheses.
  • mPEG(30k) indicates poly(ethylene glycol) monomethyl ether with a molecular weight of approximately 30 kDa. This polymer may, by the way, be composed of approximately 680 ⁇ 100 ethylene glycol units.
  • n is 90 and the molecular weight is 3991 Da, i.e. approx 4 kDa.
  • mPEG(20k) has an average molecular weight of 20 kDa and an average n of 454.
  • the Peg for use according to the present invention may be linear, or branched.
  • the Peg for use according to the invention is a) polydisperse, or b) monodisperse.
  • the polydispersity index of the Peg for use according to the invention is i) below 1.06, ii) below 1.05, iii) below 1.04, iv) below 1.03, or v) between 1.02 and 1.03.
  • prodrug includes biohydrolyzable amides and biohydrolyzable esters, carbonates, carbamates, ureas, thiocarbonates, thiocarbamates, thioureas, guanidines, cyanoguanidines, dithiocarbonates, dithiocarbamates, disulfides, acetals, aminals, thioaminals, thioacetals, oximes, and hydrazones, and also encompasses a) compounds in which the biohydrolyzable functionality in such a prodrug is encompassed in the compound according to the present invention, and b) compounds which may be oxidized or reduced biologically at a given functional group to yield drug substances according to the present invention. Examples of these functional groups include 1 ,4- dihydropyridine, N-acyl-1 ,4-dihydropyridine, 1 ,4-cyclohexadiene, thioethers, and the like
  • biohydrolyzable ester is an ester of a drug substance (in casu, a compound according to the invention) which either a) does not interfere with the biological activity of the parent substance but confers on that substance advantageous properties in vivo such as duration of action, onset of action, and the like, or b) is biologically inactive but is readily converted in vivo to the biologically active principle.
  • the advantages can, for example, be increased solubility or oral availability of the biohydrolyzable ester.
  • lower alkyl esters e.g., C 1 -C 4
  • lower acyloxyalkyl esters lower alkoxyacyloxyalkyl esters
  • alkoxyacyloxy esters alkyl acylamino alkyl esters
  • choline esters e.g., choline esters
  • biohydrolyzable amide is an amide of a drug substance (in casu, a compound according to the present invention) which either a) does not interfere with the biological activity of the parent substance but confers on that substance advantageous properties in vivo such as duration of action, onset of action, and the like, or b) is biologically inactive but is readily converted in vivo to the biologically active principle.
  • the advantages can, for example, be increased solubility or oral availability of the biohydrolyzable amide.
  • Many examples of such are known in the art and include by way of example lower alkyl amides, ⁇ -amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.
  • salts are intended to indicate salts which are not harmful to the patient.
  • Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts.
  • Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like.
  • suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like.
  • compositions include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 66, 2 (1977), which is incorporated herein by reference.
  • metal salts include lithium, sodium, potassium, magnesium salts and the like.
  • ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like.
  • solvate is a complex of defined stoichiometry formed by a solute and a solvent.
  • Solvents may be, by way of example, water, ethanol, or acetic acid.
  • growth hormone is intended to indicate a protein which exhibits growth hormone activity in vivo or in vitro.
  • a protein which exhibits an activity above 20%, such as above 40%, such as above 60%, such as above 80% of that of hGH in a conventional growth hormone functional assay such as the assay of Example 1 1 is defined as a growth hormone compound.
  • prolactin is intended to indicate a protein which exhibits prolactin activity in vivo or in vitro.
  • a protein which exhibits an activity above 20%, such as above 40%, such as above 60%, such as above 80% of that of prolactin in a conventional prolactin functional assay is defined as a prolactin compound.
  • GLP-1 (7-37) refers to the human GLP-1 sequence.
  • GLP-1 peptide as used herein means GLP-1 (7-37), a GLP-1 analogue, a GLP-1 derivative, or a derivative of a GLP-1 analogue. In one embodiment the GLP-1 peptide is an insulinotropic agent.
  • Exendin-4 is a hormone in the saliva of the GiIa monster which displays properties similar to human GLP-1.
  • the term "exendin peptide” as used herein means exendin-4, an exendin-4 analogue, an exendin-4 derivative, or a derivative of an exendin-4 analogue. This definition in particular includes Exenatide, which is a synthetic version of exendin-4 and marketed as Byetta.
  • the CAS number is exendin-4 is 141732-76-5, the ATC code A10BX04, the formula C 18-J H ⁇ N 50 O 60 S, and the molecular mass 4186.6 Da.
  • the exendin peptide is an exendin such as "exendin-4(1-
  • insulinotropic agent means a compound which is an agonist of the human GLP-1 receptor, i.e. a compound which stimulates the formation of cAMP in a suitable medium containing the human GLP-1 receptor.
  • the potency of an insulinotropic agent is determined by calculating the EC 50 value from a dose-response curve.
  • Compounds of formula (I) and (V) wherein AH m and AH n represents growth hormone or a growth hormone analogue, and which exert growth hormone activity, may be used in the treatment of diseases or conditions which will benefit from an increase in the amount of circulating growth hormone.
  • growth hormone peptide as used herein means growth hormone, a growth hormone analogue, a growth hormone derivative, or a derivative of a growth hormone analogue.
  • a preferred example of a growth hormone peptide is the human growth hormone (hGH).
  • GLP-1 , exendine, or a peptide which exerts GLP-1-like activity may be used in the treatment of diseases or conditions which will benefit from an increase in the amount of circulating
  • Compounds of formula (I) and (V) wherein AH m and AH n , respectively, represents a prolactin antagonist may be used in the treatment of diseases or conditions which will benefit from a decrease in the amount of circulating prolactin.
  • blood coagulation factor peptide means a blood coagulation factor, a blood coagulation factor analogue, a blood coagulation factor derivative, or a derivative of a blood coagulation factor analogue.
  • a preferred example of a blood coagulation factor is factor VIII (FVIII), in particular the human factor VIII, which may be designated hFVIII.
  • Compounds of formula (I) and (V) wherein AH m and AH n , respectively, represents gastrin or a peptide which exerts gastrin-like activity may be used in the treatment of diseases or conditions which will benefit from an increase in the amount of circulating gastrin.
  • Compounds of formula (I) and (V) wherein AH m and AH n , respectively, represents a growth hormone-releasing hormone antagonist may be used in the treatment of diseases or conditions which will benefit from a decrease in the amount of circulating growth hormone-releasing hormone, such as cancer.
  • peptides which may be transient pegylated according to the invention are: Human insulin, coagulation factors including factor VII, factor VIII, factor IX, factor X, factor XIII, parathyroid hormone, human follicle stimulating hormone, platelet-derived growth factor (PDGF), transforming growth factor ⁇ (TGF- ⁇ ), transforming growth factor ⁇ (TGF- ⁇ ), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), a somatomedin such as insulin growth factor I (IGF-I), insulin growth factor Il (IFG-II), erythropoietin (EPO), thrombopoietin (TPO) or angiopoietin, interferon, pro-urokinase, urokinase, tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 , plasminogen activator inhibitor 2, von Willebrandt factor, a cytokine, e.g
  • interleukin such as interleukin (IL) 1 , IL-I Ra, IL-2, IL-4, IL- 5, IL-6, IL-9, IL-11 , IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-20 or IL-21
  • IL interleukin
  • CFS colony stimulating factor
  • stem cell factor such as GM-CSF
  • tumor necrosis factor such as TNF- ⁇ , lymphotoxin- ⁇ , lymphotoxin- ⁇ , CD40L, or CD30L
  • protease inhibitor e.g.
  • aprotinin an enzyme such as superoxide dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, catalase, uricase, bilirubin oxidase, trypsin, papain, alkaline phosphatase, ⁇ -glucoronidase, purine nucleoside phosphorylase or batroxobin, an opioid, e.g. endorphins, enkephalins or non-natural opioids, a hormone or neuropeptide, e.g.
  • an opioid e.g. endorphins, enkephalins or non-natural opioids
  • a hormone or neuropeptide e.g.
  • calcitonin glucagon, gastrins, adrenocorticotropic hormone (ACTH), cholecystokinins, lutenizing hormone, gonadotropin-releassing hormone, chorionic gonadotropin, corticotrophin-releasing factor, vasopressin, oxytocin, antidiuretic hormones, thyroid-stimulating hormone, thyrotropin-releasing hormone, relaxin, prolactin, peptide YY, Y2 receptor agonists, Y4 receptor agonists , mixed Y2/Y4 receptor agonists, neuropeptide Y, pancreatic polypeptide, leptin, CART (cocaine and amphetamine regulated transcript), a CART related peptide, perilipin, natriuretic peptides, adrenomedullin, endothelin, secretin, amylin, vasoactive intestinal peptide (VIP), pituary
  • any of the above mentioned peptides can be provided in the form of an analogue wherein one or more of the amino acids have been substituted by another amino acid, or it may be deleted and/or additions of amino acids have occurred.
  • one amino acid has been amended.
  • a maximum of two amino acids has been amended.
  • a maximum of three amino acids has been amended.
  • a maximum of four amino acids has been amended.
  • a maximum of five amino acids has been amended.
  • a maximum of six amino acids has been amended.
  • Analogues of the peptides are intended to maintain their biological effect. The efficiency however may be altered, ie. larger, smaller or unamended.
  • C 1-6 -alkyl in the present context designates a branched or straight hydrocarbon group having from 1 to 6 carbon atoms. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, ferf-butyl, n- pentyl, isopentyl, neopentyl, ferf-pentyl, n-hexyl, isohexyl and the like.
  • C 2 - 6 -alkenyl represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one double bond.
  • groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, iso-propenyl, 1 ,3- butadienyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, 1-pentenyl, 2-pentenyl, 3- pentenyl, 4-pentenyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 2,4-hexadienyl, 5- hexenyl and the like.
  • C 1-6 -alkoxy refers to the radical -O-C 1-6 -alkyl where C 1-6 -alkyl is as defined above. Representative examples are methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, sec-butoxy, ferf-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.
  • C 1-6 -alkylthio refers to the radical
  • C 1-6 -alkyl where C 1-6 -alkyl is as defined above.
  • Representative examples are methylthio, ethylthio, n-propylthio, isopropylthio, butylthio, sec-butylthio, tert-butylthio, pentylthio, isopentylthio, n-hexylthio, 2-hexylthio and the like.
  • C 1-6 -alkanoyl denotes a group -C(O)H or -C(O)-C 1-5 - alkyl(ene). Representative examples are formyl, acetyl, propionyl, butyryl, valeryl, hexanoyl and the like.
  • '"C ⁇ -alkenoyl denotes a group -C(O)-C 2-5 -alkenyl.
  • Cs ⁇ -cycloalkyl represents a carbocyclic group having from 3 to 8 carbon atoms. Representative examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
  • C 3-8 -CyClOaIkOXy or cycloalkylthio as used herein represents a carbocyclic group having from 3 to 8 carbon atoms as defined above attached to -O- , or -S-, respectively.
  • C x-y -alkylene represent a divalent C- x-y -alkyl ie. -CH 2 -, -CH 2 CH 2 -,- CH 2 CH 2 CH 2 -, - CH 2 CH 2 CH 2 CH 2 -, -CH(CH3)CH 2 - etc.
  • the alkylene is a C 1-6 -alkylene.
  • the alkylene is a C 2 - 10 -alkylene.
  • aryl as used herein is intended to include carbocyclic aromatic ring systems such as phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, indenyl, pentalenyl, azulenyl, and the like.
  • Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 1 ,2,3,4-tetrahydronaphthyl, 1 ,4-dihydronaphthyl and the like.
  • aryloxy as used herein denotes a group -O-aryl, wherein aryl is as defined above.
  • Conjugates of formula (I) are biodegradable, and do only lead to the formation of carbon dioxide, the native protein and a Peg-derivative, but not to an organic small molecule byproduct:
  • conjugates of formula (I) may be suitable for therapy.
  • an aprotic solvent such as MeCN, DCM, CCI 4 , CHCI 3 , toluene, or the like
  • Y represents a leaving group for nucleophilic displacement, such as for instance halide, 1-imidazolyl, O-(N-hydroxysuccinimidyl), or 0-(N- hydroxy)benzotriazolyl
  • the stability of the corresponding derivatives of formula I may depend on the type of N-terminal amino acid.
  • the N-terminal amino acid contains a nucleophilic functional group, as for instance in histidine, serine, threonine or cysteine, then the stability of the corresponding compound of formula I may be lower than that of a similar compound devoid of such an amino acid.
  • the nucleophilic side chain may promote a cleavage of the metabolically labile bond to generate a cyclic derivative, as sketched below:
  • peptides or proteins devoid of acylable lysine side chains and containing an N-terminal amino acid with a nucleophilic side chain may lead to compounds of formula I with shorter halflives than peptides or proteins which do not contain such an N-terminal amino acid, and may yield, upon biodegradation, not the original peptide or protein but instead a cyclic, carbonylated analogue of this peptide or protein.
  • GLP-1 (7-37) analogues contain an N-terminal histidine.
  • One possibility to avoid the above-mentioned cyclization reaction is to extend the GLP-1 analogue with a dipeptide which will be readily cleaved from the GLP-1 analogue as soon as the reversible linker to Peg has been cleaved.
  • peptides ending with H- Gly-Pro-... are effectively cleaved by the protease DPPIV, which is present in humans, to yield a peptide devoid of the last two amino acids (Gly-Pro).
  • the present invention can be applied to GLP-1 analogues extended at the N-terminus with two additional amino acids, for instance with Gly-Pro.
  • A' represents a peptide-derived radical, produced by n-fold removal of one hydrogen atom from the N-terminal amino group or from the amino group of a lysine-side chain of said peptide, and n is any number in the range of 1-20 X represents O or S,
  • Z represents a bond, O, NMe, S, CH 2 , CH(OH), CH 2 -CH(NHAcyl), or CH(OE), wherein E is C- ⁇ - 6 -alkyl, C 1-6 -alkanoyl, C 2-6 -alkenoyl
  • R 2 represents O, N(R 8 ) wherein R 8 is selected from H, C 1-6 -alkyl, C 3-8 -cycloalkyl,
  • R 4 represents linear or branched mPeg(1 k-60k) (mPeg er methoxyPeg), optionally linked to
  • the invention provides compounds according to, e.g., particular embodiment no. 50.
  • the invention provides compounds according to particular embodiment no. 50 wherein Z represents O, CH 2 , CH(OH), CH 2 -CH(NH(acyl), or CH(OE), wherein E is C 1-6 - alkyl or C 1-6 -alkanoyl, C 2-6 -alkenoyl.
  • the invention provides compounds according to, e.g., particular embodiment no. 50 and/or any of the above embodiments wherein R 2 represents NH, N-Me, or O.
  • the invention provides compounds according to, e.g., particular embodiment no. 50 and/or any of the above embodiments wherein n is any number selected from the range of 1- 10.
  • the invention provides compounds according to, e.g., particular embodiment no. 50 and/or any of the above embodiments wherein X represents O.
  • the invention provides compounds according to, e.g., particular embodiment no. 50 and/or any of the above embodiments wherein R 5 , R 6 , and R 7 independently represent hydrogen, C 1-6 -alkyl, C 2 C 1-6 -alkoxy, C 1-6 -alkylthio.
  • the invention provides compounds according to, e.g., particular embodiment no. 50 and/or any of the above embodiments wherein R 2 represents NH or N-Me.
  • the invention provides compounds according to, e.g., particular embodiment no. 50 and/or any of the above embodiments wherein m is 1-6.
  • the invention provides an intermediate of general formula (IV)
  • Y represents a leaving group for nucleophilic displacement, such as halide, 1-imidazolyl, O-(N- hydroxysuccinimidyl), O-(N-hydroxybenzotriazolyl), 1-pyrazolyl, 1-(1 ,2,4-triazolyl), O-([1 ,2,3]- triazolo[4,5-b]pyridine-3-oxy), O-(4-oxo-4H-benzo[d][1 ,2,3]-triazin-3-oxy), 1-tetrazolyl, 2- nitrophenoxy, 4-nitrophenoxy, 2,4-dinitrophenoxy, 2,4,6-trichlorophenoxy, pentafluorophenoxy, 2-pyridyloxy, azido, O-(N-hydroxypiperidinyl), or 0-(N- hydroxypyrrolidinyl), and X, Z, and R 1"7 are as defined herein, e.
  • the invention also provides the use of a compound of formula (I) as a medicament.
  • the invention provides a biological effect of the compounds according to the invention which is determined by the peptide attached.
  • the peptide attached is a GLP-1 or an analogue of GLP-1
  • the effects of the compound according to the invention wewill be determined by GLP-1 and the assays used for measuring the effects will be known from that field. Measurements could be release of unpegylated protein, or a biological effect by the release protein.
  • the invention provides compounds with a protracted activity profile. This means that the peptide which has been conjugated according to the invention is active for a longer period of time as compared to the peptide itself (the isolated peptide), and/or as compared to an analogue or derivative of the peptide itself, such as a prior art analogue or derivative thereof.
  • a preferred GLP-1 peptide conjugate of the invention is active for a longer period of time as compared to (i) GLP-1 (7-37), (ii) a corresponding GLP-1 analogue, (iii) a corresponding GLP-1 derivative, and/or (iv) a corresponding derivative of a GLP-1 analogue, wherein the term "corresponding” refers to the peptide part ("AH") of the GLP-1 peptide conjugate of the invention, with or without a potential N-terminal extension such as Gly-Pro.
  • the two compounds are to be tested under comparable, preferably identical, conditions, e.g., in the same dosage.
  • comparative compounds can be derived from any one of the examples herein (by removing the formula XII part of the molecules).
  • preferred comparative compounds are selected from amongst the AH-part of the compound of Example 5, and the AH-part of the compounds of formulas (a)-(av), with or without Gly-Pro at the N-terminus.
  • the GLP-1 peptide conjugate of the invention is active for a period of time which is at least 120%, 140%, 160%, or at least 180% of the time period in which the comparative compound is active. In additional particular embodiments, it is active for at least twice as long a time period, or for a period of at least 3x, 4x, 5x, 6x, 7x, 8x, 9x, or at least 10x the time period in which the comparative compound is active. In still further particular embodiments, it is active for a time period of at least 11 x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or at least 2Ox the period of time in which the comparative compound is active.
  • GLP-1 activity assays are included in the experimental part.
  • a pharmacodynamic study using db/db mice in which blood glucose level is monitored as a function of time is preferred, for example as described in Example 9.
  • the biological half-life of a substance is the time required for half of that substance to be removed from an organism by either a physical or a chemical process.
  • Biological half- life is an important pharmacokinetic parameter which may be denoted by the abbreviation
  • the compounds of the invention provide peptides which exhibit half-lives of for example 50-300 h.
  • the half-life is at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, and most preferably at least 60 hours.
  • the half-life is at least 72 hours, preferably at least 84 hours, more preferably at least 96 hours, even more preferably at least 108 hours, and most preferably at least 120 hours.
  • the half-life is at least 140 hours, preferably at least 160 hours, more preferably at least 180 hours, even more preferably at least 200 hours, and most preferably at least 220 hours.
  • the half-life is at least 240 hours, preferably at least 260 hours, more preferably at least 280 hours, even more preferably at least 300 hours, and most preferably at least 320 hours.
  • the above half-lives are for human beings, but the half-lives may also be determined for mice, in which case the above ranges may be divided by 3 (i.e., 17-100 hours; at least 4, 8, 12, 15 or 20 hours; at least 24, 28, 32, 36, or 40 hours; at least 47, 53, 60, 67, or 73 hours; or at least 80, 87, 93, 100, or 107 hours).
  • the half-life may be determined using any suitable assay, e.g. any of the assays of the experimental part herein.
  • the assays of Examples 9 and/or 10 are preferred.
  • the pegylated compound is essentially inactive so that a high dosis can be administered.
  • the pegylated compound has some biological activity. The biological activity of the pegylated compound is to be determined, and the dose administered is then to be adjusted.
  • the compound administered has a biological activity which extends over days, weeks or even months.
  • the site for attachment of the linker is to be decided.
  • the unpegylated peptide needs to have a biological activity.
  • the site for pegylation and potentially the substitution of an amino acid at this particular site may be determined so that the biological activity of the modified peptide is very low.
  • the following are particular compounds of formula (V) and (I) of the invention:
  • 'exendin' represents an exendin-derived radical or polyradical, formally generated by removal of 1-3 acylable amino groups from exendin
  • hGH represents a human growth hormone-derived radical or polyradical, formally generated by removal of 1-4 acylable amino groups from human growth hormone
  • FVIII represents a human FVIII-derived radical or polyradical, formally generated by removal of 1-6 acylable amino groups from human FVIII.
  • A represents a peptide-derived radical, produced by n-fold removal of one hydrogen atom from the N-terminal amino group and/or from the amino group of one or more lysine residues of said peptide;
  • n is an integer in the range of 1-20;
  • -R 1 , -R 2 , -R 3 , and -R 4 independently represent hydrogen, hydroxy, C 1-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 1-6 alkoxy, C 1-6 alkylthio, Cs-scycloalkylthio, C 1-6 alkylamino, C 1-6 dialkylamino, arylamino, diarylamino, aryloxy, aryl, or arylthio; any two of -R 1 , -R 2 , -R 3 , and -R 4 taken together form a 5-7 membered ring; or any one of -R
  • -Z- is a valence-bond or a diradical with a molecular weight below or equal to 750 Da and chosen in a way, that a compound of the formula Vl:
  • solubility in water of at least 0.10 mg/ml at room temperature and a pH in the range of pH 3.0 to pH 11.0.
  • the compound of embodiment 1 wherein the compound of the formula Vl, which is used to define -Z-, has a solubility in water of at least 0.50, 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, or at least 45.0 mg/ml; preferably at least 10.0, 20.0, 30.0, or at least 40.0 mg/ml; more preferably at least 45.0 mg/ml; and most preferably at least 48.0 mg/ml.
  • -R 5 - represents a valence bond, -O-, -S-, or -NR 8 -, in which -R 8 represents hydrogen, C 1-6 alkyl, C 1-6 alkenyl, or aryl; and -R 6 - and -R 7 - independently represent a valence bond;
  • C 1-6 alkylene optionally independently substituted with C 1-6 alkyl, aryl, hydroxyl, C 1-6 alkoxy, C 1-6 alkylcarbonylamino, amino, carboxy, or C 1-6 alkoxycarbonyl;
  • C 2-6 alkenylene optionally independently substituted with C 1-6 alkyl, aryl, hydroxyl, C 1-6 alkoxy, C-i-ealkylcarbonylamino, amino, carboxy, or C 1-6 alkoxycarbonyl;
  • arylene optionally independently substituted with C 1-6 alkyl, aryl, hydroxyl, C 1-6 alkoxy, C-i-ealkylcarbonylamino, amino, carboxy, halogen, or C 1-6 alkoxycarbonyl; or
  • m is an integer in the range of 1-24, preferably in the range of 2-22, 3-20, 4-15, or 5-10.
  • n is an integer in the range of 1-15, more preferably in the range of 1-10, most preferably in the range of 1-6, such as 1 , 2, 3, 4, 5, or 6.
  • AH n represents a GLP-1 (7-37) peptide, an exendin-4(1-39) peptide, hGH, or FVIII.
  • n is an integer in the range of 1-5, preferably 1-4, more preferably 1-3, even more preferably 1-2; or, most preferably, n is 2.
  • -X represents a leaving group selected from chloride, imidazolyl, and 2,5-dioxopyrrolidin-1- yloxy
  • -R 1 , -R 2 , -R 3 , and -R 4 independently represent hydrogen, hydroxy, C 1-6 alkyl, C 2-6 alkenyl, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 1-6 alkoxy, C 1-6 alkylthio, Cs-scycloalkylthio, C 1-6 alkylamino, C 1-6 dialkylamino, arylamino, diarylamino, aryloxy, aryl, or arylthio; any two of -R 1 , -R 2 , -R 3 , and -R 4 taken together form a 5-7 membered ring; or any one of -R 1 , -R 2 , -R 3 , and -R 4 is covalently bound to Z, forming
  • -Z- is a valence-bond or a diradical with a molecular weight below or equal to 750 Da and chosen in a way, that a compound of the formula Vl: has a solubility in water of at least 0.10 mg/ml at room temperature and a pH in the range of pH 3.0 to pH 11.0.
  • -R 5 - represents a valence bond, -O-, -S-, or -NR 8 -, in which -R 8 represents hydrogen, C 1-6 alkyl, C 1-6 alkenyl, or aryl; -R 6 - and -R 7 - independently represent a valence bond;
  • C 1-6 alkylene optionally independently substituted with C 1-6 alkyl, aryl, hydroxyl, C 1-6 alkoxy, C 1-6 alkylcarbonylamino, amino, carboxy, or C 1-6 alkoxycarbonyl;
  • m is an integer in the range of 1-24, preferably in the range of 2-22, 3-20, 4-15, or 5-10.
  • -R 5 - represents a valence bond or -O-
  • -R 6 - and -R 7 - independently represent a valence bond or C 1-6 alkylene, optionally independently substituted with C 1-6 alkyl, hydroxyl, C 1-6 alkylcarbonylamino, or C 1-6 alkoxycarbonyl
  • X is 2,5-dioxopyrrolidin-1-yloxy.
  • step (i) the compound of formula (Vl) is dissolved in an aprotic solvent, such as MeCN, DCM, CCI 4 , CHCI 3 , or toluene.
  • an aprotic solvent such as MeCN, DCM, CCI 4 , CHCI 3 , or toluene.
  • a pharmaceutical composition comprising, as an active ingredient, at least one compound according to any one of embodiments 24-36 together with one or more pharmaceutically acceptable carriers or excipients.
  • A' represents a peptide-derived radical, produced by n-fold removal of one hydrogen atom from the N-terminal amino group or from the amino group of a lysine-side chain of said peptide, and n is any number in the range of 1-20 X represents O or S,
  • R 4 represents linear or branched mPeg(1 k-60k) , optionally linked to R 3 by a spacer which is selected from -C 2-10 -alkylene, -C 2-10 -alkylene -O-, -0-C 2- io-alkylene-0-, -C 2- io-alkylene -
  • R 5 , R 6 , and R 7 independently represent hydrogen, hydroxy, C 1-6 -alkyl, C 2-6 -alkenyl, C 3-8 - cycloalkyl, C 3-8 -CyClOaIkOXy, C 1-6 -alkoxy, C 1-6 -alkylthio, Cs-s-cycloalkylthio, C 1-6 -alkylamino,
  • HOBt N-hydroxybenzotriazole, 1-hydroxybenzotriazole
  • SuO-CO-OSu Disuccinimidyl carbonate r.t. room temperature
  • HPLC-systems from Merck-Hitachi HibarTM RT 250-4, LichrosorbTM RP 18, 5.0 ⁇ m, 4.0 x 250 mm, gradient elution, 20% to 80% acetonitrile in water within 30 min, 1.0 ml/min, detection at 254 nm
  • Waters SymmetryTM, C18, 3.5 ⁇ m, 3.0 x 150 mm, gradient elution, 5% to 90% acetonitrile in water within 15 min, 1.0 ml/min, detection at 214 nm
  • LCMS HPLC-Mass spectroscopy was performed on an Agilent LC/MSD TOF instrument, using a XTerra MA C18 5 ⁇ m 3.Ox 50 mm column.
  • the HPLC was performed with gradient of 5-90% a buffer of 0.1 % trifluoroacetic acid in acetonitril in a 0.1 % solution of trifluoroacetic acid in water over a period of 7.5 min at a flow of 1.5 ml/min.
  • a Zorbax 5 mm 300 SB-C3 may be used, using the same gradient as described above at a flow of 1 ml/min.
  • V A phenol of formula Vl, prepared from commercially available compounds using standard methods well known to those skilled in the art, is dissolved in an aprotic solvent, such as MeCN, DCM, CCI 4 , CHCI 3 , toluene, or the like, and treated with a carbonic acid derivative Xl, wherein X represents a leaving group for nucleophilic displacement, such as for instance halide, 1-imidazolyl, O-(N-hydroxysuccinimidyl), or O-(N-hydroxy)benzotriazolyl, to yield the intermediate XII.
  • an aprotic solvent such as MeCN, DCM, CCI 4 , CHCI 3 , toluene, or the like
  • Xl represents a leaving group for nucleophilic displacement, such as for instance halide, 1-imidazolyl, O-(N-hydroxysuccinimidyl), or O-(N-hydroxy)benzotriazolyl
  • an aprotic solvent such as MeCN, DCM, CCI 4 , CHCI 3 , toluene, or the like
  • Y represents a leaving group for nucleophilic displacement, such as for instance halide, 1-imidazolyl, O-(N-hydroxysuccinimidyl), or 0-(N- hydroxybenzotriazolyl
  • Homovanillic acid 55 mg, 0.3 mmol was mixed with DCM (7.5 ml), HOBt hydrate
  • Homovanillic acid (254 mg, 1.4 mmol) was mixed with HOBt hydrate (324 mg, 2.1 mmol), DCM (20 ml), and EDAC (345 mg, 1.8 mmol), and after 5 min were added mPeg(30k)-NH 2 (4.0 g, 140 ⁇ mol) and DIPEA (1.0 ml). After 23 h aminomethylpolystyrene (4.6 g; 0.8 mmol/g) was added, and the mixture was stirred for 24 h. The mixture was filtered, the resin rinsed well with DCM, and to the combined filtrates was added an ion-exchange resin (Amberlyst 15, 7.24 g, prewashed with DCM + MeOH).
  • Boc-Gly-Pro-OH (85 mg, 312 ⁇ mol) was dissolved in DMF (1 ml), and HOBt hydrate (85 mg, 56 ⁇ mol) and N,N'-diisopropylcarbodiimide (40 ⁇ l) were added. The mixture was stirred at room temperature for 0.5 h. Portions (200 ⁇ l) of the resulting solution were added to a mixture of N ⁇ 26( ⁇ -(hexadecanoyl)glutamyl)[Arg34]GLP-1 (7-37) (225 mg), water (1.25 ml), DMF (1.25 ml), and DIPEA (100 ⁇ l). After each addition the mixture was analyzed by LCMS.
  • the Boc group was removed by treatment of the purified peptide (approx 100 mg) with a mixture of TFA, triisopropylsilane, and water (95/2.5/2.5; 10 ml) at room temperature for 0.5 h. The mixture was concentrated under reduced pressure and coevaporated once with a mixture of MeCN and water. Drying under reduced pressure yielded 100 mg of the title compound.
  • Example 6 3-(4-Hydroxy-3,5-dimethylphenyl)propanoylamino 2 kDa mPEG Step 1 : (£)-3-(4-Hydroxy-3,5-dimethylphenyl)acrylic acid
  • Example 7 Transient pegylation of hGH with N-(mPeg(30k)yl) 4-(0-(N- hydroxysuccinimidyl)carbonyloxy)-3-methoxyphenylacetamide
  • Step 1 Transient pegylation
  • the recovered material was then buffer shifted to 2OmM HEPES buffer pH7.5 before testing the stability of the product.
  • Step 3 Stability testing The stability was tested by HPLC, using a Phenomenex Jupiter C4 5 ⁇ 300A
  • the eluents were: A: H 2 CVTFA 0.1 %, and B: CH 3 CN/TFA 0.07%.
  • the gradient started at 25% B and ended at 100%B after 19min.
  • the flow was 1 ml/min. Products were detected at 28On m.
  • hGH peak was eluting at 13.9min. Peaks at retention times 12.4min and 13.0min were assigned to dipegylated hGH and monopegylated hGH respectively.
  • Example 8 Transient pegylation of FVIII with N-(mPeg(30k)yl) 4-(O-(N- hydroxysuccinimidyl)carbonyloxy)-3-methoxyphenylacetamide
  • Buffer A 5OmM Hepes buffer pH7.5, 1OmM CaCI2, 0.02% Tween 80, 0.5M NaCI
  • Buffer B 5OmM Hepes buffer pH7.5, 1OmM CaCI2, 0.02% Tween 80, 0.15M NaCI,
  • Buffer D buffer C + 1 M NaCI
  • the reagent in solution in buffer A (3 ⁇ l, 408 ⁇ g, 13.6nmoles) was added to a FVIII solution (24 ⁇ g, 14.0 ⁇ l, 136pmoles) in buffer A.
  • the reaction mixture was incubated for 30min at 25°C.
  • a second portion of the reagent was added (3 ⁇ l,408 ⁇ g).
  • the reaction mixture was further incubated for 30min.
  • reaction mixture was buffer shifted to buffer C on a PRO-SPIN spin column CS- 800 (Princeton Separations, USA).
  • An ion exchange step was then performed on a Vivapure Q Mini M device
  • the reaction products were eluted with the elution buffer D.
  • the protein recovery was 20%.
  • the recovered product was analyzed by SDS gel and showed the appearance of bands of higher molecular weight as expected for a pegylated product.
  • FVIII is a large protein of more than 2000 amino acid residues and with several interconnected peptide-chains. It includes several pegylation sites, and it is estimated that pegylation occurs at at least 5, maybe up to 10, different sites.
  • the gel was a NuPage 3-8% Tris-acetate gel (Cat no EA 03755, Invitrogen, USA).
  • the electrophoresis was run on a Power Ease 500 (Invitrogen, USA).
  • the electrophoresis was run for 70min with a voltage of 150V.
  • the Silver Quest silver staining kit (Cat no LC6070, Invitrogen, USA) was used to stain the gel.
  • the protein mixture used for molecular weight references was the Hi Mark HMW Standard (Cat no LC 5688, Invitrogen, USA).
  • Example 9 Pharmacodynamic study using db/db mice The following compound according to the invention:
  • E F I AWLVR G R G- ⁇ OH was administered s.c. to diabetic mice in a dosage corresponding to 300 nmol/kg.
  • mice Male db/db mice were from Taconic, Denmark and shipped at the age of 8-10 weeks. From the time of arrival, the mice were housed under standard conditions but at 24°C. The mice were kept 10 per cage until experimentation with free access to standard chow (Altromin 1324, Brogaarden APS., Denmark) and tap water at a normal day: light cycle (light on from 6 am to 6 pm). The mice are used for 1 experiment per week for 3 weeks. After this, the mice were euthanized. After an acclimatisation period of 1 week, the blood glucose was measured by sampling from the tail tip capillary.
  • mice 5 ⁇ l blood was sampled in heparinised glass capillary tubes and immediately suspended in 250 ⁇ l EBIO buffer solution (Eppendorf, Germany) in an 1.5 ml Eppendorf tube. The blood glucose concentration was measured by the glucose oxidase method at an EBIO Plus Auto analyser (Eppendorf, Germany).
  • EBIO Plus Auto analyser Eppendorf, Germany.
  • the blood glucose values were followed up to 96 hours (time 1 , 3, 6, 24, 48, 72, and 96 h).
  • GLP-1 peptides are determined as described in the experimental section of J. of Med. Chem., 2000, vol. 43, no. 9, p. 1664-1669 (Knudsen et al), see in particular the section headed Pharmacokinetic Experiments at p. 1668-9.
  • blood samples from human beings or other animals such as mice and rat may be used in the alternative.
  • the first part of the pharmacokinetic screening is a single dose subcutaneous administration of 2 nmol/kg of a GLP-1 peptide in question to three minipigs weighing 8-12 kg. Blood samples are drawn from each animal at predose, 0.5, 1 , 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 hours post-injection. All blood samples are stabilised with a special stabilisation buffer consisting of: EDTA (di-sodium) 0.18 M, Aprotinin 15000 KIE/ml, Val-Pyr 0.30 mM, pH adjusted to 7.4 in order to prevent enzymatic degradation of the GLP-1 peptide.
  • EDTA di-sodium
  • Aprotinin 15000 KIE/ml Aprotinin 15000 KIE/ml
  • Val-Pyr 0.30 mM pH adjusted to 7.4 in order to prevent enzymatic degradation of the GLP-1 peptide.
  • Plasma is collected from each stabilised blood sample by centrifugation (4 0 C, 10 min., 1270 G (4000 rpm), and analysed for the content of GLP-1 peptide by ELISA assays, as described below.
  • Two different ELISA assays may be used for the plasma analysis: "The "Total assay” using the antibody combination F1/Ra2135 detecting both N-terminally intact 7-37GLP-1 peptides and N-terminal enzymatically degraded 9-37GLP-1 peptides with a limit of detection (LOD) of 35 pM and a dynamic analytical range of 35-30000 pM; and the "Aib-intact assay” using the antibody combination F1/GLP162-3F15, detecting an Aib stabilised N-terminal of the GLP-1 peptide, if present, with a LOD of 45 pM and a dynamic analytical range of 45-30000 pM.
  • LOD limit of detection
  • All plasma concentration-time profiles may be analysed pharmacokinetically and parameters such as the following calculated if data so permit: t max , C max , AUC, AUC/Dose, AUCo /oExtrap0
  • a second part of the pharmacokinetic screening may be conducted on those compounds with an initial terminal half-life of 60-70 hours or more.
  • This screening consists of a single dose intravenous and subcutaneous administration of 2 nmol/kg to six minipigs for each route of administration.
  • the blood sampling schedule is extended from 0-120 hours to 0-432 and 0-504 hours after intravenous and subcutaneous administration respectively. This is in order to increase the precision and accuracy of the pharmacokinetic parameter estimates, especially the terminal half-life, AUC and the derived parameters clearance and volume of distribution, and to estimate the bioavailability after subcutaneous administration.
  • the assay is a two-site assay with simultaneous incubation of the analyte with catcher and detector antibody. A ready to use chemiluminescent substrate is used to maximize signal.
  • the assay neither recognizes endogen GLP-1 (7-37) nor DPPIV cleaved GLP-1 (9- 37).
  • Reference plasma Is prepared from pooled EDTA plasma without Valine Pyrrolidide and Aprotinin from fasting animals.
  • the pooled EDTA plasma is incubated at 37°C for 4 hours to remove traces of GLP-1 and after incubation Valine Pyrrolidide and Aprotinin are added. Buffers:
  • Coating buffer PBS is used as coating buffer, 1OmM sodium phosphate and 145mM sodium chloride adjusted to pH 7.4.
  • Washing buffer PBS with 0.05% (v/v) Tween 20
  • Assay buffer PBS with 0.05% (v/v) Tween 20, 10g/L BSA and 10mg/L anti-TNP
  • Streptavidin buffer Washing buffer with an additional 0.5M NaCI.
  • Substrate Ready-to-use substrate SuperSignal ELISA Femto (Pierce, cat.no. 37075).
  • Standards are prepared from a 25 ⁇ M stock solution of a desired standard compound.
  • the peptide is serially diluted into reference plasma to make standards with final concentrations of 30000-10000-3333-11 11-370-123-41 and 0 pM.
  • Standards are stored in Micronic tubes in 100 ⁇ L aliquots at -20 0 C. Instead of standards, the activity may be determined relative to a comparative compound and the activity expressed relatively to the comparative compound. Assay procedure:
  • Crystal 2000 Microplates black are coated with monoclonal antibody GLPbI -7F1 (available from Novo Nordisk A/S, Novo AIIe, DK-2880 Bagsvaerd, Denmark), 100 ⁇ L of 5 ⁇ g/mL in PBS overnight at 4°C.
  • GLPbI -7F1 available from Novo Nordisk A/S, Novo AIIe, DK-2880 Bagsvaerd, Denmark
  • Plates are washed 5 times with washing buffer in an automated plate washer (SkanWasher, Skatron) and allowed to stand for at least 30min. with washing buffer to block remaining sites. 20 ⁇ L of sample or standard is added to each well in duplicate immediately followed by 100 ⁇ L GLP162-3F15 biotinylated (available from Novo Nordisk A/S, Novo AIIe, DK-2880 Bagsvaerd, Denmark), 1 ⁇ g/mL in assay buffer. Plates are incubated for 2 hours at room temperature on a plate shaker followed by 5 wash cycles as previously described.
  • streptavidin-peroxidase solution 100 ⁇ L of streptavidin-peroxidase solution (KPL, code 14-30-00, 1 :20000 in streptavidin buffer) is added to each well and incubated for 1 hour at room temperature on a plate shaker. Plates are washed as previously described and after emptying 100 ⁇ l_ of SuperSignal femto is added. Plates are put on a shaker for 1 minute and measured in Orion Luminometer (Berthold). Data may be transferred to MultiCalc and standard curves calculated using the weighted 4PL method. Sample concentrations are calculated from the standard curve.
  • the assay is a two-site assay with simultaneous incubation of the analyte with catcher and detector antibody.
  • the assay recognizes N-terminally cleaved GLP-1 up to GLP- 1 (12-37). Buffers:
  • Coating buffer PBS, 1OmM sodium phosphate and 145mM sodium chloride adjusted to pH 7.4.
  • Washing buffer PBS with 0.05% (v/v) Tween 20
  • Assay buffer PBS with 0.05% (v/v) Tween 20, 10g/L BSA and 10mg/L anti-TNP.
  • Streptavidin buffer Washing buffer with an additional 0.5M NaCI.
  • Standards are prepared from a 25 ⁇ M stock solution of a desired standard.
  • the peptide is serially diluted into reference plasma to make standards with final concentrations of 30000-10000-3333-11 11-370-123-41 and 0 pM.
  • Standards are stored in Micronic tubes in 100 ⁇ L aliquots at -20 0 C. Instead of standards, the activity may be determined relative to a comparative compound and the activity expressed relatively to the comparative compound. Assay procedure:
  • 100 ⁇ l_ of streptavidin-peroxidase solution (Amersham Biosciences, code RPN4401V, 1 :8000 in assay buffer) is added to each well and incubated for 1 hour at room temperature on a plate shaker. Plates are washed as previously described and after emptying 100 ⁇ l_ of TMB is added and after 5 minutes the reaction is stopped with 100 ⁇ l_ H 3 PO 4 .
  • Plates are measured in Victor Multilabel Reader (Wallac). Data may be transferred to MultiCalc and standard curves calculated using the weighted 4PL method. Sample concentrations are calculated from the standard curve.
  • Growth hormone (GH) activity may be determined as the activity of a compound on the growth hormone receptor (GHR), as described in the following:
  • BAF-3 cells (a murine pro-B lymphoid cell line derived from the bone marrow) are originally IL-3 dependent for growth and survival.
  • IL-3 activates JAK-2 and STAT which are the same mediators GH is activating upon stimulation.
  • STAT the same mediators GH is activating upon stimulation.
  • the cell line After transfection of the human growth hormone receptor the cell line is turned into a growth hormone-dependent cell line. This clone can be used to evaluate the effect of different growth hormone samples on the survival of the BAF-3GHR.
  • the BAF-3GHR cells are grown in starvation medium (culture medium without growth hormone) for 24 h at 37 °C in an atmosphere containing 5% CO 2 .
  • the cells are washed and resuspended in starvation medium and seeded in plates.
  • test compound such as a hGH-analogue or hGH in different concentrations or control are added to the cells.
  • the plates are incubated for 68 h at 37 C in an atmosphere containing 5% CO 2 .
  • AlamarBlue® is added to each well and the cells are then incubated for further 4 h.
  • the AlamarBlue® is a redox indicator, and is reduced by reactions innate to cellular metabolism and, therefore, provides an indirect measure of viable cell number.
  • the metabolic activity of the cells is measured in a fluorescence plate reader.
  • the absorbance in the samples is expressed in percent of cells not stimulated with the compound in question, e.g. a hGH-analogue to be tested, or e.g. hGH as control. From the concentration-response curves the activity (amount of a compound that stimulates the cells with 50%) can be calculated.
  • Example 12 Assay for determining in vitro stability
  • the plasma stability of the compounds of formula (I) and (V) of the present invention can be assessed either by measuring directly the regeneration of peptide AH m in plasma (in vitro or in vivo) or in a plasma-like buffer, for instance with the aid of an ELISA assay, or by measuring the biological activity of said peptide AH m in vivo or in vitro as a function of time, as described in the literature (see, e.g., Greenwald et al., Bioconjugate Chem. 2003, 14, 395- 403; Greenwald et al., J. Med. Chem. 2003, 46, 5021-5030; Peleg-Shulman et al., J. Med. Chem. 2004, 47, 4897-4904; Zhao et al., Bioconjugate Chem. 2006, 17, 341-351 ; US 2005/0197290; WO 2005/053749; WO 2005/099768).

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Abstract

La présente invention concerne de nouveaux peptides à pégylation biodégradable transitoire et de formule générale V, ainsi qu'un procédé pour leur préparation, et des intermédiaires utiles pour leur préparation. Les conjugués de GLP-1, l'exendine, l'hormone de croissance, et le facteur VIII de coagulation sanguine constituent des exemples de ces composés. L'invention concerne également l'utilisation de ces peptides en tant que médicaments, ainsi que les compositions pharmaceutiques correspondantes. Ces nouveaux composés donnent des médicaments à durée d'action prolongée.
PCT/EP2008/053658 2007-03-28 2008-03-27 Composés peptidiques à pégylation biodégradable transitoire Ceased WO2008116913A2 (fr)

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US13/720,956 US9340544B2 (en) 2007-03-28 2012-12-19 Purinyl derivatives and their use as potassium channel modulators

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008148839A3 (fr) * 2007-06-08 2009-08-13 Ascendis Pharma As Conjugués polymères transitoires longue durée de l'exendine
US9133276B2 (en) 2010-09-17 2015-09-15 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an exendin linker conjugate
US9138462B2 (en) 2009-07-31 2015-09-22 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an insulin linker conjugate
US9265723B2 (en) 2009-07-31 2016-02-23 Sanofi-Aventis Deutschland Gmbh Long acting insulin composition
US10227444B2 (en) 2016-06-01 2019-03-12 International Business Machines Corporation Degradable polyurethanes containing thioaminal groups

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6413507B1 (en) * 1999-12-23 2002-07-02 Shearwater Corporation Hydrolytically degradable carbamate derivatives of poly (ethylene glycol)
EP1608680A2 (fr) * 2003-03-24 2005-12-28 Novo Nordisk A/S Derives de glp-2
WO2006110776A2 (fr) * 2005-04-12 2006-10-19 Nektar Therapeutics Al, Corporation Compositions a base de polymere et conjugues d'agents antimicrobiens
KR20080108147A (ko) * 2006-03-31 2008-12-11 백스터 인터내셔널 인코포레이티드 페질화된 인자 ｖｉｉｉ

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008148839A3 (fr) * 2007-06-08 2009-08-13 Ascendis Pharma As Conjugués polymères transitoires longue durée de l'exendine
AU2008258548B2 (en) * 2007-06-08 2014-07-10 Sanofi-Aventis Deutschland Gmbh Long-acting transient polymer conjugates of exendin
US9353170B2 (en) 2007-06-08 2016-05-31 Sanofi-Aventis Deutschland Gmbh Long-acting transient polymer conjugates of exendin
US9138462B2 (en) 2009-07-31 2015-09-22 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an insulin linker conjugate
US9265723B2 (en) 2009-07-31 2016-02-23 Sanofi-Aventis Deutschland Gmbh Long acting insulin composition
US9457066B2 (en) 2009-07-31 2016-10-04 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an insulin linker conjugate
US9133276B2 (en) 2010-09-17 2015-09-15 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an exendin linker conjugate
US10227444B2 (en) 2016-06-01 2019-03-12 International Business Machines Corporation Degradable polyurethanes containing thioaminal groups

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