EP1853321A2 - Insulinotropic agents conjugated with structurally well defined branched polymers - Google Patents

Insulinotropic agents conjugated with structurally well defined branched polymers

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Publication number
EP1853321A2
EP1853321A2 EP06708301A EP06708301A EP1853321A2 EP 1853321 A2 EP1853321 A2 EP 1853321A2 EP 06708301 A EP06708301 A EP 06708301A EP 06708301 A EP06708301 A EP 06708301A EP 1853321 A2 EP1853321 A2 EP 1853321A2
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EP
European Patent Office
Prior art keywords
glp
xaa
lys
ethoxy
aib
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EP06708301A
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German (de)
French (fr)
Inventor
Carsten Behrens
Jesper Lau
János Tibor KODRA
Mikael Kofod-Hansen
Thomas Kruse Hansen
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Novo Nordisk AS
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Novo Nordisk AS
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Publication of EP1853321A2 publication Critical patent/EP1853321A2/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • 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
    • A61K47/595—Polyamides, e.g. nylon
    • 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
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/641—Branched, dendritic or hypercomb peptides
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • A61P9/12—Antihypertensives

Definitions

  • This invention relates generally to methods of treating humans suffering from diabetes mellitus. More specifically, the present invention relates to insulinotropic agents conjugated with structurally well defined branched polymers. The branched polymers are composed of monomer building blocks. Furthermore, this invention relates to the use of such conjugated insulinotropic agents, for example, by pulmonary delivery for systemic absorption through the lungs to reduce or eliminate the need for administering other insulinotropic agents by injection as well as to pharmaceutical compositions comprising these compounds and to the use of the compounds for the treatment of diseases related to diabetes.
  • Diabetes mellitus is a disease affecting approximately 6% of the world's population. Furthermore, the population of most countries is aging and diabetes is particularly common in aging populations. Often, it is this population group which experiences difficulty or unwillingness to self-administer insulin by injection. In the United States, approximately 5% of the population has diabetes and approximately one-third of those diabetics self-administer one or more doses of insulin per day by subcutaneous injection. This type of intensive therapy is necessary to lower the levels of blood glucose. High levels of blood glucose, which are the result of low or absent levels of endogenous insulin, alter the normal body chemistry and can lead to failure of the microvascular system in many organs. Untreated diabetics often undergo amputations and experience blindness and kidney failure. Medical treatment of the side effects of diabetes and lost productivity due to inadequate treatment of diabetes is estimated to have an annual cost of about $40 billion in the United States alone.
  • GLP-1 glucagon-like peptide-1
  • Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesized La. in the L-cells in the distal ileum, in the pancreas and in the brain.
  • GLP-1 is an important gut hormone with regulatory function in glucose metabolism and gastrointestinal secretion and metabolism. GLP-1 stimulates insulin secretion in a glucose-dependant manner, stimulates insulin biosynthesis, promotes beta cell rescue, decreases glucagon secretion, gastric emptying and food intake.
  • PCT publications WO 98/08871 and WO 99/43706 disclose stable derivatives of GLP-1 analogues, which have a lipophilic substituent. These stable derivatives of GLP-1 analogues have a protracted profile of action compared to the corresponding GLP-1 analogues.
  • Exendin-4 is a 39 amino acid residue peptide isolated from the venom of Heloderma suspectum, and this peptide shares 52% homology with GLP-1 (7-37) in the overlapping region.
  • Exendin-4 is a potent GLP-1 receptor agonist which has been shown to stimulate insulin release and ensuing lowering of the blood glucose level when injected into dogs.
  • the group of exendin-4(1 -39), certain fragments thereof, analogs thereof and derivatives thereof, are potent insulinotropic agents.
  • the group of exendin-4(1 -39), insulinotropic fragments thereof, insulinotropic analogs thereof and insulinotropic derivatives thereof are potent insulinotropic agents.
  • GLP-1 and exendins are that an extensive amount of variants have been synthesized and studied in particular in relation the plasma half-life. Low plasma half- lifes may be due to chemical stability towards peptidases (mainly dipeptidyl aminopeptidase IV) and to renal clearance.
  • peptidases mainly dipeptidyl aminopeptidase IV
  • these analogues and derivatives of insulionotropic peptides lack a satisfactory bioavailability when administered by the pulmonary route, i.e. when administered to the lower respiratory tract such as through the bronchioles or alveoli.
  • WO 00/66629 discloses modified exendin agonists which have been coupled to polyethyleneglycol via a lysine residue to decrease renal clearance.
  • WO 03/40309 discloses peptide acting as both GLP-1 receptor agonists and glucagon receptor antagonists.
  • the disclosed peptides are two peptides which have been coupled to polyethyleneglycol via a C-terminal cysteine residue.
  • WO 2004/093823 discloses polyethylene glycolated GLP-1 peptides.
  • GLP-1 peptides Pulmonary administration of GLP-1 peptides have been disclosed in WO 01/51071 and WO 00/12116.
  • the insulinotropic peptides derived from GLP-1 and Exendin-4 stimulates insulin release only when plasma glucose levels are high, and therefore the risk of hypoglycaemic events is reduced.
  • the peptides are particularly useful for patients with diabetes who no longer respond to OHA ' s (oral hyperglycaemic agents) and who should from a strict medical point of view be administered insulin. Patients and to some extent also doctors are often not keen on initiating insulin treatment before this is absolutely necessary, presumably because of the fear of hypoglycaemic events or the fear of injections/needles.
  • OHA ' s oral hyperglycaemic agents
  • peptides of therapeutic interest such as hormones, soluble receptors, cytokines, enzymes etc. often have short circulation half-life in the body as a result of proteolytic degradation, clearance by the kidney or liver, or in some cases the appearance of neutralizing antibodies. This generally reduces the therapeutic utility of peptides.
  • peptides can be enhanced by grafting organic chain-like molecules onto them. Such grafting can improve pharmaceutical properties such as half life in serum, stability against proteolytical degradation, and reduced immunogenicity.
  • the organic chain-like molecules often used to enhance properties are polyethylene glycol-based or polyethylene based chains, i.e., chains that are based on the repeating unit - CH 2 CH 2 O-.
  • PEG polyethylene glycol-based or polyethylene based chains
  • the abbreviation "PEG” is used for polyethyleneglycol.
  • the techniques used to prepare PEG or PEG-based chains involve a poorly-controlled polymerisation step which leads to preparations having a wide spread of chain lengths about a mean value. Consequently, peptide conjugates based on PEG grafting are generally characterised by broad range molecular weight distributions. Kochendoefer et al.
  • the mass growth of the polymer will in this case follow an exponential curve, with an exponent determined by the furcation number, for example, bifurcated monomers provides 2nd power growth, trifurcated monomers provides 3rd power growth, etc.
  • the type of polymers obtained by this procedure has been well described in the literature (S. M. Grayson and J.M.J. Frechet, Chem. Rev. 2001 , IPJ., 3819) and are commonly known as dendrimers.
  • Biodegradable 4th generation polyester dendrimers based on 2,2-bis(hydroxymethyl)- propionic acid and capped with polyethyleneoxide via a carbamate linkage has recently been reported (E.R.Gillies and J. M. J. Frechet, J. Amer. Chem. Soc, 2002, 124, 14137-14146).
  • the architecture of this system bears a close resemblance to the system described by Kochendoefer et al. as described above, as the dendritic part of the structure is used to generate a polyhydroxy scaffold that function as attachment points for the polyethyleneoxide tails.
  • Kochendoefer et al. as described above, as the dendritic part of the structure is used to generate a polyhydroxy scaffold that function as attachment points for the polyethyleneoxide tails.
  • a large degree of dispersity is introduced from each polyethyleneoxide tail, as only the core structure is chemically well defined.
  • the present invention provides a new class of branched polymers conjugated to an insulinotropic agent. These new compounds have the general formula I mentioned below with the definitions mentioned below.
  • the compounds of formula I contain a controlled number of monomer building blocks (designated Yb and Yt, below).
  • This invention also provides the use of a conjugate as above as a medicament.
  • polypeptide and peptide as used herein means a compound composed of at least five constituent amino acids connected by peptide bonds.
  • the constituent amino acids may be from the group of the amino acids encoded by the genetic code and they may 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 for example, hydroxyproline, ⁇ -carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine.
  • Synthetic 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), ⁇ -alanine, 3- aminomethyl benzoic acid, anthranilic acid.
  • analogue as used herein referring to a polypeptide 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.
  • GLP-1 (7-37)Lys designates a GLP-1 (7-37) analogue wherein the naturally occuring lysine at position 34 has been substituted with arginine and wherein a lysine has been added to the terminal amino acid residue, i.e. to the GIy 37 . All amino acids for which the optical isomer is not stated is to be understood to mean the L-isomer.
  • Human GLP-1 is hydrolysed to GLP-1 (7-37) and GLP-1 (7-36)-amide which are both insulinotropic peptides.
  • [Gly 8 ]GLP-1 (7-37) designates an analogue of GLP-1 (7-37) formally derived from GLP-1 (7-37) by substituting the naturally occurring amino acid residue in position 8 (Ala) by GIy.
  • (N ⁇ 34 -tetradecanoyl)[Lys 34 ]GLP-1 (7-37) designates GLP-1 (7-37) wherein the ⁇ -amino group of the Lys residue in position 34 has been tetradecanoylated.
  • analogues of GLP-1 or exendin-4 has maximum of 15 amino acids has been added, deleted or exchanged compared to the native sequence, In aspects of the invention maximum of 12 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 10 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 8 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 6 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 4 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 2 amino acids has been added, deleted or exchanged.
  • 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 N ⁇ 26 -((4S)-4-(hexadecanoylamino)- butanoyl)[Arg 34 , Lys 26 ]GLP-1 -(7-37).
  • 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 (one such medium disclosed below).
  • the potency of an insulinotropic agent is determined by calculating the EC 50 value from the dose-response curve as described below.
  • Baby hamster kidney (BHK) cells expressing the cloned human GLP-1 receptor (BHK- 467-12A) were grown in DMEM media with the addition of 100 ILVmL penicillin, 100 ⁇ g/mL streptomycin, 5% fetal calf serum and 0.5 mg/mL Geneticin G-418 (Life
  • Plasma membranes were prepared from the cells by homogenisation with an Ultraturrax in buffer 1 (20 mM HEPES-Na, 10 mM EDTA, pH 7.4). The homogenate was centrifuged at 48,000 x g for 15 min at 4 0 C. The pellet was suspended by homogenization in buffer 2 (20 mM HEPES-Na, 0.1 mM EDTA, pH 7.4), then centrifuged at 48,000 x g for 15 min at 4 0 C. The washing procedure was repeated one more time. The final pellet was suspended in buffer 2 and used immediately for assays or stored at -8O 0 C.
  • the functional receptor assay was carried out by measuring cyclic AMP (cAMP) as a response to stimulation by the insulinotropic agent.
  • cAMP formed was quantified by the AlphaScreenTM cAMP Kit (Perkin Elmer Life Sciences). Incubations were carried out in half- area 96-well microtiter plates in a total volume of 50 ⁇ L buffer 3 (50 mM Tris-HCI, 5 mM HEPES, 10 mM MgCI 2 , pH 7.4) and with the following addiditions: 1 mM ATP, 1 ⁇ M GTP, 0.5 mM 3-isobutyl-1 -methylxanthine (IBMX), 0.01 % Tween-20, 0.1 % BSA, 6 ⁇ g membrane preparation, 15 ⁇ g/mL acceptor beads, 20 ⁇ g/mL donor beads preincubated with 6 nM biotinyl-cAMP.
  • buffer 3 50 mM Tris-HCI, 5 mM HEPES, 10
  • GLP-1 peptide as used herein means GLP-1 (7-37) (SEQ ID No 1 ), a
  • GLP-1 (7-37) analogue a GLP-1 (7-37) derivative or a derivative of a GLP-1 (7-37) analogue.
  • the GLP-1 peptide is an insulinotropic agent.
  • exendin-4 peptide means exendin-4(1 -39) (SEQ ID No 2), an exendin-4(1 -39) analogue, an exendin-4(1 -39) derivative or a derivative of an exendin- 4(1 -39) analogue.
  • the exendin-4 peptide is an insulinotropic agent.
  • DPP-IV protected as used herein referring to a polypeptide means a polypeptide which has been chemically modified in order to render said compound resistant to the plasma peptidase dipeptidyl aminopeptidase-4 (DPP-IV).
  • DPP-IV enzyme in plasma is known to be involved in the degradation of several peptide hormones, for example, GLP-1 , GLP-2, Exendin-4 etc.
  • GLP-1 peptide hormones
  • GLP-2 GLP-2
  • Exendin-4 etc.
  • a DPP-IV protected peptide is more resistant to DPP-IV than GLP-1 (7-37) or Exendin-4(1 -39). Resistance of a peptide to degradation by dipeptidyl aminopeptidase IV is determined by the following degradation assay :
  • Peptides and their degradation products may be monitored by their absorbance at 220 nm (peptide bonds) or 280 nm (aromatic amino acids), and are quantified by integration of their peak areas related to those of standards.
  • the rate of hydrolysis of a peptide by dipeptidyl aminopeptidase IV is estimated at incubation times which result in less than 10% of the peptide being hydrolysed.
  • any skilled art worker for example, a physician, knows when and which dosages to administer of the GLP-1 analogue.
  • covalent attachment means that the polymeric molecule and the GLP-1 is either directly covalently joined to one another, or else is indirectly covalently joined to one another through an intervening moiety or moieties, such as bridge, spacer, or linkage moiety or moieties.
  • branched polymer or “dendritic polymer” or “dendritic structure” “or dendrimer” means an organic polymer assembled from a selection of monomer building blocks of which, some contains branches.
  • conjuggate or “conjugate GLP-1 ", is intended to indicate a heterogeneous
  • polydispersity is used to indicate the purity of a polymer.
  • polydispersity index (PDI) is the ratio of M w to M n wherein M w is Z(M 1 2 N 1 )ZZ(M 1 N 1 ) and M n is Z(M 1 N 1 )/ Z(N 1 ), wherein M 1 is the molecular weight of the individual molecules present in the mixture, and N 1 is the number of molecules represented by a certain molecular weight.
  • the PDI provides a rough indication of the breadth of the distribution of the specific polymers present in a mixture. If the PDI of a certain polymer is 1 , said polymer has a purity of 100%. For small generations of polymers, for example 1 -3 generation, it may be more convenient to indicate the purity that to indicate a PDI. However, for longer polymers, it may be more convenient to use PDI.
  • the term "monodisperse” is, herein, used for a polymer having a PDI of less than 1 .09. In an embodiment it is less than 1 .08. In an embodiment it is less than 1 .07 and at least 1 .
  • the term "structurally well defined" in connection with a product indicates that the product has a high purity of a specific, chemically well-defined compound. In an embodiment such a purity is above about 80%. In an embodiment it is above about 90%. In an embodiment it is above about 95%. In an embodiment it is above about 97.5%.
  • Immunogenicity of a polymer modified GLP-1 refers to the ability of the polymer modified GLP-1 , when administrated to a human, to elicit an immune response, whether humoral, cellular, or both.
  • attachment group is intended to indicate a functional group on the GLP-1 or linker modified GLP-1 capable of attaching a polymer molecule either directly or indirectly through a linker.
  • Useful attachment groups are, for example, amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate.
  • reactive functional group means by way of illustration and not limitation, any free amino, carboxyl, thiol, alkyl halide, acyl halide, chloroformiate, aryloxycarbonate, hydroxy or aldehyde group, carbonates such as the p-nitrophenyl, or succinimidyl; carbonyl imidazoles, carbonyl chlorides; carboxylic acids that are activated in situ; carbonyl halides, activated esters such as N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, esters of such as those comprising 1 ,2,3-benzotriazin-4(3H)-one, phosphoramidites and H- phosphonates, phosphortriesters or phosphordiesters activates in situ, isocyanates or isothiocyanates, in addition to groups such as -NH 2 , -OH, -N 3 , -NHR' or -OR' (where R' is a
  • protected functional group means a functional group which has been protected in a way rendering it essential non-reactive.
  • protection groups used for amines include but are not limited to tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, azides etc.
  • carboxyl group other groups become relevant such as tert-butyl, or more generally alkyl groups.
  • Appropriate protection groups are known to the skilled person, and examples can be found in Green & Wuts "Protection groups in organic synthesis", 3 rd Edition, Wiley-interscience.
  • cleavable moiety is intended to mean a moiety that is capable of being selectively cleaved to release the branched polymer linker or branched polymer linker GLP-1 from for example, a solid support.
  • the term "generation” refers to a single uniform layer, created by reacting one or more identical functional groups on an organic molecule with a particular monomer building block. Dendrimer synthesis demands a high level of synthetic control which is achieved through stepwise reactions, building the dendrimer up one monomer layer, or "generation,” at a time. Each dendrimer consists of a multifunctional core molecule with a dendritic wedge attached to each functional site. The core molecule is referred to as "generation 0". Each successive repeat unit along all branches forms the next generation, “generation 1 ", “generation 2,” etc. until the terminating generation.
  • the number of reactive surface groups available for reaction is 2 m , where m is an integer of 1 , 2, 3 ... 8 representing the particular generation.
  • the number of reactive groups is 3 m
  • the number of reactive groups is n m .
  • the number of reactive groups in a particular layer or generation can be calculated recursively knowing the layer position and the number of branches of the individual monomers.
  • the term "functional in vivo half-life” is used in its normal meaning, i.e., the time at which 50% of the biological activity of the GLP-1 or conjugate is still present in the body/target organ, or the time at which the activity of the GLP-1 or conjugate is 50% of its initial value.
  • "serum half-life” may be determined, i.e., the time at which 50% of the GLP-1 or conjugate molecules circulate in the plasma or bloodstream prior to being cleared. Determination of serum-half-life is often more simple than determining functional half-life and the magnitude of serum-half-life is usually a good indication of the magnitude of functional in vivo half-life.
  • serum half-life alternatives include plasma half-life, circulating half-life, circulatory half-life, serum clearance, plasma clearance, and clearance half-life.
  • the GLP-1 or conjugate is cleared by the action of one or more of the reticuloendothelial system (RES), kidney, spleen, or liver, by tissue factor, SEC receptor, or other receptor- mediated elimination, or by specific or unspecific proteolysis.
  • RES reticuloendothelial system
  • tissue factor tissue factor
  • SEC receptor or other receptor- mediated elimination
  • specific or unspecific proteolysis Normally, clearance depends on size (relative to the cut-off for glomerular filtration), charge, attached carbohydrate chains, and the presence of cellular receptors for the GLP-1 .
  • the functionality to be retained is normally selected from procoagulant, proteolytic, co-factor binding or receptor binding activity.
  • the functional in vivo half-life and the serum half-life may be determined by any suitable method known in the art.
  • the term "increased" as used about the functional in vivo half-life or plasma half-life is used to indicate that the relevant half-life of the GLP-1 or conjugate is statistically significantly increased relative to that of a reference molecule.
  • the relevant half- life may be increased by at least about 10% or at least 25%, such as by at least about 50%, for example, by at least about 100%, 150%, 200%, 250%, or 500%.
  • halogen means fluoro, chloro, bromo or iodo.
  • the term "heavy atom” as used herein means an atom having a molar weight equal to or larger than carbon, for example, C, N, O and S.
  • alkyl represents a saturated, branched or straight hydrocarbon group having from 1 to 18 carbon atoms. In an embodiment it is from 1 to 10 carbon atoms. In an embodiment it is from 1 to 6 carbon atoms with one, two, three, or four bonds, respectively.
  • Typical groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, terf-butyl, pentyl, and hexyl.
  • Specific alkylene, alkantriyl, and alkantetrayl groups include the corresponding divalent, trivalent, and tetravalent radicals.
  • alkenyl and alkenylene refer to a C 2 - 6 -alkenyl and C 2 - 6 -alkenylene, respectively, and represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one double bond and having one or two bonds, respectively.
  • Typical C 2 - 6 -alkenyl groups include, but are not limited to, ethenyl, 1 -propenyl, 2-propenyl, isopropenyl, 1 ,3-butadienyl, 1 -butenyl, 2-butenyl, 1 -pentenyl, 2-pentenyl, 1 -hexenyl, 2- hexenyl, 1 -ethylprop-2-enyl, 1 ,1 -(dimethyl)prop-2-enyl, 1 -ethylbut-3-enyl, and 1 ,1 - (dimethyl)but-2-enyl.
  • Examples of C 2 - 6 -alkenylen groups include the corresponding divalent radicals.
  • alkynyl or “alkynylene” refer to a C 2 . 6 -alkynyl or C 2 . 6 -alkynylene, representing a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one triple bond and having one or two bonds, respectively.
  • Typical C 2 . 6 -alkynyl or C 2 . 6 -alkynylene representing a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one triple bond and having one or two bonds, respectively.
  • 6 -alkynyl groups include, but are not limited to, 1 -propynyl, 2-propynyl, isopropynyl, 1 ,3-butadynyl, 1 - butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 1 -hexynyl, 2-hexynyl, 1 -ethylprop-2-ynyl, 1 ,1 -(di- methyl)prop-2-ynyl, 1 -ethylbut-3-ynyl, 1 ,1 -(dimethyl)but-2-ynyl, and C 2 - 6 -alkynylene groups include the corresponding divalent radicals.
  • alkyleneoxy or "alkoxy” refer to "Ci-e-alkoxy” or Ci-e-alkyleneoxy representing the radical -O-Ci- 6 -alkyl or -O-Ci- 6 -alkylene, respectively, wherein
  • Ci- 6 -alkyl(ene) is as defined above. Representative examples are methoxy, ethoxy, n- propoxy, isopropoxy, butoxy, sec-butoxy, terf-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.
  • alkylenethio alkenylenethio
  • alkynylenethio refer to the corresponding thio analogues of the oxy-radicals as defined above. Representative examples are methylthio, ethylthio, propylthio, butylthio, pentylthio, hexylthio, and the corresponding divalent radicals and the corresponding alkenyl and alkynyl derivatives also defined above.
  • -diyl and “-triyl” is used and refers to different alkyl, alkenyl, alkynyl, cycloalkyl or aromatic radicals with two and three attachment points, respectively.
  • alkantrioxy refers to an alkantriyl moiety with one oxy (-O-) attached to each of the three alkantriyl bonds.
  • Representative examples are propantrioxy, tert-butyltrioxy ect.
  • cycloalkyl refers to C 3 - 8 -cycloalkyl representing a monocyclic, carbocyclic group having from 3 to 8 carbon atoms.
  • Representative examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
  • cycloalkenyl refers to C 3 -8-cycloalkenyl representing a monocyclic, carbocyclic, non-aromatic group having from 3 to 8 carbon atoms and at least one double bond.
  • Representative examples are cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl and the like.
  • polyalkoxy designates alkoxy-alkoxy-alkoxy-alkoxy etc. where the number of carbon atoms in each of the alkoxy moieties is the same or different. In an embodiment they are the same.
  • polyalkoxyalkyl and polyalkoxyalkylcarbonyl designates (polyalkoxy)- alkyl and (polyalkoxy)-alkyl-CO-, respectively.
  • polyalkoxydiyl designates alkoxy- alkoxy-alkoxy-alkoxy etc having two free bonds.
  • poly means many. In an embodiment it is a numbering the range from 2 to 24. In an embodiment it is from 2 to 12. In an embodiment it is 3, 4 or 5.
  • oxyalkyl is -O-alkyl-, i.e. a divalent radical.
  • aryl as used herein is intended to include carbocyclic aromatic ring systems such as phenyl, biphenylyl, 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 hydro- genated derivatives are 1 ,2,3,4-tetrahydronaphthyl, 1 ,4-dihydronaphthyl and the like.
  • arenetriyl and “arenetetrayl” are moieties identical with aryl as defined above with the proviso that in arenetriyl and arenetetrayl there are not one but three and four, respectively, free bonds. With the same proviso, examples of arenetriyl and arenetetrayl are as mentioned for aryl above.
  • heteroaryl as used herein is intended to include heterocyclic aromatic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1 ,2,3- triazolyl, 1 ,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, 1 ,3,5- triazinyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4- oxadiazolyl, 1 ,2,3-thiadiazolyl, 1 ,2,4
  • Heteroaryl is also intended to include the partially hydrogenated derivatives of the heterocyclic systems enumerated above.
  • Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydro- benzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.
  • heteroaryl-Ci. 6 -alkyl denotes heteroaryl as defined above and Ci-6-alkyl as defined above.
  • aryl-Ci. 6 -alkyl and "aryl-C 2 - 6 -alkenyl” as used herein denotes aryl as defined above and Ci- 6 -alkyl and C 2 -6-alkenyl, respectively, as defined above.
  • Fmoc is:
  • alkyleneaminocarbonylalkylamino [-alkylene-NH-CO-alkylene-NH-, for example, -NH-- CH 2 CH 2 -CO-NH-CH 2 CH 2 CH 2 CH 2 -], alkylenecarbonylamino(polyalkoxy)alkylamino [-alkylene- CO-NH-(polyalkoxy)-alkylene-NH-], alkyleneoxyalkyl [-alkylene-O-alkylene-, for example, - CH 2 CH 2 -O-CH 2 CH 2 -], carbonylalkylamino [-CO-alkylene-NH-, for example, -NHCH 2 CH 2 C(O)- ], carbonylalkylcarbonylamino(polyalkoxy)
  • treatment means the prevention, management and care of a patient for the purpose of combating a disease, disorder or condition.
  • the term is intended to include the prevention of the disease, delaying of the progression of the disease, disorder or condition, the alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition.
  • the patient to be treated is a mammal, in particular a human being.
  • excipient means the chemical compounds which are normally added to pharmaceutical compositions, for example, buffers, tonicity agents, preservatives and the like.
  • an effective amount means a dosage which is sufficient to be effective for the treatment of the patient compared with no treatment.
  • pharmaceutical composition means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative, and optionally a tonicity modifier and/or a stabilizer.
  • pharmaceutical excipients such as buffer, preservative, and optionally a tonicity modifier and/or a stabilizer.
  • a pharmaceutical composition is also known in the art as a pharmaceutical formulation.
  • the present invention relates to branched polymers attached to GLP-1 which branched polymers are made up of a precise number of monomer building blocks.
  • the monomer building blocks may be oligomerised either on solid support or in solution using suitable monomer protection and activation strategies.
  • a branched polymer attached to GLP- 1 is herein also designated a conjugated GLP-1 or an GLP-1 conjugate.
  • Using the methods described below it is possible to prepare a conjugated GLP-1 wherein the branched polymer is structural well defined.
  • the compounds of this invention are monodisperse.
  • compounds of the general formula I below having a purity above 50%. In an embodiment it is above 75%. In an embodiment it is above 90%.
  • this invention relates to a product containing a single, specific compound of formula I in such a high purity.
  • An embodiment of this invention provides an GLP-1 conjugate as described above, which is represented by the general formula I:
  • ITA represents an insulinotropic agent from which a hydrogen has been removed from an alpha-amino group present in the insulinotropic agent, or from an epsilon amino group present in lysine at any position in the insulinotropic agent, for the 1 st generation of bifurcated compounds
  • Y1 is Yb; Y2 is Z; r, q, p, and s are all zero; and n is 2; for the 2 nd generation of bifurcated compounds,
  • Y1 and Y2 are Yb; Y3 is Z; r, q, and p are all zero; s is 4; and n is 2; for the 3 rd generation of bifurcated compounds,
  • Y1 , Y2, and Y3 are all Yb; Y4 is Z; r and q are zero; p is 8; s is 4; and n is 2; for the 4 th generation of bifurcated compounds, Y1 , Y2, Y3, and Y4 are all Yb; Y5 is Z; r is zero; q is 16; p is 8; s is 4; and n is 2; and for the 5 th generation of bifurcated compounds,
  • Y1 , Y2, Y3, Y4, and Y5 are all Yb; Y6 is Z; r is 32; q is 16, p is 8; s is 4; and n is 2; wherein
  • Yb is -A-L r X 3
  • Y1 is Yt
  • Y2 is Z
  • r, q, p, and s are all zero
  • n is 3
  • for the 2 nd generation of trifurcated compounds Y1 and Y2 are Yt
  • Y3 is Z
  • r, q, and p are all zero
  • s is 9
  • n is 3
  • for the 3 rd generation of trifurcated compounds
  • Y1 , Y2, and Y3 are all Yt; Y4 is Z; r and q are zero; p is 27; s is 9; and n is 3; and for the 4 th generation of trifurcated compounds,
  • Y1 , Y2, Y3, and Y4 are all Yt; Y5 is Z; r is zero; q is 81 ; p is 27; s is 9; and n is 3;
  • X 3 is a nitrogen atom, alkantriyl, arenetriyl, alkantrioxy, an aminocarbonyl moiety of the formula -CO-N ⁇ , an acetamido moiety of the formula -CH 2 CO-N ⁇ or a moiety of the formula: -CO-NH-Q-NH-CO- I wherein Q is alkantriyl;
  • X 4 is alkantetrayl or arenetetrayl
  • L 1 is a valence bond, oxy, alkylene, alkyleneoxyalkyl, polyalkoxydiyl, (polyalkoxy)alkylcarbonyl, oxyalkyl or (polyalkoxy)alkyl wherein the terminal alkyl moiety of the last 3 moieties is connected to A ;
  • Li is connected to A in the oxy- part of the last three moieties.
  • L 2 is a valence bond, oxy, alkylene, alkyleneoxyalkyl, polyalkoxydiyl, (polyalkoxy)alkylcarbonyl, oxyalkyl or (polyalkoxy)alkyl wherein the terminal alkyl moiety of the last 3 moieties is connected to B;
  • L 2 is connected to B in the other end of the divalent radicals.
  • L 3 represents a valence bond, alkylene, oxy, polyalkoxydiyl, oxyalkyl, alkylamino, carbonylalkylamino, alkylaminocarbonylalkylamino, carbonylalkyl- carbonylamino(polyalkoxy)alkylamino, carbonylalkoxyalkylcarbonylamino(polyalkoxy)- alkylamino, alkylcarbonylamino(polyalkoxy)alkylamino, carbonyl(polyalkoxy)alkylamino or carbonylalkoxyalkylamino wherein the terminal carbonyl, alkyl and oxy moiety of the last 10 moieties, is connected to the ITA group, optionally via the L 4 moiety; In an embodiments of the invention the moieties are connected in the other end of the divalent radical.
  • n is zero, 1 , 2 or 3;
  • Z is hydrogen, alkyl, alkoxy, hydroxyalkyl, polyalkoxy, oxyalkyl, acyl, polyalkoxyalkyl, or polyalkoxyalkylcarbonyl.
  • L 1 , L 2 , L 3 and L 4 all shall be interpreted as divalent radicals, X 3 is a trivalent radical and X 4 is a tetravalent radical.
  • ITA, Y1 , Y2, L3, L4, m, Yb and Z each are as defined above.
  • the 2 nd generation bifurcated compounds can be illustrated by the formula Ib ITA-l_4-(L 3 ) m -Y1 (Y2(Y3)4)2 (Ib)
  • r is zero. In another embodiment of this invention, r and q are each zero. In another embodiment of this invention, n is 2 (for bifurcated compounds) or 3 (for trifurcated compounds). In another embodiment of this invention, s is 4 (for bifurcated compounds) or 9 (for trifurcated compounds). In another embodiment of this invention, s is 4, and p is 8 (for bifurcated compounds) or s is 9, and p is 27 (for trifurcated compounds). As mentioned above, compounds of formula I contains one or more Yb moieties. If a compound of formula I contains more than one Yb moiety, those moieties may be the same or different. In an embodiment of this invention, all Yb moieties are identical.
  • the Yb moieties from the same level are identical, but the Yb moieties (or moiety) in one level are (is) different from the Yb moieties (or moiety) in another level, each level being identified by the suffixes n, s, p, q and r, respectively, in formula I.
  • the two B moieties are the same or different. In an embodiment such two B moieties are the same.
  • the specific moiety Yb the two L 2 moieties are the same or different. In an embodiment such two L 2 moieties are the same. This, similarly, applies for the Yt moieties and the B and L 2 moieties present therein.
  • it relates to bifurcated compounds, in another embodiment, it relate to trifurcated compounds.
  • the two or three L 2 moieties present in any Yb or Yt moiety, respectively, may be the same or different.
  • the two or three L 2 moieties present in any Yb or Yt moiety, respectively, are the same.
  • the major part of the non insulinotropic agent of compounds of formula I can be build from compounds of formula IVb or IVc having the following formula:
  • the nature of the covalent bond formed by reaction between the groups A' and B' depends upon the selection of A' and B', and include, as indicated above, amide bonds, carbamate bonds, phosphate ester bonds, thiophosphate ester bonds, and phosphit bonds.
  • B' is selected from the group consisting of -NH 2 , -OH, -N 3 , - NHR' and -OR'; where R' is a protection group, that facilitates stepwise monomer oligomerization as used in, for example, peptide chemistry and oligonucleotide chemistry.
  • Non-limiting examples of protecting groups includes 9-fluorenylmethoxycarbonyl (designated Fmoc), terf-butoxycarbonyl (designated Boc), phthaloyl, triphenylmethyl, and substituted triphenylmethyl, trihaloacetyl such as trifluoroacetyl or trichloroacetyl, pixyl, trimethylsilyl, terf-butyldimethylsilyl, and terf-butyldiphenylsilyl.
  • Other examples of appropriate protection groups are known to the skilled person, and suggestions can be found in Green & Wuts "Protection groups in organic synthesis", 3 rd edition, Wiley-interscience.
  • X 3 may be a branched, trivalent organic radical (linker).
  • X 3 is of hydrophilic nature. In an embodiment, it includes a multiply-functionalised alkyl group containing up to 18. In an embodiment it contains from 1 to about 10 carbon atoms. In another embodiment, X 3 is a single nitrogen atom. In another embodiment, X 3 is alkantriyl. In another embodiment, X 3 is propan-1 ,2,3-triyl. In another embodiment X 3 is an alkantrioxy.
  • X 3 is alkantriyl, alkantrioxy or a moiety of the formula: -CO-NH-Q-NH- CO-, wherein Q is alkantriyl; and, furthermore, X 3 can be an aminocarbonyl moiety of the formula -CO-N ⁇ or an acetamido moiety of the formula -CH 2 CO-N ⁇ and, in another embodiment X 3 has one of the following formulas: the two last moieties being (R) and (S)-1 ,5-bis(aminocarbonyl)pentyl.
  • X 4 is symmetrical. In an embodiment X 4 is benzen-1 ,3,4,5-tetrayl. In another embodiment of this invention, X 3 or X 4 is symmetrically.
  • L 1 and L 2 are alkylene and -((CH 2 ) m O) n -, where m' is 2, 3, 4, 5, or 6, and n' is an integer from O to 10.
  • L 1 and L 2 are of hydrophilic nature.
  • L 1 , L 2 or both are valence bonds.
  • L 1 is -CH 2 (OCH 2 CH 2 ) n -OCH 2 C(O)-, where n" is an integer from O to 10.
  • L 1 and L 2 are independently selected from water soluble organic divalent radicals.
  • either L 1 or L 2 or both are divalent organic radicals containing about 1 to 5 PEG (-CH 2 CH 2 O-) groups.
  • L 1 and L 2 are each, independently of each other, a tri, tetra or pentaethylenglycol moiety, i.e., (-CH 2 CH 2 O) 3 , (-CH 2 CH 2 O) 4 or (-CH 2 CH 2 O) 5 .
  • L 1 is oxy (-0-) or oxymethyl (-OCH 2 -)
  • L 2 is a
  • L 1 is a valence bond, oxy, alkyleneoxyalkyl, oxyalkyl or
  • L 1 is a valence bond, -O- or one of the following three moieties: -OCH 2 -, -CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 - and -CH 2 OCH 2 -.
  • L 1 is a valence bond, oxy, alkylene, polyalkoxydiyl or oxyalkyl.
  • L 1 is (polyalkoxy)alkylcarbonyl, oxyalkyl or (polyalkoxy)alkyl wherein the terminal alkyl moiety, preferably, is connected to A .
  • the moieties are connected in the other end of the divalent radical.
  • L 2 is alkylene, alkyleneoxyalkyl, polyalkoxydiyl, or
  • L 2 is one of the following four moieties: moieties: -CH 2 - CH 2 OCH 2 CH 2 O-, -CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 2 -, -CH 2 CH 2 OCH 2 CH 2 - or -CH 2 CH 2 -.
  • L 2 is a valence bond, oxy, alkylene, polyalkoxydiyl or oxyalkyl.
  • L 2 is (polyalkoxy)alkylcarbonyl, oxyalkyl or (polyalkoxy)alkyl wherein the terminal alkyl moiety, preferably, is connected to B.
  • the moieties are connected in the other end of the divalent radical.
  • X 3 is a nitrogen atom, alkantriyl, arenetriyl or a moiety of the formula: -CO-NH-Q-NH-CO-
  • Q is 1 ,1 ,5-pentatriyl.
  • m is an integer or 1 , 2 or 3. In another embodiment of this invention, m is an integer. In another embodiment of this invention, m is 1 , 2 or 3.
  • L 3 is alkylene, polyalkoxydiyl, oxy, oxyalkyl, and a valence bond
  • L 4 is a bond
  • L 3 as a hole is aminoalkenyl or aminopolyalkoxydiyl derivatives such as aminoalkyloxyiminoarylcarbonyl or aminopolyalkoxyiminocarbonyl.
  • L 3 is alkylene, polyalkoxydiyl, oxy, oxyalkyl, and a valence bond
  • L 4 is a bond
  • L 3 as a hole is aminoalkenyl or aminopolyalkoxydiyl derivatives such as aminoalkyloxyiminomethylarylcarbonyl or aminopolyalkoxyiminoacetyl.
  • L 3 is alkylamino, carbonylalkylamino or alkylaminocarbonyl- alkylamino.
  • L 3 is one of the following three moieties: -C(O)CH 2 CH 2 NH-, - CH 2 CH 2 CH 2 CH 2 NHC(O)CH 2 CH 2 NH- or -CH 2 CH 2 CH 2 CH 2 NH- .
  • L 3 is a valence bond or a divalent linker radical such as those illustrated by the following six formulae:
  • each end of the divalent radicals can be attached to the ITA group.
  • the ITA group is attached via the carbonyl group.
  • L 4 and the adjacent L 3 is a divalent linker radical such as those illustrated by the following eight formulae:
  • each ends of the divalent radicals can be connected to the ITA group.
  • the carbonyl group is attached to ITA group.
  • L 4 is a valence bond.
  • L 4 is syn or anti forms of one of the moieties of the formulae:
  • L 4 is syn and anti forms of the moieties of the formulae:
  • A is -CO-, -C(O)O-, -P(O)O - or -P(S)O -.
  • B is -NH- or -0-.
  • Z is hydrogen, alkyl, acyl or polyalkoxyalkyl- carbonyl and in an embodiment Z is H-, CH 3 OCH 2 CH 2 OCH 2 CH 2 OCH 2 C(O)-, CH 3 - or C 6 H 5 C(O)-. In an embodiment Z is hydrogen or one of the three groups: CH 3 OCH 2 CH 2 OCH 2 - CH 2 OCH 2 C(O)-, CH 3 - and C 6 H 5 C(O)-.
  • A' is a carboxyl group
  • B' is a protected amino group which after deprotection may be coupled to a new monomer of same structure via its carboxy group to form an amide. Larger polymers can be assembled in a repetitive manner as is known from standard oligopeptide synthesis.
  • A' is p-nitrophenylcarbonate (-C(O)-O-pC 4 H 6 NO 2 ), carbonylimidazol (-C(O)-lm), - COOH or -C(O)CI.
  • B' is N 3 -, FmocNH- or a group of one of the formulae:
  • L 3 is aminoalkyl, the carbon atom thereof is connected to the part of formula I containing the ITA-L 4 moiety.
  • L 3 is oxyalkyl, the carbon atom thereof is connected to the part of formula I containing the ITA-L 4 moiety.
  • L 1 is polyalkoxydiyl, polyalkoxyalkyl or oxyalkyl, the terminal alkylene moiety thereof is, connected to the A moiety.
  • L 2 is polyalkoxydiyl, or oxyalkyl, the oxy part thereof is connected to the X 3 and X 4 moiety.
  • A' is a phosphoramidite and B' is a hydroxyl group suitable protected, which upon deprotection can be coupled to another monomer of the same type to form a phosphite triester which subsequently is oxidised to form a stable phosphate triester or thiophosphate triester.
  • B' is a hydroxyl group suitable protected, which upon deprotection can be coupled to another monomer of the same type to form a phosphite triester which subsequently is oxidised to form a stable phosphate triester or thiophosphate triester.
  • A' is a reactive carbonate such as nitrophenyl carbonate
  • B' is an amino group. In an embodiment this is in its protected form.
  • larger polymers can be assembled in a repetitive manner as is known from standard oligocarbamate synthesis.
  • A' is an acyl halide such as -COCI or -COBr
  • B' is an amino group. In an embodiment this is in its protected form.
  • A is one of the three moieties: -CO-, -P(O)O- and -P(S)O-.
  • B is oxy or the moiety -NH-.
  • the branched polymer of the compounds of this invention has a molecular weight of above about 500 Da. In an embodiment it is above about 3 kDa. In an embodiment it is above about 5 KDa.
  • the branched polymer of the compounds of this invention has a molecular weight of below about 10 kDa. In an embodiment it is below about 7 kDa.
  • the compounds of this invention have an isoelectric point between about 3 and about 7.
  • the compounds of this invention have a net negative charge under physiological conditions.
  • the monomer building blocks of the formula A'-Li-X 3 -(L 2 -B') 2 is
  • the monomer building blocks of the formula A'-Li-X 3 -(L 2 -B') 2 is
  • L 3 is a divalent linker radical such as the following three formula:
  • Z is a capping agent that can react with a terminal amino group or hydroxy group.
  • Z include the following three examples: o
  • the major part of the non ITA part of the compounds of formula I is build from monomers of the formula A'-L r X 3 -(L 2 -B') 2 :
  • the major part of the non ITA part of the compounds of formula I is build from monomers of the formula A'-L r X 4 - (U-BV
  • ITA is GLP-1 from any natural species and salts thereof, active derivatives of GLP-1 , or GLP-1 analogues, from which a hydrogen atom has been removed as mentioned above.
  • ITA is any of the GLP-1 molecules mentioned specifically in the example below, from which a hydrogen atom has been removed as mentioned above.
  • the insulinotropic agent is derived from a peptide having a length between 27 and 45 amino acid residues in which 22 out of the first 28 amino acid residues are identical to those found in corresponding positions in GLP-1 (7-37) (SEQ ID No. 1 ) or in corresponding positions in Exendin-4(1 -39) (SEQ ID No. 2).
  • the insulinotropic agent is derived from a peptide having a length between 28 and 45 amino acid residues in which 22 out of the first 28 amino acid residues are identical to those found in corresponding positions in GLP-1 (7- 37) or in corresponding positions in Exendin-4(1 -39).
  • the insulinotropic agent is selected from a peptide comprising the amino acid sequence of the formula (II):
  • Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy- histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
  • Xaa 8 is Ala, D-AIa, GIy, VaI, Leu, He, Lys, Aib, (1 -aminocyclopropyl) carboxylic acid, (1 - aminocyclobutyl) carboxylic acid, (1 -aminocyclopentyl) carboxylic acid, (1 -aminocyclohexyl) carboxylic acid, (1 -aminocycloheptyl) carboxylic acid, or (1 -aminocyclooctyl) carboxylic acid;
  • Xaaie is VaI or Leu;
  • Xaais is Ser, Lys or Arg
  • Xaaig is Tyr or Gln
  • Xaa 2 o is Leu or Met
  • Xaa 22 is GIy, GIu or Aib;
  • Xaa 23 is GIn, GIu, Lys or Arg;
  • Xaa 25 is Ala or VaI
  • Xaa 26 is Lys, GIu or Arg;
  • Xaa 30 is Ala, GIu or Arg; Xaa 33 is VaI or Lys;
  • Xaa 34 is Lys, GIu, Asn or Arg;
  • Xaa 35 is GIy or Aib
  • Xaa 36 is Arg, GIy or Lys
  • Xaa 37 is GIy, Ala, GIu, Pro, Lys, amide or is absent; Xaa 38 is Lys, Ser, amide or is absent.
  • Xaa 39 is Ser, Lys, amide or is absent;
  • Xaa 40 is GIy, amide or is absent
  • Xaa 4 i is Ala, amide or is absent;
  • Xaa 42 is Pro, amide or is absent;
  • Xaa 43 is Pro, amide or is absent;
  • Xaa 4 4 is Pro, amide or is absent;
  • Xaa 45 is Ser, amide or is absent;
  • Xaa 46 is amide or is absent ; provided that if Xaa 3 s, Xaa 3 g, Xaa4o, Xaa4i, Xaa42, Xaa43, Xaa44, Xaa45 or Xaa46 is absent then each amino acid residue downstream is also absent.
  • the insulinotropic agent is a peptide comprising the amino acid sequence of formula (III):
  • Xaa 7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy- histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
  • Xaa 8 is Ala, D-AIa, GIy, VaI, Leu, He, Lys, Aib, (1 -aminocyclopropyl) carboxylic acid, (1 - aminocyclobutyl) carboxylic acid, (1 -aminocyclopentyl) carboxylic acid, (1 -aminocyclohexyl) carboxylic acid, (1 -aminocycloheptyl) carboxylic acid, or (1 -aminocyclooo
  • Xaais is Ser, Lys or Arg
  • Xaa 22 is GIy, GIu or Aib;
  • Xaa 23 is GIn, GIu, Lys or Arg;
  • Xaa 26 is Lys, GIu or Arg;
  • Xaa 30 is Ala, GIu or Arg
  • Xaa 34 is Lys, GIu or Arg;
  • Xaa 35 is GIy or Aib; Xaa 36 is Arg or Lys;
  • Xaa 37 is GIy, Ala, GIu or Lys
  • Xaa 38 is Lys, amide or is absent.
  • the insulinotropic agent comprises no more than fifteen amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ), or no more than ten amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ). In another embodiment of the invention the insulinotropic agent comprises no more than six amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ).
  • the insulinotropic agent comprises no more than 4 amino acid residues which are not encoded by the genetic code.
  • the insulinotropic agent comprises an Aib residue as the second amino acid residue from the N-terminal.
  • the N-terminal amino acid residue (position 7 in formulae Il and III) of said insulinotropic agent is selected from the group consisting of D- histidine, desamino-histidine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ - acetyl-histidine , ⁇ -fluoromethyl-histidine, ⁇ -methyl-histidine, 3-pyridylalanine, 2- pyridylalanine and 4-pyridylalanine.
  • the insulinotropic agent is selected from the group consisting of [Arg 34 ]GLP-1 (7-37), [Arg 26 ' 34 ]GLP-1 (7-37)l_ys, [Lys 36 Arg 26 ' 34 ]GLP-1 (7-36), [Aib 8 ' 22 ' 35 ]GLP-1 (7-37),
  • the insulinotropic agent comprises at least one Aib residue.
  • the insulinotropic agent contains two Aib residues.
  • the insulinotropic agent comprises a serine residue at position 18 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 12 relative to Exendin-4(1 -39).
  • the insulinotropic agent comprises a tyrosine residue at position 19 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 13 relative to Exendin-4(1 -39).
  • the insulinotropic agent comprises a glycine residue at position 22 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 16 relative to Exendin-4(1 -39).
  • the insulinotropic agent comprises a glutamine residue at position 23 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 17 relative to Exendin-4(1 -39).
  • the insulinotropic agent comprises a lysine residue at position 26 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 20 relative to Exendin-4(1 -39).
  • the insulinotropic agent comprises a glutamate residue at position 27 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 21 relative to Exendin-4(1 -39).
  • the insulinotropic agent is exendin-4(1 -39). In another embodiment of the invention the insulinotropic agent is ZP-10, i.e. [Ser 38 Lys 39 ]Exendin-4(1 -39)LysLysLysLysLys-amide (SEQ ID No. 5).
  • the insulinotropic agent is attached the branched polymer via the amino acid residue in position 25 to 45 relative to the amino acid sequence SEQ ID No 1 .
  • the insulinotropic agent is attached to the branched polymer via an amino acid residue selected from one of the 10 C-terminal amino acid residues.
  • the insulinotropic agent is attached to the branched polymer via the amino acid residue in position 23, 26, 34, 36 or 38 relative to the amino acid sequence SEQ ID No: 1 .
  • the insulinotropic agent is attached to the branched polymer via the amino acid residue in position 17, 20, 28, 30 or 32 relative to the amino acid sequence SEQ ID No: 2.
  • the insulinotropic agent is attached to the branched polymer via the C-terminal amino acid residue. In another embodiment of the invention the insulinotropic agent is attached to the branched polymer via a carboxyl group, an amino group, a keto group, a hydroxyl group, a thiol group or a hydrazide group.
  • the insulinotropic agent is attached to the branched polymer via a the epsilon-amino group on a lysine residue. In another embodiment of the invention the insulinotropic agent comprises only one lysine residue.
  • the insulinotropic agent comprises only one lysine residue which is the C-terminal amino acid residue of said insulinotropic agent.
  • the compound according to the present invention has an
  • the GLP-1 analogs can be produced by classical peptide synthesis, for example, solid phase peptide synthesis using t-Boc or F-Moc chemistry or other well established techniques., see the examples and for example, Houben-Weyl, Methods of organic Chemistry, Volume E 22a, E 22b and E 22 c; Green and Wuts, "Protecting Groups in Organic Synthesis", Jogn Wiley & Sons, 1999. These methods are preferred when the insulinotropic agent is a peptide comprising non-natural amino acid residues.
  • the polypeptides can also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide, after which the resulting peptide is recovered from the culture and then derivatized to the compound of formula (I).
  • the medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (for example, in catalogues of the American Type Culture Collection).
  • the peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration.
  • the proteinaceous components of the supernatant are isolated by filtration, column chromatography or precipitation, for example, microfiltation, ultrafiltration, isoelectric precipitation, purification by a variety of chromatographic procedures, for example, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.
  • chromatographic procedures for example, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.
  • intracellular or periplasmic products the cells isolated from the culture medium are disintegrated or permeabilised and extracted to recover the product polypeptide or precursor thereof.
  • the DNA sequence encoding the therapeutic polypeptide may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the peptide by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989).
  • the DNA sequence encoding the polypeptide may also be prepared synthetically by established standard methods, for example, the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981 ), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805.
  • the DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
  • the DNA sequence may be inserted into any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, for example, a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is an expression vector in which the DNA sequence encoding the polypeptide is operably linked to additional segments required for transcription of the DNA, such as a promoter.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the peptide of the invention in a variety of host cells are well known in the art, cf. for instance Sambrook et al., supra.
  • the DNA sequence encoding the polypeptide may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences.
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, for example, a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, for example, ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • a selectable marker for example, a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, for example, ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
  • the selectable marker is not antibiotic resistance, for example, antibiotic resistance genes in the vector are excised when the vector is used for large scale manufacture.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the peptide.
  • the secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.
  • the host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells.
  • suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
  • Branched polymers can in general be assembled from the monomer building blocks described above using one of two fundamentally different oligomerisation strategies called the divergent approach and the convergent approach.
  • the branched polymers are assembled by an iterative process of synthesis cycles, where each cycle use suitable activated, reactive bi or trifurcated monomer building blocks, them self containing functional end groups - allowing for further elongation (i.e. polymer "growth").
  • the functional end groups usually needs to be protected in order to prevent self polymerisation and a deprotection step will in such cases be needed in order to generate a functional end group necessary for further elongation.
  • One such cycle of adding an activated (reactive) monomer building block and subsequent deprotection in the iterative process completes a generation.
  • the divergent approach is illustrated in reaction scheme 4 using solution phase chemistry and in reaction scheme 3 using solid phase chemistry.
  • the branched polymer therefore is assembled by the convergent approach described in US patent 5,041 ,516.
  • the convergent approach to build macromolecules involves building the final molecule by beginning at its periphery, rather than at its core as in the divergent approach. This avoids problems, such as incomplete formation of covalent bonds, typically associated with the reaction at progressively larger numbers of sites.
  • the convergent approach for assembly 2nd generation branched polymer is illustrated in reaction scheme 1 and reaction scheme 2 using a specific example involving one of the monomer building blocks.
  • Rigidity of the branched polymer can be controlled by the design of the particular monomer, for example by using a rigid core structure (X 3 or X 4 ) or by using rigid linker moieties (L 1 and L 2 ).
  • adjustment of the rigity is obtained by using the rigid monomer in one or more specific layers intermixed with monomers of more flexible nature.
  • the overall hydrophilic nature of the polymer is controllable. This is achieved by choosing monomers with more hydrophobic core structure (X 3 or X 4 ) or more hydrophobic linker moieties (L 1 and L 2 ), in one or more of the dendritic layers.
  • a different monomer is used in the outer terminal layer (Z) of the branched polymer, which in the final GLP-1 conjugate will be exposed to the surrounding environment.
  • Some of the monomers described here have protected amine functions as terminal end groups (B'), which after a deprotection step, and under physiological conditions, i.e. neutral physiological buffered to a pH value around 7.4, will be protonated, causing the overall structure to be polycationically charged.
  • neutral structures can be made by capping with various acylating reagents.
  • One example as depicted in reaction scheme 5 uses CH3(OCH 2 CH 2 ) 2 CH 2 COOH for capping the final layer (Z) of a dendritic structure, that otherwise would be terminated in amines.
  • branched polymers which imitates the natural occurring glycopeptides, which commonly has multiple anionic charged sialic acids as termination groups on the antenna structure of their N-glycans.
  • glycans can be imitated with respect to their poly anionic nature.
  • reaction scheme 6 where the branched polymer is capped with succinic acid mono terf-butyl esters which upon deprotection with acids render a polymer surface that is negatively charged under physiological conditions.
  • the assembly of monomers into polymers may for example be conducted either on solid support as described by NJ. Wells, A. Basso and M. Bradley in Biopolymers 47, 381 - 396 (1998), or in an appropriate organic solvent by classical solution phase chemistry, for example, as described by Frechet et al. in U.S. patent 5,041 ,516.
  • the branched polymer is assembled on a solid support derivatised with a suitable linkage in an iterative divergent process as described above and illustrated in reaction scheme 3.
  • solid phase protocols useful for conventional peptide synthesis can conveniently be adapted.
  • Applicably standard solid phase techniques such as those described in the literature (see Fields, editor, Solid phase peptide synthesis, in Meth Enzymol 289) can be conducted either by use of suitable programmable instruments (for example, ABI 430A) or similar home build machines, or manually using standard filtration techniques for separation and washing of support.
  • This type of solid support oxidation is typically achieved with iodine/water or peroxides such as but not limited to tert- butyl hydrogenperoxid and 3-chloroperbenzoic acid and requires that the monomers with or without protection resist oxidation condition.
  • iodine/water or peroxides such as but not limited to tert- butyl hydrogenperoxid and 3-chloroperbenzoic acid and requires that the monomers with or without protection resist oxidation condition.
  • the phosphor amidite methodology also allows for convenient synthesis of thiophosphates by simple replacement of the iodine with elementary sulfur in pyridine or organic thiolation reagents such as 3H-1 ,2-benzodithiole-3- one-1 ,1 -dioxide (see, for example, M. Dubber and J.M.J. Frechet in Bioconjugate chem. 2003, 14, 239-246).
  • the resin attached branched polymer when complete, can then be cleaved from the resin under suitable conditions. It is important, that the cleavable linker between the growing polymer and the solid support is selected in such way that it will stay intact during the oligomerisation process of the individual monomers, including any deprotection steps, oxidation or reduction steps used in the individual synthesis cycle, but when desired under appropriate conditions can be cleaved leaving the final branched polymer intact.
  • the skilled person will be able to make suitable choices of linker and support, as well as reaction conditions for the oligomerisation process, the deprotection process, and optionally oxidation process, depending on the monomers in question.
  • Resins derivatised with appropriate functional groups that allows for attachment of monomer units and later act as cleavable moieties, are commercial available (see, for example, the catalogue of Bachem and NovoBiochem).
  • the branched polymer is synthesised on a resin with a suitable linker, which upon cleavage generates a branched polymer product furnished with a functional group that directly can act as an attachment group in a subsequent solution phase conjugation process to the insulionotropic agent (ITA) as described below or, alternatively, by appropriate chemical means can be converted into such an attachment group.
  • ITA insulionotropic agent
  • the dendritic branched polymers of a certain size and compositions is synthesised using classical solution phase techniques.
  • the branched polymer is assembled in an appropriate solvent, by sequential addition of suitable activated monomers to the growing polymer. After each addition, a deprotection step may be needed before construction of the next generation can be initiated. It may be desirable to use excess of monomer in order to reach complete reactions. In an embodiment, the removal of excess monomer takes advantages of the fact that hydrophilic polymers have low solubility in diethyl ether or similar types of solvents.
  • the growing polymer can thus be precipitated leaving the excess of monomers, coupling reagents, by-products etc. in solution. Phase separation can then be performed by simple decantation. In embodiments by centrifugation followed by decantation.
  • Polymers can also be separated from by-products by conventional chromatographic techniques on, for example, silica gel, or by the use of HPLC or MPLC systems under either normal or reverse phase conditions as described by P. R. Ashton in et al. in J.Org.Chem. 1998, 63, 3429-3437.
  • the considerably larger polymer can be separated from low molecular components, such as excess monomers and by-products, using size exclusion chromatography, optionally in combination with dialysis as described by E.R.Gillies and J.M.J. Frechet in J. Am. Chem. Soc. 2002, 124, 14137-14146.
  • a convergent solution phase synthesis is used.
  • solution phase also makes it possible to use the convergent approach for assembly of branched polymers as described above and further reviewed by S.M.Grayson and J. M. J. Frechet in Chem. Rev. 2001 , IQl, 3819-3867.
  • protection groups for the functional moiety depends on the actually functional group. For example, if A' in general formula IVb or IVc is a carboxyl group, a terf-butyl ester derivate that can be removed by TFA would be an appropriate choice.
  • Suitable protection groups are known to the skilled person, and other examples can be found in Green & Wuts "Protection groups in organic synthesis", 3 rd edition, Wiley-interscience. The convergent assembly of branched polymers is illustrated in reaction scheme 1 and reaction scheme 2. The rection schemes can be found below.
  • a tert- butyl ester functionality (A') is prepared by reaction of a suitable precurser with terf-butyl ⁇ - bromoacetate.
  • the terminal end groups (B') are manipulated in such way that they allow for the acylation of step (iii) with a carboxylic acid that is converted into an acyl halid in step (iv).
  • the terf-butyl ester functionality (A') is removed creating an end (B') capped monomer.
  • This end capped monomer serves as starting material for preparing the second generation product in reaction scheme 2, where two equivalents are used in an acylation reaction with the product of step (ii) in reaction scheme 1.
  • the product of this reaction is a new terf-butyl ester, which after deprotection can re-enter in the initial step of reaction scheme 2 in an iterative manner creating higher generation materials.
  • the branched polymer must be provided with a reactive handle, i.e., furnished with a reactive functional group, examples of which include carboxylic acids, primary amino groups, hydrazides, O-alkylated hydroxylamines, thiols, succinates, succinimidyl succinates, succimidyl proprionates, succimidyl carboxymethylates, hydrazides arylcarbonates and aryl carbamates such as nitrophenylcarbamates and nitrophenyl carbonates, chlorocarbonates, isothiocyanates, isocyanates, malemides, and activated esters such as:
  • the conjugation of the branched polymer to ITA is conducted by conventional methods, known to the skilled artisan.
  • the skilled person will be aware that the activation method and/or conjugation chemistry (for example, choice of reaction groups ect.) to be use depends on the attachment group(s) selected on the ITA (for example, amino groups, hydroxyl groups, thiol groups ect.) and the branched polymer (for example, succimidyl proprionates, nitrophenylcarbonates, malimides, vinylsulfones, haloacetates ect.).
  • suitable attachment moieties on the branched polymer such as those mentioned above, are created after the branched polymer has been assembled using conventional solution phase chemistry.
  • Embodiments of this invention illustrating different ways to create nucleophilic attachment moieties on a branched polymer containing a carboxylic acid group are listed in reaction scheme 7.
  • insulinotropic agent may initially be acylated with formyl derivated carboxyl acids, for example, using activation such as N-hydroxysuccinimide esters, 1 - hydroxybenzotriazol esters and the like.
  • the resulting ITA carrying an aldehyde functionality may then in turn be condensed with mono-, oligo- or polymeric building blocks of the invention suitable derivatized as, for example, O-substituted hydroxylamines, hydrazines or hydrazides, by mixing the two components in an aqueous media, optionally containing organic co-solvents at neutral, acid or alkaline pH.
  • L 4 is a valence bond
  • the divalent radical L 3 in the general formula I contains an oxime group.
  • Representative non limiting examples of the moiety L 4 plus the adjacent L 3 include (as syn and anti forms):
  • L 4 includes (as syn and anti forms):
  • insulinotropic agent may be derivatized with a moiety that after a chemical reaction, such as, for example, a periodate oxidation, may generate an ITA molecule containing an aldehyde functionality.
  • the ITA carrying an aldehyde functionality may then as above be condensed with mono-, oligo- or polymeric building blocks of the invention similarly derivatized as, for example, O-substituted hydroxylamines, hydrazines or hydrazides, by mixing the two components in an aqueous media, optionally containing organic co-solvents at neutral, acid or alkaline pH.
  • a particular example is an initial acylation of ⁇ -amino group on lysine with serine, followed by a periodate cleavage to generate glyoxyl derived ITA.
  • representative non limiting examples of the divalent L 4 moiety plus the adjacent L 3 moiety include (as syn and anti forms):
  • L 3 may also be a divalent radical according to the definitions, and L 4 may be an oxyiminoalkylcarbonyl group.
  • Representative non limiting examples includes (as syn and anti forms):
  • the biologically active ITA is reacted with the activated branched polymers in an aqueous reaction medium which is optionally buffered, depending upon the pH requirements of the ITA.
  • the optimum pH value for the reaction is generally between about 6.5 and about 8. In an embodiment about 7.4 for most ITA analogues.
  • the optimum reaction conditions for the insulinotropic agent (ITA) stability, reaction efficiency, etc. is within level of ordinary skill in the art.
  • the temperature range is from about 4 0 C to about 37 0 C.
  • the temperature of the reaction medium cannot exceed the temperature at which the insulinotropic agent (ITA) may denature or decompose.
  • the insulinotropic agent (ITA) is reacted with an excess of the activated branched polymer.
  • the conjugate is recovered and purified such as by diafiltration, column chromatography including size exclusion chromatography, ion-exchange chromatograph, affinity chromatography, electrophoreses, or combinations thereof, or the like.
  • the method of conjugation is based upon standard chemistry, which is performed in the following manner.
  • the branched polymer has an aminooxyacetyl group attached during synthesis, for example, by acylation of diaminoalkyl linked aminooxyacetic acid as depicted in reaction scheme 7.
  • the ITA has a terminal serine or threonine residue, which is oxidised to a glyoxylyl group under mild conditions with periodate according to Rose in J. Am. Chem. Soc. 1994, V ⁇ 6, 30-33, and European patent 0243929.
  • an aldehyde function may be introduced by acylating an exposed amino group such as an epsilon amino group of a lysin residue with an acylating moiety containing an aldehyde or a temporarily protected aldehyd group.
  • the aminooxy component of the branched polymer and the aldehyde component of the ITA are mixed in approximately equal proportions at a concentration in the range from about 1 to about 10 mM in aqueous solution at mildly acid conditions, for example at a pH value in the range from about 2 to about 5, especially at around room temperature, and the conjugation reaction (in this case oximation) is followed by reversed phase high pressure liquid chromatography (HPLC) and electrospray ionisation mass spectrometry (ES-MS).
  • HPLC high pressure liquid chromatography
  • ES-MS electrospray ionisation mass spectrometry
  • the reaction speed depends on concentrations, pH value, and steric factors but is normally at equilibrium within a few hours, and the equilibrium is greatly in favour of conjugate (Rose, et al., Bioconjugate Chemistry 1996, 7, 552-556).
  • ITA analogues are purified, for example, by reversed phase HPLC (Rose, J Am. Chem.Soc, supra and Rose, et al., Bioconjugate Chemistry, supra).
  • the method of conjugation is performed in the following manner:
  • the branched polymer is synthesised on the Sasrin or Wang resin (Bachem) as depicted in reaction scheme 3.
  • the branched polymer is cleaved from the resin by repeated treatment with TFA in dichloromethane and the solution of cleaved polymer is neutralised with pyridine in methanol.
  • the carboxyl group which was connected to the resin is activated (for example, with HBTU, TSTU or HATU) and coupled to a nucleophilic group (such as an amino group, i.e., an epsilon amino group on the side chain of lysin) on the insulinotropic agent (ITA) by standard techniques of peptide chemistry.
  • ITA insulinotropic agent
  • the modified target molecule or material can be purified from the reaction mixture by one of numerous purification methods that are well known to those of ordinary skill in the art such as size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, preparative isoelectric focusing, etc.
  • purification methods such as size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, preparative isoelectric focusing, etc.
  • General methods and principles for macromolecule purification, particularly peptide purification can be found, for example, in "Protein Purification: Principles and Practice” by Seeres, 2 nd edition, Springer-Verlag, New York, NY, (1987), which is incorporated herein by reference.
  • GLP-1 analogues or GLP-1 derivatives used for preparing the compounds of this invention are known (see L. B. Knudsen et al., J. Med. Chem. 2000, 43, 1664-1669, for a series of non-limited examples) and other can be prepared analogously with the preparation of the known compounds or by other methods which will be obvious for the skilled art worker.
  • insulinotropic agent includes GLP-1 analogues, which are suitable for conjugation with the branched polymers. It is to be understood that insulinotropic agent and analogues not specifically mentioned but having suitable properties are also intended and are within the scope of the present invention.
  • water soluble polymers are provided. These are important as agents for enhancing the properties of the GLP-1 analogues. For example, by conjugating water soluble polymers to GLP-1 analogues to increased solubility.
  • the attachment of a branched polymer to GLP-1 analogues, that have inherent immunogenic properties provides conjugates with decreased immune response compared to the immune response generated by the non conjugated GLP-1 analogues, or an increased pharmacokinetic profile, an increased shelf-life, and an increased biological half-life.
  • This invention provides GLP-1 analogues which are modified by the attachment of the hydrophilic water soluble branched polymers without substantially reducing or interfering with the biologic activity of the non modified GLP-1 analogues.
  • This invention GLP-1 analogues modified by the structurally well defined polymers, which are essentially homogeneous compounds, wherein the number of generations of the branched polymer is well defined.
  • This invention provides conjugates which have maintained the biological activity of the non conjugated GLP-1 .
  • the conjugated GLP-1 has improved characteristics compared to the non-conjugated GLP-1.
  • the branched polymers conjugated to certain parts of GLP-1 reduce the bioavailability, the potency, and the efficacy or the activity of GLP- 1 . Such reduction can be desirable in drug delivery systems based on the sustain release principle.
  • a sustain release principle in which the branched polymer is used in connection with a linker that can be cleaved under physiological conditions, thereby releasing the bio-active GLP-1 slowly from the branched polymer, is contemplated.
  • the GLP-1 may not be biological active before the branched polymer is removed.
  • the cleavable linker is a small peptide that can function as a substrate for, for example, proteases present in the blood serum.
  • the polymer conjugation is designed so as to produce the optimal molecule with respect to the number of polymer molecules attached, the size, and composition (for example, number of generations and particular monomer used in each generation), and the attachment site(s) on GLP-1 .
  • the particular molecular weight of the branched polymer to be used may, for example, be chosen on the basis of the desired effect to be achieved. For instance, if the primary purpose of the conjugate is to achieve a conjugate having a high molecular weight (for example, to reduce renal clearance), it is usually desirable to conjugate as few high molecular branched polymer molecules as possible to obtain the desirable molecular weight.
  • a branched polymer prepared as described herein is conjugated to GLP-1 .
  • this produces a conjugate with increased pulmonal bioavailability.
  • this produce a conjugate with increased pulmonary duration of action.
  • a branched polymer as described herein is used to shield immunogenic epitopes on biopharmaceutical GLP-1 obtained from non-human sources.
  • a branched water soluble polymer is conjugated to GLP-1 that in its unmodified state and under physiological conditions has a low solubility.
  • the in vivo half life of certain GLP-1 conjugates of this invention is improved by more than 10%. In an embodiment, the in vivo half life of certain GLP-1 conjugates is improved by more than 25%. In an embodiment, the in vivo half life of GLP-1 conjugates is improved by more than 50%. In an embodiment, the in vivo half life of certain GLP-1 conjugates is improved by more than 75%. In an embodiment, the in vivo half life of certain GLP-1 conjugates is improved by more than 100%. In another embodiment, the in vivo half life of certain GLP-1 increased 250 % upon conjugation of a branched polymer.
  • the functional in vivo half life of certain GLP-1 conjugates of this invention is improved by more than 10%. In another embodiment, the functional in vivo half life of certain GLP-1 conjugates is improved by more than 25%. In another embodiment, the functional in vivo half life of certain GLP-1 conjugates is improved by more than 50%. In another embodiment, the functional in vivo half life of certain GLP-1 conjugates is improved by more than 75%. In another embodiment, the functional in vivo half life of certain GLP-1 conjugates is improved by more than 100%. In another embodiment, the functional half life of certain GLP-1 is increased 250 % upon conjugation of a branched polymer.
  • GLP-1 analogues in solution are very poor. Therefore, in one embodiment of this invention, well defined water soluble branched polymers as described herein can conjugate GLP-1 analogues and stabilize the GLP-1 by minimizing structural transformations such as refolding and maintain GLP-1 activity. In a related embodiment, the shelf-half life of GLP-1 is improved upon conjugation to a branched polymer as described herein.
  • the insulinotropic agent is a DPPIV protected peptide.
  • the insulinotropic agent has an EC 50 of less than 1 nM as determined by the functional receptor assay disclosed herein. In another embodiment of the invention the insulinotropic agent has an EC 50 of less than 300 pM, less than 200 pM or less than 100 pM as determined by the functional receptor assay disclosed herein.
  • Reaction scheme 2 Second generation with protected focal point
  • Reaction scheme 3 Solid phase synthesis of a second generation branched polymer
  • Reaction scheme 4 Divergent synthesis of a second generation material in solution
  • Reaction scheme 5 illustration of end capping of a second generation polymer using a Me(PEG)2CH2COOH acid.
  • Reaction scheme 6 illustration of end capping of a second generation polymer attatched to a solid support or an insulinotropic agent (R) using succinic acid mono tert butyl ester to create a poly anionic glyco mimic polymer.
  • Reaction scheme 7 Formation of suitable reactive handles for polymer conjugation to ITA molecules. Illustrated for a second generation polymer material.
  • the conjugated GLP-1 analogues of this invention of formula I can, for example, be administered subcutaneously, orally, or pulmonary.
  • the compounds of formula I are formulated analogously with the formulation of known GLP-1 analogues.
  • the compounds of formula I are administered analogously with the administration of known GLP-1 analogues and, generally, the physicians are familiar with this procedure.
  • the compounds of formula I are formulated analogously with the formulation of other medicaments which are to be administered orally.
  • the compounds of formula I are administered analogously with the administration of known oral medicaments and, principally, the physicians are familiar with such procedure.
  • pulmonary products the following details are given:
  • the conjugated GLP-1 analogues of this invention may be administered by inhalation in a dose effective manner to increase circulating GLP-1 levels and/or to lower circulating glucose levels. Such administration can be effective for treating disorders such as diabetes or hyperglycemia. Achieving effective doses of GLP-1 requires administration of an inhaled dose of more than about 0.5 ⁇ g/kg to about 50 ⁇ g/kg of conjugated GLP-1 of this invention. A therapeutically effective amount can be determined by a knowledgeable practitioner, who will take into account factors including GLP-1 level, blood glucose levels, the physical condition of the patient, the patient's pulmonary status, or the like. According to the invention, conjugated GLP-1 of this invention may be delivered by inhalation to achieve rapid absorption thereof.
  • inhalation can result in pharmacokinetics comparable to subcutaneous administration of GLP-1 analogues.
  • Inhalation of a conjugated GLP-1 of this invention leads to a rapid rise in the level of circulating GLP-1 followed by a rapid fall in blood glucose levels.
  • Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.
  • conjugated GLP-1 of this invention may be delivered by any of a variety of inhalation devices known in the art for administration of a therapeutic agent by inhalation. These devices include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. In an embodiment conjugated GLP-1 of this invention is delivered by a dry powder inhaler or a sprayer.
  • an inhalation device for administering conjugated GLP-1 of this invention is advantageously reliable, reproducible, and accurate.
  • the inhalation device should deliver small particles, for example, less than about 10 ⁇ m, for example about 1 -5 ⁇ m, for good respirability.
  • Some specific examples of commercially available inhalation devices suitable for the practice of this invention are TurbohalerTM (Astra), Rotahaler ® (Glaxo), Diskus ® (Glaxo), SpirosTM inhaler (Dura), devices marketed by Inhale Therapeutics, AEFtxTM (Aradigm), the Ultravent ® nebulizer (Mallinckrodt), the Acorn II ® nebulizer (Marquest Medical Products), the Ventolin ® metered dose inhaler (Glaxo), the Spinhaler ® powder inhaler (Fisons), or the like.
  • the formulation of conjugated GLP-1 of this invention, the quantity of the formulation delivered, and the duration of administration of a single dose depend on the type of inhalation device employed.
  • the frequency of administration and length of time for which the system is activated will depend mainly on the concentration of GLP-1 conjugate in the aerosol. For example, shorter periods of administration can be used at higher concentrations of GLP-1 conjugate in the nebulizer solution.
  • Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods to deliver the desired amount of GLP-1 conjugate.
  • Devices such as powder inhalers deliver active agent until a given charge of agent is expelled from the device. In this type of inhaler, the amount of conjugated GLP-1 of this invention in a given quantity of the powder determines the dose delivered in a single administration.
  • the particle size of conjugated GLP-1 of this invention in the formulation delivered by the inhalation device is critical with respect to the ability of GLP-1 to make it into the lungs. In an embodiment into the lower airways or alveoli. In an embodiment the conjugated GLP-1 of this invention is formulated so that at least about 10% of the GLP-1 conjugate delivered is deposited in the lung. In an embodiment about 10 to about 20%, or more. It is known that the maximum efficiency of pulmonary deposition for mouth breathing humans is obtained with particle sizes of about 2 ⁇ m to about 3 ⁇ m. When particle sizes are above about 5 m ⁇ , pulmonary deposition decreases substantially. Particle sizes below about 1 ⁇ m cause pulmonary deposition to decrease, and it becomes difficult to deliver particles with sufficient mass to be therapeutically effective.
  • particles of GLP- 1 conjugate delivered by inhalation have a particle size less than about 10 ⁇ m. In an embodiments in the range of about 1 ⁇ m to about 5 ⁇ m.
  • the formulation of GLP-1 conjugate is selected to yield the desired particle size in the chosen inhalation device.
  • conjugated GLP-1 of this invention is prepared in a particulate form with a particle size of less than about 10 ⁇ m. In an embodiment about 1 to about 5 ⁇ m. In an embodiment the particle size is effective for delivery to the alveoli of the patient's lung. In an embodiment the dry powder is largely composed of particles produced so that a majority of the particles have a size in the desired range. In an embodiment at least about 50% of the dry powder is made of particles having a diameter less than about 10 ⁇ m. Such formulations can be achieved by spray drying, milling, or critical point condensation of a solution containing GLP-1 conjugate and other desired ingredients. Other methods also suitable for generating particles useful in the current invention are known in the art.
  • the particles are usually separated from a dry powder formulation in a container and then transported into the lung of a patient via a carrier air stream.
  • a dry powder inhaler typically, the force for breaking up the solid is provided solely by the patient's inhalation.
  • One suitable dry powder inhaler is the TurbohalerTM manufactured by Astra (S ⁇ dertalje, Sweden).
  • air flow generated by the patient's inhalation activates an impeller motor which deagglomerates the monomeric GLP-1 analogue particles.
  • the Dura SpirosTM inhaler is such a device.
  • Formulations of conjugated GLP-1 of this invention for administration from a dry powder inhaler typically include a finely divided dry powder containing GLP-1 conjugate, but the powder can also include a bulking agent, carrier, excipient, another additive, or the like.
  • Additives can be included in a dry powder formulation of GLP-1 conjugate, for example, to dilute the powder as required for delivery from the particular powder inhaler, to facilitate processing of the formulation, to provide advantageous powder properties to the formulation, to facilitate dispersion of the powder from the inhalation device, to stabilize the formulation (for example, antioxidants or buffers), to provide taste to the formulation, or the like.
  • the GLP-1 conjugate can be mixed with an additive at a molecular level or the solid formulation can include particles of the GLP-1 conjugate mixed with or coated on particles of the additive.
  • Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like.
  • an additive such as a bulking agent
  • an additive is present in an amount effective for a purpose described above, often at about 50% to about 90% by weight of the formulation.
  • Additional agents known in the art for formulation of a protein such as GLP-1 analogue protein can also be included in the formulation.
  • a spray including conjugated GLP-1 of this invention can be produced by forcing a suspension or solution of GLP-1 conjugate through a nozzle under pressure.
  • the nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size.
  • An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed.
  • particles of GLP- 1 conjugate delivered by a sprayer have a particle size less than about 10 ⁇ m. In an embodiment in the range of about 1 ⁇ m to about 5 ⁇ m.
  • Formulations of conjugated GLP-1 of this invention suitable for use with a sprayer typically include GLP-1 conjugate in an aqueous solution at a concentration of about 1 mg to about 20 mg of GLP-1 conjugate per ml of solution.
  • the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant. In an embodiment the formulations contain zinc.
  • the formulation can also include an excipient or agent for stabilization of the GLP-1 conjugate, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in formulating GLP-1 conjugates include albumin, protamine, or the like.
  • Typical carbohydrates useful in formulating GLP-1 conjugates include sucrose, mannitol, lactose, trehalose, glucose, or the like.
  • the GLP-1 conjugate formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the GLP-1 conjugate caused by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation.
  • the surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like.
  • Conjugated GLP-1 of this invention can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
  • a nebulizer such as jet nebulizer or an ultrasonic nebulizer.
  • a compressed air source is used to create a high-velocity air jet through an orifice.
  • a low-pressure region is created, which draws a solution of GLP-1 conjugate through a capillary tube connected to a liquid reservoir.
  • the liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol.
  • a range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer.
  • an ultrasonic nebulizer high- frequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer. This energy is transmitted to the formulation of GLP-1 conjugate either directly or through a coupling fluid, creating an aerosol including the GLP-1 conjugate.
  • particles of GLP-1 conjugate delivered by a nebulizer have a particle size less than about 10 ⁇ m. In an embodiment in the range of about 1 ⁇ m to about 5 ⁇ m.
  • Formulations of GLP-1 conjugate suitable for use with a nebulizer, either jet or ultrasonic typically include GLP-1 conjugate in an aqueous solution at a concentration of about 1 mg to about 20 mg of GLP-1 conjugate per ml of solution.
  • the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant.
  • the formulation can also include an excipient or agent for stabilization of the GLP-1 conjugate, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in formulating GLP-1 conjugates include albumin, protamine, or the like.
  • Typical carbohydrates useful in formulating GLP-1 conjugates include sucrose, mannitol, lactose, trehalose, glucose, or the like.
  • the GLP-1 conjugate formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the GLP-1 conjugate of this invention caused by atomization of the solution in forming an aerosol.
  • Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation
  • the surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as GLP-1 analogue protein can also be included in the formulation.
  • a propellant, an GLP-1 conjugate of this invention, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol.
  • a formulation of GLP-1 conjugate of this invention produced by various methods known to those of skill in the art, including jet-milling, spray drying, critical point condensation, or the like.
  • the metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.
  • Formulations of a GLP-1 conjugate of this invention for use with a metered-dose inhaler device will generally include a finely divided powder containing GLP-1 conjugate of this invention as a suspension in a non aqueous medium, for example, suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1 ,1 ,1 ,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.
  • the propellant is a hydrofluorocarbon.
  • the surfactant can be chosen to stabilize the GLP-1 conjugate of this invention as a suspension in the propellant, to protect the active agent against chemical degradation, and the like.
  • Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like.
  • solution aerosols are using solvents such as ethanol. Additional agents known in the art for formulation of a protein such as GLP-1 analogue protein can also be included in the formulation.
  • the present invention also relates to a pharmaceutical composition or formulation including an GLP-1 conjugate of this invention and suitable for administration by inhalation.
  • an GLP-1 conjugate of this invention can be used for manufacturing a formulation or medicament suitable for administration by inhalation.
  • the invention also relates to methods for manufacturing formulations including an GLP-1 conjugate of this invention in a form that is suitable for administration by inhalation.
  • a dry powder formulation can be manufactured in several ways, using conventional techniques. Particles in the size range appropriate for maximal deposition in the lower respiratory tract can be made by micronizing, milling, spray drying, or the like.
  • a liquid formulation can be manufactured by dissolving an GLP-1 conjugate of this invention in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients.
  • this invention relates to a method of administering a conjugated insulin of formula Il comprising administering an effective amount of the conjugated insulin of formula Il to a patient in need thereof by pulmonary means;
  • said conjugated insulin of formula Il is inhaled through the mouth of said patient.
  • this invention also relates to the following embodiments: a) The method as described herein, wherein the conjugated GLP1 analogue of formula I is delivered to a lower airway of the patient. b) The method as described herein, wherein the conjugated GLP1 analogue of formula I is deposited in the alveoli. c) The method as described herein, wherein the conjugated GLP1 analogue of formula I is administered as a pharmaceutical formulation comprising the conjugated GLP1 analogue of formula I in a pharmaceutically acceptable carrier. d) The method as described herein, wherein the formulation is selected from the group consisting of a solution in an aqueous medium and a suspension in a non-aqueous medium.
  • the device is selected from the group consisting of a nebulizer, a metered-dose inhaler, a dry powder inhaler, and a sprayer, m) The method as described herein, wherein the device is a dry powder inhaler, n) The method as described herein, wherein the device is a nebulizer, o) The method as described herein, wherein the device is a metered-dose inhaler, p) The method as described herein, wherein the device is a sprayer.
  • One object of the present invention is to provide a pharmaceutical formulation comprising a compound according to the present invention which is present in a concentration from about 0.1 mg/ml to about 25 mg/ml, and wherein said formulation has a pH from 2.0 to 10.0.
  • the formulation may further comprise a buffer system, preservative(s), isotonicity agent(s), chelating agent(s), stabilizers and surfactants.
  • the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water. Such formulation is typically a solution or a suspension.
  • the pharmaceutical formulation is an aqueous solution.
  • aqueous formulation is defined as a formulation comprising at least 50 %w/w water.
  • aqueous solution is defined as a solution comprising at least 50 %w/w water
  • aqueous suspension is defined as a suspension comprising at least 50 %w/w water.
  • the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use.
  • the pharmaceutical formulation is a dried formulation (for example, freeze-dried or spray-dried) ready for use without any prior dissolution.
  • the invention in a further aspect relates to a pharmaceutical formulation
  • a pharmaceutical formulation comprising an aqueous solution of a compound according to the present invention, and a buffer, wherein said compound is present in a concentration from 0.1 mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0.
  • the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention the pH of the formulation is from about 3.0 to about 7.0. In another embodiment of the invention the pH of the formulation is from about 5.0 to about 7.5. In another embodiment of the invention the pH of the formulation is from about 7.5 to about 9.0. In another embodiment of the invention the pH of the formulation is from about 7.5 to about 8.5. In another embodiment of the invention the pH of the formulation is from about 6.0 to about 7.5. In another embodiment of the invention the pH of the formulation is from about 6.0 to about 7.0.
  • the pH of the formulation is from about 3.0 to about 9.0, and said pH is at least 2.0 pH units from the isoelectric pH of compound of the present invention.
  • the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginin, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof.
  • Each one of these specific buffers constitutes an alternative embodiment of the invention.
  • the formulation further comprises a pharmaceutically acceptable preservative.
  • the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p- hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p- hydroxybenzoate, benzethonium chloride, chlorphenesine (3p-chlorphenoxypropane-1 ,2-diol) or mixtures thereof.
  • the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of the invention.
  • the use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • the formulation further comprises an isotonic agent.
  • the isotonic agent is selected from the group consisting of a salt (for example, sodium chloride), a sugar or sugar alcohol, an amino acid (for example, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (for example, glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3- propanediol, 1 ,3-butanediol) polyethyleneglycol (for example, PEG400), or mixtures thereof.
  • a salt for example, sodium chloride
  • a sugar or sugar alcohol an amino acid (for example, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol
  • Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.
  • the sugar additive is sucrose.
  • Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galacititol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol additive is mannitol.
  • the sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of the invention.
  • the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml.
  • the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of the invention.
  • the use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • the formulation further comprises a chelating agent.
  • the chelating agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof.
  • the chelating agent is present in a concentration from 0.1 mg/ml to 5mg/ml.
  • the chelating agent is present in a concentration from 0.1 mg/ml to 2mg/ml.
  • the chelating agent is present in a concentration from 2mg/ml to 5mg/ml.
  • Each one of these specific chelating agents constitutes an alternative embodiment of the invention.
  • the use of a chelating agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • the formulation further comprises a stabiliser.
  • a stabilizer in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.
  • compositions of the invention are stabilized liquid pharmaceutical compositions whose therapeutically active components include a polypeptide that possibly exhibits aggregate formation during storage in liquid pharmaceutical formulations.
  • aggregate formation is intended a physical interaction between the polypeptide molecules that results in formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution.
  • a liquid pharmaceutical composition or formulation once prepared is not immediately administered to a subject. Rather, following preparation, it is packaged for storage, either in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject.
  • dried form is intended the liquid pharmaceutical composition or formulation is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and PoIIi (1984) J. Parenteral Sci. Technol.
  • compositions of the invention may further comprise an amount of an amino acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition.
  • amino acid base is intended an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. Where a combination of amino acids is used, all of the amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their salt forms.
  • amino acids to use in preparing the compositions of the invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid, and glutamic acid.
  • Any stereoisomer i.e., L, D, or DL isomer
  • a particular amino acid for example, glycine, methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof
  • a particular amino acid for example, glycine, methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof
  • the L-stereoisomer is used.
  • Compositions of the invention may also be formulated with analogues of these amino acids.
  • amino acid analogue is intended a derivative of the naturally occurring amino acid that brings about the desired effect of decreasing aggregate formation by the polypeptide during storage of the liquid pharmaceutical compositions of the invention.
  • Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine
  • suitable methionine analogues include S-ethyl homocysteine and S-butyl homocysteine
  • suitable cystein analogues include S-methyl-L cystein.
  • the amino acid analogues are incorporated into the compositions in either their free base form or their salt form.
  • the amino acids or amino acid analogues are used in a concentration, which is sufficient to prevent or delay aggregation of the protein.
  • methionine or other sulphur containing amino acids or amino acid analogous
  • methionine may be added to inhibit oxidation of methionine residues to methionine sulfoxide when the polypeptide acting as the therapeutic agent is a polypeptide comprising at least one methionine residue susceptible to such oxidation.
  • inhibit is intended minimal accumulation of methionine oxidized species over time. Inhibiting methionine oxidation results in greater retention of the polypeptide in its proper molecular form.
  • any stereoisomer of methionine (L, D, or DL isomer) or combinations thereof can be used.
  • the amount to be added should be an amount sufficient to inhibit oxidation of the methionine residues such that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Generally, this can be achieved by adding methionine such that the ratio of methionine added to methionine residues ranges from about 1 :1 to about 1000:1 , such as 10:1 to about 100:1 .
  • the formulation further comprises a stabiliser selected from the group of high molecular weight polymers or low molecular compounds.
  • the stabilizer is selected from polyethylene glycol (for example, PEG 3350), polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates thereof (for example, HPC, HPC-SL, HPC-L and
  • HPMC HPMC
  • cyclodextrins cyclodextrins
  • sulphur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol
  • salts for example, sodium chloride
  • compositions may also comprise additional stabilizing agents, which further enhance stability of a therapeutically active polypeptide therein.
  • Stabilizing agents of particular interest to the present invention include, but are not limited to, methionine and EDTA, which protect the polypeptide against methionine oxidation, and a nonionic surfactant, which protects the polypeptide against aggregation associated with freeze-thawing or mechanical shearing.
  • the formulation further comprises a surfactant.
  • the surfactant is selected from a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (for example, poloxamers such as Pluronic ® F68, poloxamer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, for example, Tween-20, Tween-40, Tween-80 and Brij- 35), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lecitins and phospholipids (for example, phosphatidyl serine, phosphatidyl choline, phosphatidyl
  • Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (for example human serum albumin, gelatin or proteins) and a zwitterion (for example an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine).
  • additional ingredients should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.
  • compositions containing a compound according to the present invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen.
  • topical sites for example, skin and mucosal sites
  • sites which bypass absorption for example, administration in an artery, in a vein, in the heart
  • sites which involve absorption for example, administration in the skin, under the skin, in a muscle or in the abdomen.
  • Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment.
  • routes of administration for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to Formula (I), and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is suited for pulmonary administration.
  • the present invention relates to the use of a compound of formula (I) for the preparation of a pulmonary medicament.
  • compositions of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants.
  • solutions for example, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses,
  • compositions of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the compound, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof.
  • carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, polyvinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions there of, well known to those skilled in the art of phase behaviour in lipid-water systems, polymeric micelles, multiple emulsions, self- emulsifying, self-microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.
  • polymers for example cellulose and derivatives, polysaccharides, for example dextran and derivative
  • compositions of the current invention are useful in the formulation of solids, semisolids, powder and solutions for pulmonary administration of the compound, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art.
  • compositions of the current invention are specifically useful in the formulation of controlled, sustained, protracting, retarded, and slow release drug delivery systems. More specifically, but not limited to, compositions are useful in formulation of parenteral controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), well known to those skilled in the art. I In an embodiment controlled release and sustained release systems are administered subcutaneous.
  • examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles,
  • Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co- cystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenization, encapsulation, spray drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes.
  • General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D.L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, EJ. , ed. Marcel Dekker, New York, 2000).
  • Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • parenteral administration can be performed by means of an infusion pump.
  • a further option is a composition which may be a solution or suspension for the administration of the compound according to the present invention in the form of a nasal or pulmonal spray.
  • the pharmaceutical compositions containing the compound of the invention can also be adapted to transdermal administration, for example, by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, for example, buccal, administration.
  • stabilized formulation refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.
  • physical stability of the protein formulation as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces.
  • Physical stability of the aqueous protein formulations is evaluated by means of visual inspection and/or turbidity measurements after exposing the formulation filled in suitable containers (for example, cartridges or vials) to mechanical/physical stress (for example, agitation) at different temperatures for various time periods. Visual inspection of the formulations is performed in a sharp focused light with a dark background.
  • the turbidity of the formulation is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a formulation showing no turbidity corresponds to a visual score 0, and a formulation showing visual turbidity in daylight corresponds to visual score 3).
  • a formulation is classified physical unstable with respect to protein aggregation, when it shows visual turbidity in daylight.
  • the turbidity of the formulation can be evaluated by simple turbidity measurements well-known to the skilled person. Physical stability of the aqueous protein formulations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. In an embodiment the probe is a small molecule.
  • Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.
  • hydrophobic patch probes that bind to exposed hydrophobic patches of a protein.
  • the hydrophobic patches are generally buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature.
  • these small molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthroline or the like.
  • spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.
  • chemical stability of the protein formulation refers to chemical covalent changes in the protein structure leading to formation of chemical degradation products with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure.
  • chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation can most probably not be completely avoided and increasing amounts of chemical degradation products is often seen during storage and use of the protein formulation as well- known by the person skilled in the art.
  • Most proteins are prone to deamidation, a process in which the side chain amide group in glutaminyl or asparaginyl residues is hydrolysed to form a free carboxylic acid.
  • a “stabilized formulation” refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.
  • a formulation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.
  • the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 6 weeks of usage and for more than 3 years of storage.
  • the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 4 weeks of usage and for more than 3 years of storage. In a further embodiment of the invention the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 4 weeks of usage and for more than two years of storage.
  • the pharmaceutical formulation comprising the compound is stable for more than 2 weeks of usage and for more than two years of storage.
  • the present invention relates to the use of a compound according to the invention for the preparation of a medicament.
  • a compound according to the invention is used for the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers.
  • a compound according to the invention is used for the preparation of a medicament for delaying or preventing disease progression in type 2 diabetes.
  • a compound according to the invention is used for the preparation of a medicament for decreasing food intake, decreasing ⁇ -cell apoptosis, increasing ⁇ -cell function and ⁇ -cell mass, and/or for restoring glucose sensitivity to ⁇ -cells.
  • the treatment with a compound according to the present invention may also be combined with combined with a second or more pharmacologically active substances, for example, selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.
  • a second or more pharmacologically active substances for example, selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.
  • Examples of these pharmacologically active substances are: Insulin, sulphonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenosis, glucose uptake modulators, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the ⁇ - cells; Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide; ⁇ -blockers such as alprenolol, atenolol,
  • GLP-1 conjugates were generally analysed using following HPLC systems:
  • HPLC Method A: The RP-analysis was performed using a Waters 2690 systems fitted with a Waters 996 diode array detector. UV detections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6 mm x 250 mm 5 ⁇ C-18 silica column (The Seperations Group, Hesperia), which was eluted at 1 ml/min at 42 0 C. The column was equilibrated with 10% of a 0,5 M ammonium sulfate, which was adjusted to pH 2.5 with 4M sulfuric acid. After injection, the sample was eluted by a gradient of 0% to 60% acetonitrile in the same aqueous buffer during 50 min.
  • LC-MS (Method A) analysis was performed on HP1 100 MSD equipped with binary pump, column compartment, diode array detector and a single quadropole massspectrometer detector. The analysis was performed at 40 C on Waters Xterra MS C-18 X 3 mm column with a linear gradient from 10% aqueous acetonitrile - 100% acetonitrile containing 0,01 %TFA running over 7.5 min. UV detection at 210nm and MS scanning range from 100-1000 amu.
  • LC-MS (Method B): was performed on a setup consisting of Hewlett Packard series 1100 G1312A Bin Pump, Hewlett Packard series 1100 Column compartment, Hewlett Packard series 1 100 G1315A DAD diode array detector, Hewlett Packard series 1100 MSD and Sedere 75 Evaporative Light Scattering detectorcontrolled by HP Chemstation software.
  • the HPLC pump was connected to two eluent reservoirs containing: (A) 1 OmM NH 4 OH in water and (B) 10mM NH 4 OH in 90% acetonitrile.
  • the analysis was performed at 23° C by injecting an appropriate volume of the sample (preferably 20 ⁇ l) onto the column which is eluted with a gradient of A and B.
  • the HPLC conditions, detector settings and mass spectrometer settings used was as follows: Column Waters Xterra MS C-18 X 3 mm id 5 urn Gradient 5% - 100% acetonitrile linear during 6.5 min at 1 .5ml/min Detection 210 nm (analogue output from DAD) ELS (analogue output from ELS) MS ionisation mode API-ES. Scan 100-1000 amu step 0.1 amu
  • CDI Carbonyldiimidazole
  • CH.CN Acetonitrile
  • DBU 1 ,8-Diazabicyclo[5,4,0]undec-7-ene
  • DIPEA ⁇ /, ⁇ /-Diisopropylethylamine
  • HOBt 1 -Hydroxybenzotriazole ivDde: 1 -(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl
  • TIS triisopropylsilane
  • TLC Thin Layer Chromatography
  • the protected peptidyl resin was synthesized according to the Fmoc strategy on an Applied Biosystems 431 A peptide synthesizer in 0.25 mmol or 1 .0 mmol scale using the manufacturer supplied FastMoc UV protocols which employ HBTU (2-(1 H-benzotriazol-1 -yl)- 1 ,1 ,3,3 tetramethyluronium hexafluorophosphate) or HATU (O-(7-azabenzotriazol-1 -yl)- 1 ,1 ,3,3-tetra-methyluronium hexafluorophosphate) mediated couplings in NMP (N-methyl pyrrolidone), and UV monitoring of the deprotection of the Fmoc protection group.
  • HBTU 2-(1 H-benzotriazol-1 -yl)- 1 ,1 ,3,3 tetramethyluronium hexafluorophosphate
  • HATU O-(7-
  • the starting resin used for the synthesis of the GLP-1 peptide amides was Rink-Amide resin and either Wang or chlorotrityl resin was used for GLP-1 peptides with a carboxy C-terminal.
  • the protected amino acid derivatives used were standard Fmoc-amino acids (supplied from for example, Anaspec, or Novabiochem) supplied in preweighed cartridges suitable for the ABI433A synthesizer with the exception of unnatural aminoacids such as Fmoc-Aib-OH (Fmoc-aminoisobutyric acid).
  • the N terminal amino acid was Boc protected at the alpha amino group (for example, Boc-His(Boc)OH was used for peptides with His at the N- terminal).
  • the epsilon amino group of the lysine which has to be further derivatized was either protected with Mtt, Mmt, Dde, ivDde, or Boc, depending on the route for attachment of the albumin binding moiety and spacer.
  • the resin (0.25 mmol) was placed in a manual shaker/filtration apparatus and treated with 2% TFA in DCM (20 ml, 5-10 min repeated 6-12 times) to remove the Mtt or Mmt group and wash with DCM (2x20 ml), 10%MeOH and 5% DIPEA in DCM (2x20ml) and N-methyl pyrrolidone (4x20 ml).
  • the branched polymers (4 molar equivalents relative to resin bound peptide) was dissolved in NMP/DCM (1 :1 ). Hydroxybenzotriazole (HOBt) (4 molar equivalents relative to resin bound peptide) and diisopropylcarbodiimide (4 molar equivalents relative to resin bound peptide) was added and the solution was stirred for 15 min. The solution was added to the resin and diisopropyethylamine (4 molar equivalents relative to resin bound peptide) was added. The resin was shaken 24 hours at room temperature. The resin was was washed with twice with NMP, twice with NMP/DCM (1 :1 ) and twice with DCM.
  • HOBt Hydroxybenzotriazole
  • diisopropylcarbodiimide (4 molar equivalents relative to resin bound peptide) was added and the solution was stirred for 15 min.
  • the solution was added to the resin and diisopropyethylamine (4 molar equivalents
  • the peptide was cleaved from the resin by stirring for 180 min at room temperature with a mixture of trifluoroacetic acid, water and triisopropylsilane (95:2.5:2.5). The cleavage mixture was filtered and the filtrate was concentrated to an oil by a stream of nitrogen. The crude peptide was precipitated from this oil with 45 ml diethyl ether and washed 3 times with 45 ml diethyl ether.
  • the peptide products is purified using one of the protocol described below (unless described otherwise in the example):
  • Preparative HPLC protocol in a TFA (acid) based solvent system The crude peptide was purified by semipreparative HPLC on a 20 mm x 250 mm column packed with 7 ⁇ C-18 silica. After drying the crude peptide was dissolved in 5 ml 50% acetic acid H 2 O and diluted to 20 ml with H 2 O and injected on the column which then was eluted with a gradient of 40-60 % CH 3 CN in 0.1 % TFA 10 ml/min during 50 min at 40 0 C. The peptide containing fractions were collected. The purified peptide was lyophilized after dilution of the eluate with water.
  • Preparative HPLC protocol in a ammonium sulphate (basic) based solvent system The crude peptide was purified by semipreparative HPLC on a 20 mm x 250 mm column packed with 7 ⁇ C-18 silica. The column was equilibrated with 40% CH 3 CN in 0.05M (NH 4 ) 2 SO 4 , which was adjusted to pH 2.5 with concentrated H 2 SO 4 .
  • the crude peptide was dissolved in 5 ml 50% acetic acid H 2 O and diluted to 20 ml with H 2 O and injected on the column which then was eluted with a gradient of 40% - 60% CH 3 CN in 0.05M (NH 4 ) 2 SO 4 , pH 2.5 at 10 ml/min during 50 min at 40 0 C.
  • the peptide containing fractions were collected and diluted with 3 volumes of H 2 O and passed through a Sep-Pak ® C18 cartridge (Waters part. #:51910 ) which has been equilibrated with 0.1 % TFA. It was then eluted with 70% CH 3 CN containing 0.1 % TFA and the purified peptide was isolated by lyophilisation after dilution of the eluate with water.
  • the final product obtained was characterised by analytical RP-HPLC (retention time) and by LCMS.
  • the RP-HPLC analysis was performed using UV detection at 214 nm and a Vydac 218TP54 4.6mm x 250mm 5 ⁇ C-18 silica column (The Separations Group, Hesperia,
  • 2-(2-Chloroethoxy)ethanol (100.00 g; 0.802 mol) was dissolved in dichloromethane (100 ml) and a catalytic amount of boron trifluoride etherate (2.28 g; 16 mmol) was added.
  • the clear solution was cooled to 0 5 C, and epibromohydrin (104.46 g; 0.762 mol) was added dropwise maintaining the temperature at 0 5 C.
  • the clear solution was stirred for an additional 3h at 0 5 C, then solvent was removed by rotary evaporation.
  • Trichloroacetylchloride (1 ,42 g, 7.85 mmol) was dissolved in THF (10 ml), and the solution was cooled to 0 5 C.
  • a solution of 1 ,3-bis[2-(2-azidoethoxy)ethoxy]propan-2-ol (1 .00 g; 3.3 mmol) and triethylamine (0,32 g, 3.3 mmol) in THF (5 ml) was slowly added drop wise over 10 min. Cooling was removed, and the resulting suspension was stirred for 6h at ambient temperature. The mixture was filtered, and the filtrate was evaporated to give a light brown oil. The oil was treated twice with acetonitrile following evaporation, and the product was used without further purification.
  • ⁇ /, ⁇ /-Bis(2-hydroxyethyl)-0-tert-butylcarbamate is dissolved in a polar, non-protic solvent such as THF or DMF.
  • Sodium hydride 60 % suspension in mineral oil
  • ⁇ /-(2-Bromoethyl)phthalimide is added slowly to the solution.
  • the mixture is stirred until the reaction is complete.
  • the reaction is quenched by slow addition of methanol.
  • Ethylacetate is added.
  • the solution is washed with aqueous sodium hydrogencarbonate.
  • the organic phase is dried, filtered, and subsequently concentrated under vacuum as much as possible.
  • the crude compound is purified by standard column chromatography.
  • N,N-Bis(2-(2-phthalimidoethoxy)ethyl)-O-tert-butylcarbamate is dissolved in a polar solvent such as ethanol. Hydrazine (or another agent known to remove the phthaloyl protecting group) is added. The mixture is stirred at room temperature (or if necessary elevated temperature) until the reaction is complete. The mixture is concentrated under vacuum as much as possible. The crude compound is purified by standard column chromatography or if possible by vacuum destination.
  • N,N-Bis(2-(2-aminoethoxy)ethyl)-O-tert-butylcarbamate is dissolved in a mixture of aqueous sodium hydroxide and THF or in a mixture of aqueous sodium hydroxide and acetonitrile.
  • Benzyloxychloroformate is added. The mixture is stirred at room temperature until the reaction is complete. If necessary, the volume is reduced in vacuo.
  • Ethyl acetate is added. The organic phase is washed with brine. The organic phase is dried, filtered, and subsequently concentrated in vacuo as much as possible.
  • the crude compound is purified by standard column chromatography.
  • 3,6,9-Trioxaundecanoic acid is dissolved in dichloromethane.
  • a carbodiimide for example NjN-dicyclohexylcarbodiimide or N,N-diisopropylcarbodiimide
  • the solution is stirred over night at room temperature.
  • the mixture is filtered.
  • the filtrate can be concentrated in vacuo if necessary.
  • the acylation of amines with the formed intramolecular anhydride is known from literature (for example Cook, R. M.; Adams, J. H.; Hudson, D. Tetrahedron Lett., 1994, 35, 6777-6780 or Stora, T.; Dienes, Z.; Vogel, H.; Duschl, C.
  • a solution of diglycolic anhydride in a non-protic solvent such as dichloromethane or N, N- dimethylformamide is added dropwise to a solution of bis(2-(2-phthalimidoethoxy)ethyl)amine in a non-protic solvent such as dichloromethane or N,N-dimethylformamide.
  • the mixture is stirred until the reaction is complete.
  • the crude compound is purified by extraction and subsequently standard column chromatography.
  • the oil was mixed with acetonitrile (250 ml) and the mixture was heated to reflux briefly. The solution was kept in fridge over night. The formed crystals were isolated by filtration. The isolated bright yellow crystals were dried in vacuum oven.
  • Phenyl chloroformate (54.1 g, 500 mmol) was added dropwise to a mixture of benzyl alcohol (78.3 g, 500 mmol), dichloromethane (90 ml) and pyridine (50 ml) in a 1 l-f lask with condenser and addition funnel. The mixture was stirred for 1 h. Water (125 ml) was added. The phases were separated. The organic phase was washed with dilute sulfuric acid (2 M, 2x125 ml). Brine had to be added in the final wash in order to obtain good separation. The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. The crude compound was vacuum destilled to yield a colourless liquid. Yield:104.3 g, 91 %
  • Benzyl phenylcarbonate (25,1 g, 1 10 mmol) was added dropwise to a solution of diethylenetriamine (5,16 g, 50 mmol) in dichloromethane (100 ml). The mixture was stirred for at least 20 h. The organic phase was washed with phosphate buffer (0.025 M K 2 HPO 4 ,
  • the formed compound was mixed with bis-(2-benzyloxycarbonylaminoethyl)ammonium chloride (2.8 g, 6.87 mmol) and ⁇ /, ⁇ /, ⁇ /', ⁇ /'-tetramethylguanidine (791 mg, 6.87mmol)(250 ml) in ⁇ /, ⁇ /-di- methylformamide (27 ml).
  • the resulting mixture was stirred for 20 h.
  • the mixture was concentrated in vacuo. Ethyl acetate (150 ml) and aqueous sodium hydrogencarbonate (5 % w/w, 150 ml) were added. The phases were separated.
  • the organic phase was extracted with aqueous sodium hydrogencarbonate (5 % w/w, 2 x 100 ml). The combined aqueous extracts were mixed with ethyl acetate (200 ml). Concentrated hydrochloric acid was added to the mixture until pH was 2-3. The phases were separated immidiately. The aqueous phase was extracted with ethyl acetate (2 x 200 ml). The combined organic extracts were dried with magnesium sulphate, filtered, and concentrated in vacuo to yield colourless syrup. Yield: 2.17 g, 55 %
  • Solid Phase Oligomerisation The reactions described below are all performed on polystyrene functionalised with the Wang linker. The reactions will in general also work on other types of solid supports, as well as with other types of functionalised linkers.
  • Solid phase azide reduction The reaction is known (Schneider, S. E. et al. Tetrahedron, 1998, 54(50) 15063-15086) and can be performed by treating the support bound azide with excess of triphenyl phosphine in a mixture of THF and water for 12-24 hours at room temperature.
  • trimethylphosphine in aqueous THF as described by Chan, T. Y. et al Tetrahedron Lett. 1997, 38(16), 2821 -2824 can be used.
  • Reduction of azides can also be performed on solid phase using sulfides such as dithiothreitol (Meldal, M. et al. Tetrahedron Lett.
  • the reaction is known and is usually performed by reacting an activated carbonate, or a halo formiate derivative with an amine, preferable in the presence of a base.
  • This example uses the 1 ,3-bis[2-(2-azidoethoxy)ethoxy]propan-2-yl-p-nitrophenylcarbonate monomer building block prepared in example 4 in the synthesis of a second generation carbamate based branched polymer capped with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid.
  • the coupling chemistry is based on standard solid phase carbamate chemistry, and the protection methodology is based on a solid phase azide reduction step as described above.
  • Step 1 Fmoc- ⁇ -Ala-Wang resin (100 mg; loading 0.31 mmol/g BACHEM) was suspended in dichloromethane for 30 min, and then washed twice with DMF. A solution of 20% piperidine in DMF was added, and the mixture was shaken for 15 min at ambient temperature. This step was repeated, and the resin was washed with DMF (3x) and DCM (3x).
  • Step 2 Coupling of monomer building blocks: A solution of 1 ,3-bis[azidoethoxyethyl]propan- 2-yl-p-nitrophenylcarbamate (527 mg; 1 ,4 mmol, 4x) was added to the resin together with DIPEA (240 ⁇ l; 1 ,4 mmol, 4x). The resin was shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
  • Step 3 Capping with acetic anhydride: The resin was then treated with a solution of acetic anhydride, DIPEA, DMF (12:4:48) for 10 min. at ambient temperature. Solvent was removed and the resin was washed with DMF (3x) and DCM (3x).
  • Step 4 Deprotection (reduction of azido groups): The resin was treated with a solution of DTT (2M) and DIPEA (1 M) in DMF at 50 5 C for 1 hour. The resin was then washed with DMF (3x) and DCM (3x). A small amount of resin was withdrawn and treated with a solution of benzoylchloride (0.5 M) and DIPEA (1 M) in DMF for 1 h.
  • Step 1 Fmoc- ⁇ -Ala linked Wang resin (A22608, Nova Biochem, 3.00 g; with loading 0.83 mmol/g) was swelled in DCM for 20 min. then washed with DCM (2x20 ml) and NMP (2x20 ml). The resin was then treated twice with 20% piperidine in NMP (2x15 min). The resin was washed with NMP (3x20 ml) and DCM (3x20 ml).
  • Step 2 2-(1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-yloxy)acetic acid (3.70 g; 10 mmol) was dissolved in NMP (30 ml) and DhbtOH (1 .60 g; 10 mmol) and DIC (1 .55 ml; 10 mmol) was added. The mixture was stirred at ambient temperature for 30 min, and then added to the resin obtained in step 1 together with DIPEA (1.71 ml; 10 mmol). The reaction mixture was shaken for 1 .5 h, then drained and washed with NMP (5x20 ml) and DCM (3x20 ml).
  • Step 3 A solution of SnCI 2 .2H 2 O (1 1.2 g; 49.8 mmol) in NMP (15 ml) and DCM (15 ml) was then added. The reaction mixture was shaken for 1 h. The resin was drained and washed with NMP:MeOH (5x20 ml; 1 :1 ). The resin was then dried in vacuo.
  • Step 4 A solution of 2-[2-(2-methoxyethyl)ethoxy]acetic acid (1 .20 g; 6.64 mmol), DhbtOH (1 .06 g; 6.60 mmol) and DIC (1 .05 ml; 6.60 mmol) in NMP (10 ml) was mixed for 10 min, at room temperature, and then added to the 3-[2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2- yloxy)acetylamino]propanoic acid tethered wang resin (1.0 g; 0.83 mmol/g) obtained in step 3.
  • Step 5 The resin product of step 4 was treated with TFA:DCM (10 ml, 1 :1 ) for 1 hour. The resin was filtered and washed once with TFA:DCM (10 ml, 1 :1 ). The combined filtrate and washing was then taken dryness, to give a yellow oil (711 mg). The oil was dissolved in 10% acetonitril-water (20 ml), and purified over two runs on a preparative HPLC apparatus using a C18 column, and a gradient of 15-40% acetonitril-water. Fractions were subsequently analysed by LC-MS. Fractions containing product were pooled and taken to dryness. Yield: 222 mg (37%).
  • EXAMPLE 38 3-(1 ,3-Bis ⁇ 2-(2-[2-(1 ,3-bis[2-(2- ⁇ 2-[2-(2-methoxyethoxy)ethoxy]acetamino ⁇ ethoxy)ethoxy]- propan-2-yloxy)acetylamino]ethoxy)ethoxy ⁇ propan-2-yloxy)acetylamino)propanoic acid
  • This material was prepared from 3-[2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)- acetylamino]propanoic acid tethered wang resin (1 .0 g; 0.83 mmol/g), obtained in step 3 of example by repeating step 2-5, doubling the amount of reagents used. Yield: 460 mg (33%).
  • MALDI-MS ⁇ -cyano-4-hydroxycinnamic acid
  • m/z 1670 (M+Na).
  • This example uses the 2-(1 ,3-bis[azidoethoxyethyl]propan-2-yloxy)acetic acid monomer building block prepared in example 6 in the synthesis of a second generation amide based branched polymer capped with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid.
  • the coupling chemistry is based on standard solid phase peptide chemistry, and the protection methodology is based on a solid phase azide reduction step as described above.
  • Step 1 Fmoc- ⁇ -Ala-Wang resin (100 mg; loading 0.31 mmol/g BACHEM) was suspended in dichloromethane for 30 min, and then washed twice with DMF. A solution of 20% piperidine in DMF was added, and the mixture was shaken for 15 min at ambient temperature. This step was repeated, and the resin was washed with DMF (3x) and DCM (3x).
  • Step 2 Coupling of monomer building blocks: A solution of 2-(1 ,3-bis[azidoethoxyethyl]- propan-2-yloxy)acetic acid (527 mg; 1 ,4 mmol, 4x) and DhbtOH (225 mg; 1 ,4 mmol, 4x) were dissolved in DMF (5 ml) and DIC (216 ⁇ l, 1 ,4 mmol, 4x) was added. The mixture was left for 10 min (pre-activation) then added to the resin together with DIPEA (240 ul; 1 ,4 mmol, 4x). The resin was shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
  • Step 3 Capping with acetic anhydride: The resin was then treated with a solution of acetic anhydride, DIPEA, DMF (12:4:48) for 10 min. at ambient temperature. Solvent was removed and the resin was washed with DMF (3x) and DCM (3x).
  • Step 4 Deprotection (reduction of azido groups): The resin was treated with a solution of DTT (2M) and DIPEA (1 M) in DMF at 50 5 C for 1 hour. The resin was then washed with DMF (3x) and DCM (3x). A small amount of resin was withdrawn and treated with a solution of benzoylchloride (0.5 M) and DIPEA (1 M) in DMF for 1 h. The resin was cleaved with 50% TFA/DCM and the dibenzoylated product analysed with NMR and LC-MS.
  • Step 8 Capping with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid: A solution of 2-[2-(2- methoxyethoxy)ethoxy]acetic acid (997 mg; 5.6 mmol, 16x with respect to resin loading) and DhbtOH (900 mg; 5.6 mmol, 16x) were dissolved in DMF (5 ml) and DIC (864 ul, 5.6 mmol, 16x) was added. The mixture was left for 10 min (pre-activation) then added to the resin together with DIPEA (960 ul; 5.6 mmol, 16x). The resin was shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
  • Step 9 Cleavage from resin: The resin was treated with a 50% TFA - DCM solution at ambient temperature for 30 min. The solvent was collected and the resin was washed an additional time with 50% TFA - DCM. The combined filtrates were evaporated to dryness, and the residue was purified by chromatography.
  • This material was prepared from 3-[2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)- acetylamino]propanoic acid tethered wang resin (1 .0 g; 0.83 mmol/g), obtained in step 3 of example by repeating step 2-3 with 2x the amount of reagents used, then repeating step 2-5 with 4x the amount of reagent used. Yield: 84 mg (4%).
  • N-Hydroxysuccinimidyl 3-(1 ,3-bis ⁇ 2-(2-[2-(1 ,3-bis[2-(2- ⁇ 2-[2-(2-methoxyethoxy)ethoxy]acet- amino ⁇ ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy ⁇ propan-2-yloxy)acetyl- amino)propanoate 105 mg; 0.06 mmol
  • DCM dimethyl methoxyethoxyethoxy
  • DIPEA 13 ⁇ l; 0.07 mmol
  • the material was prepared from two equivalents of N-hydroxysuccimidyl 2-(1 ,3-bis[2-(2- ⁇ 2-[2- (2-methoxyethoxy)ethoxy]acetamino ⁇ ethoxy)ethoxy]propan-2-yloxy)acetate and one equivalent of terf-butyl 2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)acetate, using the protocol and purification method described in example 45.
  • Further dendritic growth may be acchieved by removing the terf-butyl group as described in example 46 and subsequent N-hydroxysuccimidyl ester formation as described in example 47 followed by coupling to terf-butyl 2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan- 2-yloxy)acetate as described in this example.
  • the (S)-2,6-Bis-(2-[2-(2-[2-((S)-2,6-bis-[2-(2-[2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-methoxyethoxy)ethoxy)acetyl- amino)ethoxy)ethoxy]ethoxy)acetylamino]hexanoylamino)ethoxy]ethoxy]acetyl- amino)hexanoic acid methyl ester can be saponified to the free acid and attached to a free amino group of ITA for example, on either ⁇ amino lysin residues or on the terminal ⁇ -amino group using an activated ester.
  • the activated ester may be produced and coupled to the amino group of the ITA peptide by standard coupling methods known in the art such as diisopropylethylamine and N-hydroxybenzotriazole or other activating conditions.
  • the tertbutyl protected carboxylic acids intermediate above may be deprotected and subsequemtly activated as OSu esters (for example, as described in example 40) for attachment to the ITA peptide.
  • the mixture was stirred for at least 20 h. Solid sodium hydrogensulfate was added until pH was 2-3. The phases were separated. The aqueous phase was extracted with dichloromethane (3 x 50 ml). The combined organic phases were washed with aqueous sodium hydrogensulfate (5 % w/w, 50 ml). The aqueous phase was extracted with dichloromethane (2 x 50 ml). The combined organic phases were dried over magnesium sulfate, filtered, and concentrated in vacuo.
  • the tertbutyl protected carboxylic acids intermediate above may be deprotected and subsequemtly activated as OSu esters (for example as described in example 47) for attachment to insulin.
  • Triphenyl chloromethane (1 Og, 35.8 mmol) was dissolved in dry pyridine, diethyleneglycol (3.43 ml_, 35.8 mmol) was added and the mixture was stirred under nitrogen overnight. Solvent removed in vacuo. Dissolved in dichloromethane (100 ml_) and washed with water. Organic phase dried over Na 2 SO 4 and solvent removed in vacuo. Crude product was purified by recrystallization from heptane/toluene (3:2) to yield the title compound.
  • 2-(2-Trityloxyethoxy)ethanol (6.65 g, 19 mmol) was dissolved in dry THF (100 ml_). 60 % NaH (0.764 mg, 19 mmol) was added slowly. The suspension was stirred for 15 min. Epibromohydrin (1 .58 ml_, 19 mmol) was added and the mixture was stirred under nitrogen at room temperature overnight. The reaction was quenched with ice, separated between diethyl ether (300 ml_) and water (300 ml_). The water fase was extracted with dichloromethane.
  • R 34 -GLP-1 (7-37) (L.B. Knudsen et al., J. Med. Chem. 2000, 43, 1664-1669. )(1 10 mg; 33 umol) was suspended in water (30 ml). To the unclear suspension was added DIPEA (156 ul; 1 .6 mmol), and the mixture was stirred for 10 min, during which time the solution turned clear. The pH was measured to 10.
  • the reaction mixture was then purified over 2 run by preparative HPLC with direct injection (20 ml and 16 ml respectively), using a C18 column (20x2 cm) with a linear gradient of 25-55% water - acetonitril and a flow of 10 ml/min, collecting 10 ml fractions.
  • R 34 -GLP-1 (7-37) (15.6 mg; 4.5 umol) was suspended in water (10 ml). To the unclear suspension was added DIPEA (86 ul; 0.88 mmol), and the mixture was stirred for 10 min, during which time the solution turned clear. The pH was measured to 10.
  • the mixture was stirred for 5 min at room temperature.
  • Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)- Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Aib-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala- Trp(Boc)-Leu-Val-Lys(Boc)-Aib-Arg(Pmc)-Lys(Dde)-Rink amide resin was prepared according to the Fmoc strategy on an Applied Biosystems 433A peptide synthesizer in 0.25 mmol scale using the manufacturer supplied FastMoc UV protocols which employ HBTU mediated couplings in NMP, and UV monitoring of the deprotection of the Fmoc protection group.
  • Aib residues and residues following Aib were coupled using HATU instead of HBTU as the coupling reagent.
  • the starting resin (438 mg) used for the synthesis was 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)- phenoxy resin (Rink amide resin) (Merck Biosciences GmbH, Germany, cat. #: 01 -12-0013) with a substitution capacity of 0.57 mmol / g.
  • the protected amino acid derivatives used were (2S)-6-[1 -(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-ethylamino]-2-(9 H-fluoren-9-ylmethoxy- carbonylamino)hexanoic acid (Fmoc-Lys(Dde)-OH), Fmoc-Arg(Pmc)-OH, Fmoc-Aib-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH,
  • Fmoc-lle-OH Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(tBu)-OH,
  • the yield was 1 .37 g of dry peptidyl resin.
  • the resin was characterized by cleaving off the crude peptide from 50 mg of this resin by treating it for 2 hours with a mixture of 14 ⁇ l TIS, 14 ⁇ l H 2 O and 0.5 ml TFA. The resin was removed by filtration and the crude peptide was isolated by precipitation and wash with Et 2 O. HPLC and LC-MS analysis was performed on the dry precipitate. Analytical results:
  • NMP:DCM 1 :1 (15 ml) twice.
  • the reaction mixture was shaken for 12 min at room temperature, and then filtered.
  • the hydrazine treatment was repeated twice. After this the resin was washed extensively with NMP, DCM and NMP.
  • the resin from 1.d is stirred for 3 h at room temperature with a mixture of 350 ⁇ l TIS, 350 ⁇ l H 2 O and 14 ml TFA.
  • the resin is removed by filtration and washed with 3 ml TFA.
  • the collected filtrates are concentrated in vacuo, to 5 ml and the crude product is precipitated by addition of 40 ml Et 2 O followed by centrifugation. The pellet is washed with 40 ml Et 2 O two times and then air dried.
  • the crude peptide is dissolved in H 2 O/AcOH (40:4) (40ml) and purified by semipreparative HPLC in 2 runs on a 25 mm x 250 mm column packed with 7 ⁇ C-18 silica.
  • the column is eluted with a gradient of CH 3 CN from 40 to 62% against 0.1% TFA / H 2 O at 10 ml/min at a temperature of 4O 0 C for 47min.
  • the peptide containing fractions are collected, diluted with 3 volumes of H 2 O and lyophilized.
  • the final product obtained is characterized by HPLC.
  • Compounds of this invention includes:
  • Dde-Lys(Fmoc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val- Arg(Pmc)-Aib-Arg(Pmc)-Gly-Rink amide resin was prepared according to the Fmoc strategy on an Applied Biosystems 433A peptide synthesizer in 0.25 mmol scale using the manufacturer supplied FastMoc UV protocols which employ HBTU mediated couplings in NMP, and UV monitoring of the deprotection of the Fmoc protection group.
  • the terminal Fmoc group was removed by treatment with 2% DBU in DMF (3x3 min), and acylated on the lysine side chain, first with Fmoc-AEEAc-OH and after Fmoc deprotection with 3-[2-(1 ,3-bis[2-(2- ⁇ 2-[2-(2-(2- methoxyethoxy)ethoxy]acetylamino ⁇ ethoxy)ethoxy]propan-2-yloxy)acetic acid.
  • the terminal Dde-group was then removed with 10% hydrazin in NMP.
  • N-terminal of the peptide was then elongated with the Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)- Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Aib-Gln(Trt)-Ala-Ala-sequence using the manufacturer supplied FastMoc UV protocols which employ HBTU mediated couplings in NMP, and UV monitoring of the deprotection of the Fmoc protection group.
  • the peptide was cleaved form the resin using 5% triisopropylsilane and 5% water inTFA.
  • the resin was filtered off, and washed with TFA.
  • the combined filtrates were reduced to a minimal volume, and the peptide was precipitated by addition of cold diethyl ether, and isolated by centrifugation.
  • the precipitate was washed trice with cold diethylether.
  • the crude peptide was purified by RP18-HPLC.
  • the column was eluted with a gradient of CH 3 CN from 36 to 60% against 0.1 % TFA / H 2 O at 10 ml/min.
  • the peptide containing fractions were collected, diluted with H 2 O and lyophilized.
  • N-epsilon26,3-[2-(1 ,3-Bis[2-(2- ⁇ 2-[2-(2-methoxyethoxy)ethoxy]acetylamino ⁇ ethoxy)ethoxy]- propan-2-yloxy)acetylamino]propanoyl[Arg34]GLP-1 -(7-37)-OH (Example 55) was dissolved in water (20 ml) and pH was adjusted to 9 with triethylamine. A 1 :1 mixture of acetic anhydride and formic acid was prepared, and 5 ul of this solution (3 eq.) were added while keeping pH at 9 with triethylamine addition.
  • the present protocol describes the methods and materials used in the development of an anaesthetized rat model for pulmonal delivery of aerosols.
  • the aerosols are generated by use of a nebulizer catheter with a well defined droplet/particle size (mean mass aerodynamic diameter, MMAD).
  • the nebulizer catheter is an extruded multi-lumen catheter that provides fine-particle, baffle-free, aerosols. It incorporates multiple (typically 4-6) gas-lumens around one liquid lumen. Each lumen extends the length of the catheter which tapers to a fine (-0.5 mm diameter) nozzle with tiny orifices at the distal tip. The intimate contact between the gas and liquid at the tip produces a fine aerosol without baffling.
  • the nebulizer catheter is guided through an endotracheal tube and is placed just above the main bronchial branch.
  • the aerosol is deposited in pulses managed from a control unit.
  • the equipment for pulmonary delivery is obtained from Trudell Medical International (London, Ontario, Canada).
  • Nebulizer catheters are supplied from the manufacturer in a number of different configurations and lengths. These different designs will accommodate a variety of different fluid and flow-rates, as well as provide aerosol particle-sizes that may be as low as 5 ⁇ m MMAD (mean mass aerodynamic diameter).
  • MMAD mean mass aerodynamic diameter
  • the Aeroprobe catheter is connected to the control system according to (2).
  • Air with a pressure of 100 psi is used as supporting gas and maximal fluid pressure, usually 98 psi.
  • a 100 ⁇ l syringe is used as reservoir.
  • the LABNeb CCS used a pulse time of 80 msec and a gas delay of 20 msec. Thus, 2.3 ml air and 0.93 ⁇ l test solution are delivered in each pulse.
  • Sprague Dawley S rats weighing between 250 and 350 g.
  • the animals are housed under standardised conditions with free access to food (Altromine 1324) and drinking water. On the experimental day the animals are used in their fed state.
  • Hypnorm® (fentanyl 0.2 mg/ml, fluansol 10 mg/ml) is diluted with sterile water 1 +1 .
  • Dormicum® (midazolam 5 mg/ml) is diluted with sterile water 1 + 1 . The two solutions are mixed 1 + 1 .
  • Surgical procedures and intratracheal administration Anaesthesia is induced by injecting subcutaneously the prepared Hyponorm/Dormicum solution 0.25 ml/100 g BW.
  • An endotracheal tube PE 240, Becton Dickinson
  • PE 240, Becton Dickinson is inserted and guided to a position about Vz cm above the branch of the two main bronchii. Any heat loss is minimised by wrapping a plastic shield round the rat.
  • test solution Before applying the test solution into the lungs, it is secured that the syringe and catheter system is free of air bubbles. Before applying the test solution endotracheal ⁇ , it is sprayed into a vial to test subsequently the amount of substance administered by the catheter. Then, the catheter is guide through the endotracheal tube leaving 1 -2 mm of the catheter tip free of the tube end and the test solution is aerosolised into the lungs of the anaesthetised rat.
  • Protraction of GLP-1 derivatives after i.v. or s.c. administration The protraction of a number GLP-1 derivatives of the invention was determined by monitoring the concentration thereof in plasma after sc administration to healthy pigs, using the methods described below. For comparison also the concentration in plasma of GLP-1 (7- 37) after sc. administration was followed. The protraction of other GLP-1 derivatives of the invention can be determined in the same way.
  • test substances were dissolved in a vehicle suitable for subcutaneous or intravenous administration.
  • concentration was adjusted so the dosing volume was approximately 1 ml.
  • the study was conducted in a suitable animal room with a room temperature set at 21 -23 0 C and the relative humidity to > 50%.
  • the room was illuminated to give a cycle of 12 hours light and 12 hours darkness. Light was from 06.00 to 18.00 h.
  • the animals were housed in pens with straw as bedding, six together in each pen.
  • the animals had free access to domestic quality drinking water during the study, but were fasted from approximately 4 pm the day before dosing until approximately 12 hours after dosing. The animals were weighed on arrival and on the days of dosing.
  • the subcutaneous injection was given on the right side of the neck, approximately 5-7 cm from the ear and 7-9 cm from the middle of the neck.
  • the injections were given with a stopper on the needle, allowing 0.5 cm of the needle to be introduced.
  • Each test substance was given to three animals. Each animal received a dose of 2 nmol/kg body weight.
  • a full plasma concentration-time profile was obtained from each animal. Blood samples were collected according to the following schedule:
  • Predose O
  • 0.17 10 minutes
  • Predose (0) 0.5, 1 , 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 hours after injection.
  • 2 ml of blood was drawn from each animal.
  • the blood samples were taken from a jugular vein.
  • the blood samples were collected into test tubes containing a buffer for stabilisation in order to prevent enzymatic degradation of the GLP-1 analogues.
  • the plasma concentration-time profiles were analysed by a non-compartmental pharmacokinetic analysis.
  • the following pharmacokinetic parameters were calculated at each occasion: AUC, AUC/Dose, AUCo /oExtrap , C max , t max , ⁇ z , ty 2 , CL, CL/f, V z , V z /f and MRT.
  • the plasma concentrations of the peptides were determined in a sandwich ELISA or by RIA using different mono- or polyclonal antibodies. Choice of antibodies depends of the GLP-1 derivatives. The time at which the peak concentration in plasma is achieved varies within wide limits, depending on the particular GLP-1 derivative selected.
  • A-TNP Nonsens antibody
  • AMDEX Streptavin-horseradish-peroxodase (Amersham RPN4401V)
  • TMB-substrate 3,3',5,5'tetramethylbenzidine ( ⁇ 0.02 %), hydrogen peroxide
  • the assay was carried out as follows (volumen/well):
  • the concentration in the samples was calculated from standard curves.
  • DB-buffer 80 mM phosphate buffer, 0.1 % Human serum albumin, 10 mM EDTA,
  • the assay was carried out in minisorp tubes 12x75 mm (volumen/tube) as follows:
  • the method is a radiometric-ligand binding assay using LEADsee/cer imaging particles.
  • the assay is composed of membrane fragments containing the GLP-1 receptor, unlabeled GLP-1 analogues, human GLP-1 labelled with 125 I and PS LEADsee/cer particles coated with wheat germ agglutinin (WGA). Cold and 125 l-labelled GLP-1 will compete for the binding to the receptor.
  • WGA wheat germ agglutinin
  • the proximity between the 125 l-molecules and the LEADsee/cer particles causes light emission from the particles.
  • the LEADseeker will image the emitted light and it will be reversibly correlated to the amount of GLP-1 analogue present in the sample.
  • GLP-1 analogues calibrators GLP-1 analogues were spiked into heat-treated plasma to produce dilution lines ranging from approximately 1 ⁇ M to 1 pM.
  • GLP-1 receptor suspension GLP-1 receptor membrane fragments were purified from baby hamster kidney (BHK) cells expressing the human pancreatic GLP-1 receptor. Stored ⁇ -80°C until use.
  • WGA-coupled polystyrene LEADseeker imaging beads (RPNQ0260, Amersham): The beads were reconstituted with GLP-1 RRA assay buffer to a concentration of 13.3 mg/mL The GLP-1 receptor membrane suspension was then added and incubated cold (2-8 0 C) at end- over-end for at least 1 hr prior to use.
  • APPARATUS Horizontal plate mixer Centrifuge with a standard swinging-bucket microtitre plate rotor assembly UltraVap - Drydown Sample Concentrator (Porvair) LEADseekerTM Multimodality Imaging System (Amersham)
  • Sample preparation Mount the MultiScreen® Solvinert filter plate on a chemical-comparable receiver plate (i.e. poly propylene plates) to collect the filtrate.
  • a chemical-comparable receiver plate i.e. poly propylene plates
  • the light emission from each wells are detected by using the LEADseekerTM Multimodality Imaging System for duration of 10 minutes.
  • Purified plasma membranes from a stable transfected cell line, BHK467-12A (tk-ts13), expressing the human GLP-1 receptor was stimulated with GLP-1 and peptide analogues, and the potency of cAMP production was measured using the AlphaScreenTM cAMP Assay Kit from Perkin Elmer Life Sciences.
  • a stable transfected cell line has been prepared at NN and a high expressing clone was selected for screening.
  • the cells were grown at 5% CO 2 in DMEM, 5% FCS, 1 % Pen/Strep and 0.5 mg/ml G418. Cells at approximate 80% confluence were washed 2X with PBS and harvested with
  • the functional receptor assay was carried out by measurering the peptide induced cAMP production by The AlphaScreen Technology.
  • the basic principle of The AlphaScreen Technology is a competition between endogenous cAMP and exogenously added biotin- cAMP.
  • the capture of cAMP is achieved by using a specific antibody conjugated to acceptor beads.
  • Formed cAMP was counted and measured at a AlphaFusion Microplate Analyzer.
  • the EC 50 values was calculated using the Graph-Pad Prisme software.

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Abstract

Novel GLP-1 conjugated with structurally well defined polymers and their therapeutically use.

Description

DERIVATIVES OF INSULINOTROPIC AGENTS CONJUGATED WITH STRUCTURALLY WELL DEFINED BRANCHED POLYMERS
FIELD OF THIS INVENTION This invention relates generally to methods of treating humans suffering from diabetes mellitus. More specifically, the present invention relates to insulinotropic agents conjugated with structurally well defined branched polymers. The branched polymers are composed of monomer building blocks. Furthermore, this invention relates to the use of such conjugated insulinotropic agents, for example, by pulmonary delivery for systemic absorption through the lungs to reduce or eliminate the need for administering other insulinotropic agents by injection as well as to pharmaceutical compositions comprising these compounds and to the use of the compounds for the treatment of diseases related to diabetes.
BACKGROUND OF THIS INVENTION Since the introduction of insulin in the 1920's, continuous efforts have been made to improve the treatment of diabetes mellitus.
Diabetes mellitus is a disease affecting approximately 6% of the world's population. Furthermore, the population of most countries is aging and diabetes is particularly common in aging populations. Often, it is this population group which experiences difficulty or unwillingness to self-administer insulin by injection. In the United States, approximately 5% of the population has diabetes and approximately one-third of those diabetics self-administer one or more doses of insulin per day by subcutaneous injection. This type of intensive therapy is necessary to lower the levels of blood glucose. High levels of blood glucose, which are the result of low or absent levels of endogenous insulin, alter the normal body chemistry and can lead to failure of the microvascular system in many organs. Untreated diabetics often undergo amputations and experience blindness and kidney failure. Medical treatment of the side effects of diabetes and lost productivity due to inadequate treatment of diabetes is estimated to have an annual cost of about $40 billion in the United States alone.
One peptide expected to become very important in the treatment of diabetes is glucagon-like peptide-1 (GLP-1 ). Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesized La. in the L-cells in the distal ileum, in the pancreas and in the brain. GLP-1 is an important gut hormone with regulatory function in glucose metabolism and gastrointestinal secretion and metabolism. GLP-1 stimulates insulin secretion in a glucose-dependant manner, stimulates insulin biosynthesis, promotes beta cell rescue, decreases glucagon secretion, gastric emptying and food intake. PCT publications WO 98/08871 and WO 99/43706 disclose stable derivatives of GLP-1 analogues, which have a lipophilic substituent. These stable derivatives of GLP-1 analogues have a protracted profile of action compared to the corresponding GLP-1 analogues. In the last decade a number of peptides have been isolated from the venom of the GiIa monster lizards (Heloderma suspectum and Heloderma horridum). Exendin-4 is a 39 amino acid residue peptide isolated from the venom of Heloderma suspectum, and this peptide shares 52% homology with GLP-1 (7-37) in the overlapping region. Exendin-4 is a potent GLP-1 receptor agonist which has been shown to stimulate insulin release and ensuing lowering of the blood glucose level when injected into dogs. The group of exendin-4(1 -39), certain fragments thereof, analogs thereof and derivatives thereof, are potent insulinotropic agents. Most importantly the group of exendin-4(1 -39), insulinotropic fragments thereof, insulinotropic analogs thereof and insulinotropic derivatives thereof.
Common to GLP-1 and exendins are that an extensive amount of variants have been synthesized and studied in particular in relation the plasma half-life. Low plasma half- lifes may be due to chemical stability towards peptidases (mainly dipeptidyl aminopeptidase IV) and to renal clearance. However, these analogues and derivatives of insulionotropic peptides lack a satisfactory bioavailability when administered by the pulmonary route, i.e. when administered to the lower respiratory tract such as through the bronchioles or alveoli. WO 00/66629 discloses modified exendin agonists which have been coupled to polyethyleneglycol via a lysine residue to decrease renal clearance.
WO 03/40309 discloses peptide acting as both GLP-1 receptor agonists and glucagon receptor antagonists. Among the disclosed peptides are two peptides which have been coupled to polyethyleneglycol via a C-terminal cysteine residue. WO 2004/093823 discloses polyethylene glycolated GLP-1 peptides.
Pulmonary administration of GLP-1 peptides have been disclosed in WO 01/51071 and WO 00/12116.
The insulinotropic peptides derived from GLP-1 and Exendin-4 stimulates insulin release only when plasma glucose levels are high, and therefore the risk of hypoglycaemic events is reduced. Thus, the peptides are particularly useful for patients with diabetes who no longer respond to OHA's (oral hyperglycaemic agents) and who should from a strict medical point of view be administered insulin. Patients and to some extent also doctors are often not keen on initiating insulin treatment before this is absolutely necessary, presumably because of the fear of hypoglycaemic events or the fear of injections/needles. Thus, there is a need for insulinotropic peptides which are sufficiently potent and which can be administered by the pulmonary route.
It has been known for a number of years that some proteins can be absorbed from the lung. In fact, administration of insulin as an inhalation aerosol to the lung was first reported by Gaensslen in 1925.
It is clear that not all proteins can be efficiently absorbed in the lungs. There are numerous factors which impact whether a protein can be effectively delivered through the lungs. Absorption through the lungs is dependent to a large extent on the physical characteristics of the particular therapeutic protein to be delivered. Efficient pulmonary delivery of a protein is dependent on the ability to deliver the protein to the deep lung alveolar epithelium. Proteins that are deposited in the upper airway epithelium are not absorbed to a significant extent. This is due to the overlying mucus which is approximately 30-40 μm thick and acts as a barrier to absorption. In addition, proteins deposited on this epithelium are cleared by mucociliary transport up the airways and then eliminated via the gastrointestinal tract. This mechanism also contributes substantially to the low absorption of some protein particles. The extent to which proteins are not absorbed and instead eliminated by these routes depends on their solubility, their size, as well as other less understood characteristics.
It is difficult to predict whether a therapeutic protein can be rapidly transported from the lung to the blood even if the protein can be successfully delivered to the deep lung alveolar epithelium. Because of the broad spectrum of peptidases which exist in the lung, a longer absorption time increases the possibility that the protein will be significantly degraded or cleared by mucociliary transport before absorption.
In addition, peptides of therapeutic interest such as hormones, soluble receptors, cytokines, enzymes etc. often have short circulation half-life in the body as a result of proteolytic degradation, clearance by the kidney or liver, or in some cases the appearance of neutralizing antibodies. This generally reduces the therapeutic utility of peptides.
It is however well recognised that the properties of peptides can be enhanced by grafting organic chain-like molecules onto them. Such grafting can improve pharmaceutical properties such as half life in serum, stability against proteolytical degradation, and reduced immunogenicity.
The organic chain-like molecules often used to enhance properties are polyethylene glycol-based or polyethylene based chains, i.e., chains that are based on the repeating unit - CH2CH2O-. Hereinafter, the abbreviation "PEG" is used for polyethyleneglycol. However, the techniques used to prepare PEG or PEG-based chains, even those of fairly low molecular weight, involve a poorly-controlled polymerisation step which leads to preparations having a wide spread of chain lengths about a mean value. Consequently, peptide conjugates based on PEG grafting are generally characterised by broad range molecular weight distributions. Kochendoefer et al. recently described (Science 2003, 299, 884-887) the design and synthesis of a homogeneous polymer modified erythropoiesis protein, and in WO 02/20033 (a PCT patent application) devised a general method for the synthesis of well defined polymer modified peptides. The building blocks used in this work were based on alternating water soluble linear long chain hydrophilic diamines and succinic acid, which were extended by sequential addition using standard peptide chemistry in solution or on solid support. An alternative and more attractive strategy for preparing large well defined polymers relies on the use of bi-, tri or multifurcated monomers which are oligomerized in a limited number of sequential synthesis steps. The mass growth of the polymer will in this case follow an exponential curve, with an exponent determined by the furcation number, for example, bifurcated monomers provides 2nd power growth, trifurcated monomers provides 3rd power growth, etc. The type of polymers obtained by this procedure has been well described in the literature (S. M. Grayson and J.M.J. Frechet, Chem. Rev. 2001 , IPJ., 3819) and are commonly known as dendrimers.
Biodegradable 4th generation polyester dendrimers based on 2,2-bis(hydroxymethyl)- propionic acid and capped with polyethyleneoxide via a carbamate linkage has recently been reported (E.R.Gillies and J. M. J. Frechet, J. Amer. Chem. Soc, 2002, 124, 14137-14146). The architecture of this system bears a close resemblance to the system described by Kochendoefer et al. as described above, as the dendritic part of the structure is used to generate a polyhydroxy scaffold that function as attachment points for the polyethyleneoxide tails. However, although impressive 12 KDa structures can be made, a large degree of dispersity is introduced from each polyethyleneoxide tail, as only the core structure is chemically well defined.
In light of the many potential applications for well defined polymer conjugated to biopharmaceuticals (for example, modifying pharmacokinetics and pharmacodynamics), there is a continuous need in the art for improving the technology for preparing well defined polymers and co-polymers in a precise well defined manner, from a precise number of monomer units.
SUMMARY OF THIS INVENTION
The present invention provides a new class of branched polymers conjugated to an insulinotropic agent. These new compounds have the general formula I mentioned below with the definitions mentioned below. The compounds of formula I contain a controlled number of monomer building blocks (designated Yb and Yt, below).
This invention also provides the use of a conjugate as above as a medicament.
DETAILED DESCRIPTION OF THIS INVENTION
In the present specification, the following terms have the indicated meaning: The term "polypeptide" and "peptide" as used herein means a compound composed of at least five constituent amino acids connected by peptide bonds. The constituent amino acids may be from the group of the amino acids encoded by the genetic code and they may 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 for example, hydroxyproline, γ-carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic 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 (α-aminoisobutyric acid), Abu (α-aminobutyric acid), Tie (tert-butylglycine), β-alanine, 3- aminomethyl benzoic acid, anthranilic acid.
The term "analogue" as used herein referring to a polypeptide 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. A simple system is often used to describe analogues: For example [Arg34]GLP-1 (7-37)Lys designates a GLP-1 (7-37) analogue wherein the naturally occuring lysine at position 34 has been substituted with arginine and wherein a lysine has been added to the terminal amino acid residue, i.e. to the GIy37. All amino acids for which the optical isomer is not stated is to be understood to mean the L-isomer. Human GLP-1 is hydrolysed to GLP-1 (7-37) and GLP-1 (7-36)-amide which are both insulinotropic peptides. Thus, for example, [Gly8]GLP-1 (7-37) designates an analogue of GLP-1 (7-37) formally derived from GLP-1 (7-37) by substituting the naturally occurring amino acid residue in position 8 (Ala) by GIy. Similarly, (Nε34-tetradecanoyl)[Lys34]GLP-1 (7-37) designates GLP-1 (7-37) wherein the ε-amino group of the Lys residue in position 34 has been tetradecanoylated. In aspects of the invention analogues of GLP-1 or exendin-4 has maximum of 15 amino acids has been added, deleted or exchanged compared to the native sequence, In aspects of the invention maximum of 12 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 10 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 8 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 6 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 4 amino acids has been added, deleted or exchanged. In aspects of the invention a maximum of 2 amino acids has been added, deleted or exchanged.
The term "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 Nε26-((4S)-4-(hexadecanoylamino)- butanoyl)[Arg34, Lys26]GLP-1 -(7-37).
The term "insulinotropic agent" as used herein 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 (one such medium disclosed below). The potency of an insulinotropic agent is determined by calculating the EC50 value from the dose-response curve as described below.
Baby hamster kidney (BHK) cells expressing the cloned human GLP-1 receptor (BHK- 467-12A) were grown in DMEM media with the addition of 100 ILVmL penicillin, 100 μg/mL streptomycin, 5% fetal calf serum and 0.5 mg/mL Geneticin G-418 (Life
Technologies). The cells were washed twice in phosphate buffered saline and harvested with Versene. Plasma membranes were prepared from the cells by homogenisation with an Ultraturrax in buffer 1 (20 mM HEPES-Na, 10 mM EDTA, pH 7.4). The homogenate was centrifuged at 48,000 x g for 15 min at 40C. The pellet was suspended by homogenization in buffer 2 (20 mM HEPES-Na, 0.1 mM EDTA, pH 7.4), then centrifuged at 48,000 x g for 15 min at 40C. The washing procedure was repeated one more time. The final pellet was suspended in buffer 2 and used immediately for assays or stored at -8O0C.
The functional receptor assay was carried out by measuring cyclic AMP (cAMP) as a response to stimulation by the insulinotropic agent. cAMP formed was quantified by the AlphaScreen™ cAMP Kit (Perkin Elmer Life Sciences). Incubations were carried out in half- area 96-well microtiter plates in a total volume of 50 μL buffer 3 (50 mM Tris-HCI, 5 mM HEPES, 10 mM MgCI2, pH 7.4) and with the following addiditions: 1 mM ATP, 1 μM GTP, 0.5 mM 3-isobutyl-1 -methylxanthine (IBMX), 0.01 % Tween-20, 0.1 % BSA, 6 μg membrane preparation, 15 μg/mL acceptor beads, 20μg/mL donor beads preincubated with 6 nM biotinyl-cAMP. Compounds to be tested for agonist activity were dissolved and diluted in buffer 3. GTP was freshly prepared for each experiment. The plate was incubated in the dark with slow agitation for three hours at room temperature followed by counting in the Fusion™ instrument (Perkin Elmer Life Sciences). Concentration-response curves were plotted for the individual compounds and EC50 values estimated using a four-parameter logistic model with Prism v. 4.0 (GraphPad, Carlsbad, CA).
The term "GLP-1 peptide" as used herein means GLP-1 (7-37) (SEQ ID No 1 ), a
GLP-1 (7-37) analogue, a GLP-1 (7-37) derivative or a derivative of a GLP-1 (7-37) analogue. In one embodiment the GLP-1 peptide is an insulinotropic agent.
The term "exendin-4 peptide" as used herein means exendin-4(1 -39) (SEQ ID No 2), an exendin-4(1 -39) analogue, an exendin-4(1 -39) derivative or a derivative of an exendin- 4(1 -39) analogue. In one embodiment the exendin-4 peptide is an insulinotropic agent.
The term "DPP-IV protected" as used herein referring to a polypeptide means a polypeptide which has been chemically modified in order to render said compound resistant to the plasma peptidase dipeptidyl aminopeptidase-4 (DPP-IV). The DPP-IV enzyme in plasma is known to be involved in the degradation of several peptide hormones, for example, GLP-1 , GLP-2, Exendin-4 etc. Thus, a considerable effort is being made to develop analogues and derivatives of the polypeptides susceptible to DPP-IV mediated hydrolysis in order to reduce the rate of degradation by DPP-IV. In one embodiment a DPP-IV protected peptide is more resistant to DPP-IV than GLP-1 (7-37) or Exendin-4(1 -39). Resistance of a peptide to degradation by dipeptidyl aminopeptidase IV is determined by the following degradation assay :
Aliquots of the peptide (5 nmol) are incubated at 37 5C with 1 μL of purified dipeptidyl aminopeptidase IV corresponding to an enzymatic activity of 5 mU for 10-180 minutes in 100 μL of 0.1 M triethylamine-HCI buffer, pH 7.4. Enzymatic reactions are terminated by the addition of 5 μL of 10% trifluoroacetic acid, and the peptide degradation products are separated and quantified using HPLC analysis. One method for performing this analysis is : The mixtures are applied onto a Vydac C18 widepore (30 nm pores, 5 μm particles) 250 x 4.6 mm column and eluted at a flow rate of 1 ml/min with linear stepwise gradients of acetonitrile in 0.1% trifluoroacetic acid (0% acetonitrile for 3 min, 0-24% acetonitrile for 17 min, 24-48% acetonitrile for 1 min) according to Siegel et al., Regul. Pept. 1999;79:93-102 and Mentlein et al. Eur. J. Biochem. 1993; 214: 829-35. Peptides and their degradation products may be monitored by their absorbance at 220 nm (peptide bonds) or 280 nm (aromatic amino acids), and are quantified by integration of their peak areas related to those of standards. The rate of hydrolysis of a peptide by dipeptidyl aminopeptidase IV is estimated at incubation times which result in less than 10% of the peptide being hydrolysed. Using results from the so-called free fat cell assay, any skilled art worker, for example, a physician, knows when and which dosages to administer of the GLP-1 analogue. The term "covalent attachment" means that the polymeric molecule and the GLP-1 is either directly covalently joined to one another, or else is indirectly covalently joined to one another through an intervening moiety or moieties, such as bridge, spacer, or linkage moiety or moieties.
The term "branched polymer", or "dendritic polymer" or "dendritic structure" "or dendrimer" means an organic polymer assembled from a selection of monomer building blocks of which, some contains branches. The term "conjugate", or "conjugate GLP-1 ", is intended to indicate a heterogeneous
(in the sense of composite or chimeric) molecule formed by covalent attachment of one or more GLP-1 analogues to one or more polymer molecules.
The term "polydispersity" is used to indicate the purity of a polymer. The term "polydispersity index" (PDI) is the ratio of Mw to Mn wherein Mw is Z(M1 2N1)ZZ(M1N1) and Mn is Z(M1N1)/ Z(N1), wherein M1 is the molecular weight of the individual molecules present in the mixture, and N1 is the number of molecules represented by a certain molecular weight. The PDI provides a rough indication of the breadth of the distribution of the specific polymers present in a mixture. If the PDI of a certain polymer is 1 , said polymer has a purity of 100%. For small generations of polymers, for example 1 -3 generation, it may be more convenient to indicate the purity that to indicate a PDI. However, for longer polymers, it may be more convenient to use PDI.
The term "monodisperse" is, herein, used for a polymer having a PDI of less than 1 .09. In an embodiment it is less than 1 .08. In an embodiment it is less than 1 .07 and at least 1 . Herein the term "structurally well defined" in connection with a product indicates that the product has a high purity of a specific, chemically well-defined compound. In an embodiment such a purity is above about 80%. In an embodiment it is above about 90%. In an embodiment it is above about 95%. In an embodiment it is above about 97.5%.
"Immunogenicity" of a polymer modified GLP-1 refers to the ability of the polymer modified GLP-1 , when administrated to a human, to elicit an immune response, whether humoral, cellular, or both.
The term "attachment group" is intended to indicate a functional group on the GLP-1 or linker modified GLP-1 capable of attaching a polymer molecule either directly or indirectly through a linker. Useful attachment groups are, for example, amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate. The term "reactive functional group" means by way of illustration and not limitation, any free amino, carboxyl, thiol, alkyl halide, acyl halide, chloroformiate, aryloxycarbonate, hydroxy or aldehyde group, carbonates such as the p-nitrophenyl, or succinimidyl; carbonyl imidazoles, carbonyl chlorides; carboxylic acids that are activated in situ; carbonyl halides, activated esters such as N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, esters of such as those comprising 1 ,2,3-benzotriazin-4(3H)-one, phosphoramidites and H- phosphonates, phosphortriesters or phosphordiesters activates in situ, isocyanates or isothiocyanates, in addition to groups such as -NH2, -OH, -N3, -NHR' or -OR' (where R' is a protection group as defined below), -0-NH2, alkynes, or any of the following: hydrazine derivatives (-NH-NH2), hydrazine carboxylate derivatives (-0-C(O)-NH-NH2), semicarbazide derivatives (-NH-C(O)-NH-NH2), thiosemicarbazide derivatives (-NH-C(S)-NH-NH2), carbonic acid dihydrazide derivatives (-NHC(O)-NH-NH-C(O)-NH-NH2), carbazide derivatives (-NH- NH-C(O)-NH-NH2), thiocarbazide derivatives (-NH-NH-C(S)-NH-NH2), aryl hydrazine derivatives (-NH-C(O)-C6H4-NH-NH2), hydrazide derivatives (-C(O)-NH-NH2), and oxylamine derivatives, such as -C(O)-O-NH2, -NH-C(O)-O-NH2 and -NH-C(S)-O-NH2.
The term "protected functional group" means a functional group which has been protected in a way rendering it essential non-reactive. Examples of protection groups used for amines include but are not limited to tert-butoxycarbonyl, 9-fluorenylmethyloxycarbonyl, azides etc. For a carboxyl group, other groups become relevant such as tert-butyl, or more generally alkyl groups. Appropriate protection groups are known to the skilled person, and examples can be found in Green & Wuts "Protection groups in organic synthesis", 3rd Edition, Wiley-interscience.
The term "cleavable moiety" is intended to mean a moiety that is capable of being selectively cleaved to release the branched polymer linker or branched polymer linker GLP-1 from for example, a solid support.
The term "generation" refers to a single uniform layer, created by reacting one or more identical functional groups on an organic molecule with a particular monomer building block. Dendrimer synthesis demands a high level of synthetic control which is achieved through stepwise reactions, building the dendrimer up one monomer layer, or "generation," at a time. Each dendrimer consists of a multifunctional core molecule with a dendritic wedge attached to each functional site. The core molecule is referred to as "generation 0". Each successive repeat unit along all branches forms the next generation, "generation 1 ", "generation 2," etc. until the terminating generation. With a dendrimer made from exclusively bifurcated monomers, the number of reactive surface groups available for reaction is 2m, where m is an integer of 1 , 2, 3 ... 8 representing the particular generation. For a dendrimer made of exclusively trifurcated monomers, the number of reactive groups is 3m, and for a dendrimer made exclusively from a multifurcated monomer with n branches, the number of reactive groups is nm. For branched polymers in which different monomers are used in each individual generation, the number of reactive groups in a particular layer or generation can be calculated recursively knowing the layer position and the number of branches of the individual monomers.
The term "functional in vivo half-life" is used in its normal meaning, i.e., the time at which 50% of the biological activity of the GLP-1 or conjugate is still present in the body/target organ, or the time at which the activity of the GLP-1 or conjugate is 50% of its initial value. As an alternative to determining functional in vivo half-life, "serum half-life" may be determined, i.e., the time at which 50% of the GLP-1 or conjugate molecules circulate in the plasma or bloodstream prior to being cleared. Determination of serum-half-life is often more simple than determining functional half-life and the magnitude of serum-half-life is usually a good indication of the magnitude of functional in vivo half-life. Alternative terms to serum half-life include plasma half-life, circulating half-life, circulatory half-life, serum clearance, plasma clearance, and clearance half-life. The GLP-1 or conjugate is cleared by the action of one or more of the reticuloendothelial system (RES), kidney, spleen, or liver, by tissue factor, SEC receptor, or other receptor- mediated elimination, or by specific or unspecific proteolysis. Normally, clearance depends on size (relative to the cut-off for glomerular filtration), charge, attached carbohydrate chains, and the presence of cellular receptors for the GLP-1 . The functionality to be retained is normally selected from procoagulant, proteolytic, co-factor binding or receptor binding activity. The functional in vivo half-life and the serum half-life may be determined by any suitable method known in the art. The term "increased" as used about the functional in vivo half-life or plasma half-life is used to indicate that the relevant half-life of the GLP-1 or conjugate is statistically significantly increased relative to that of a reference molecule. For instance the relevant half- life may be increased by at least about 10% or at least 25%, such as by at least about 50%, for example, by at least about 100%, 150%, 200%, 250%, or 500%. The term "halogen" means fluoro, chloro, bromo or iodo. The term "heavy atom" as used herein means an atom having a molar weight equal to or larger than carbon, for example, C, N, O and S.
The terms "alkyl", "alkylene", "alkantriyl", and "alkantetrayl" represents a saturated, branched or straight hydrocarbon group having from 1 to 18 carbon atoms. In an embodiment it is from 1 to 10 carbon atoms. In an embodiment it is from 1 to 6 carbon atoms with one, two, three, or four bonds, respectively. Typical groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, terf-butyl, pentyl, and hexyl. Specific alkylene, alkantriyl, and alkantetrayl groups include the corresponding divalent, trivalent, and tetravalent radicals.
The terms "alkenyl" and "alkenylene" refer to a C2-6-alkenyl and C2-6-alkenylene, respectively, and represents a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one double bond and having one or two bonds, respectively. Typical C2-6-alkenyl groups include, but are not limited to, ethenyl, 1 -propenyl, 2-propenyl, isopropenyl, 1 ,3-butadienyl, 1 -butenyl, 2-butenyl, 1 -pentenyl, 2-pentenyl, 1 -hexenyl, 2- hexenyl, 1 -ethylprop-2-enyl, 1 ,1 -(dimethyl)prop-2-enyl, 1 -ethylbut-3-enyl, and 1 ,1 - (dimethyl)but-2-enyl. Examples of C2-6-alkenylen groups include the corresponding divalent radicals.
The terms "alkynyl" or "alkynylene" refer to a C2.6-alkynyl or C2.6-alkynylene, representing a branched or straight hydrocarbon group having from 2 to 6 carbon atoms and at least one triple bond and having one or two bonds, respectively. Typical C2.6-alkynyl groups include, but are not limited to, 1 -propynyl, 2-propynyl, isopropynyl, 1 ,3-butadynyl, 1 - butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 1 -hexynyl, 2-hexynyl, 1 -ethylprop-2-ynyl, 1 ,1 -(di- methyl)prop-2-ynyl, 1 -ethylbut-3-ynyl, 1 ,1 -(dimethyl)but-2-ynyl, and C2-6-alkynylene groups include the corresponding divalent radicals.
The terms "alkyleneoxy" or "alkoxy" refer to "Ci-e-alkoxy" or Ci-e-alkyleneoxy representing the radical -O-Ci-6-alkyl or -O-Ci-6-alkylene, respectively, wherein
Ci-6-alkyl(ene) is as defined above. Representative examples are methoxy, ethoxy, n- propoxy, isopropoxy, butoxy, sec-butoxy, terf-butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy and the like.
The terms "alkylenethio", "alkenylenethio" and "alkynylenethio" refer to the corresponding thio analogues of the oxy-radicals as defined above. Representative examples are methylthio, ethylthio, propylthio, butylthio, pentylthio, hexylthio, and the corresponding divalent radicals and the corresponding alkenyl and alkynyl derivatives also defined above.
Herein, the terms "-diyl" and "-triyl" is used and refers to different alkyl, alkenyl, alkynyl, cycloalkyl or aromatic radicals with two and three attachment points, respectively.
Herein, the term "alkantrioxy" refers to an alkantriyl moiety with one oxy (-O-) attached to each of the three alkantriyl bonds. Representative examples are propantrioxy, tert-butyltrioxy ect. The term "cycloalkyl" refers to C3-8-cycloalkyl representing a monocyclic, carbocyclic group having from 3 to 8 carbon atoms. Representative examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
The term "cycloalkenyl" refers to C3-8-cycloalkenyl representing a monocyclic, carbocyclic, non-aromatic group having from 3 to 8 carbon atoms and at least one double bond. Representative examples are cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl and the like.
The term "polyalkoxy" designates alkoxy-alkoxy-alkoxy-alkoxy etc. where the number of carbon atoms in each of the alkoxy moieties is the same or different. In an embodiment they are the same. Similarly, polyalkoxyalkyl and polyalkoxyalkylcarbonyl designates (polyalkoxy)- alkyl and (polyalkoxy)-alkyl-CO-, respectively. The term polyalkoxydiyl designates alkoxy- alkoxy-alkoxy-alkoxy etc having two free bonds.
The term "poly" means many. In an embodiment it is a numbering the range from 2 to 24. In an embodiment it is from 2 to 12. In an embodiment it is 3, 4 or 5. The term "oxyalkyl" is -O-alkyl-, i.e. a divalent radical.
The term "aryl" as used herein is intended to include carbocyclic aromatic ring systems such as phenyl, biphenylyl, 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 hydro- genated derivatives are 1 ,2,3,4-tetrahydronaphthyl, 1 ,4-dihydronaphthyl and the like.
The terms "arenetriyl" and "arenetetrayl" are moieties identical with aryl as defined above with the proviso that in arenetriyl and arenetetrayl there are not one but three and four, respectively, free bonds. With the same proviso, examples of arenetriyl and arenetetrayl are as mentioned for aryl above. The term "heteroaryl" as used herein is intended to include heterocyclic aromatic ring systems containing one or more heteroatoms selected from nitrogen, oxygen and sulphur such as furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1 ,2,3- triazolyl, 1 ,2,4-triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, 1 ,3,5- triazinyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4- oxadiazolyl, 1 ,2,3-thiadiazolyl, 1 ,2,4-thiadiazolyl, 1 ,2,5-thiadiazolyl, 1 ,3,4-thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, benzothienyl, benzothiophenyl (thianaphthenyl), indazolyl, benzimidazolyl, benzthiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl and the like. Heteroaryl is also intended to include the partially hydrogenated derivatives of the heterocyclic systems enumerated above. Non-limiting examples of such partially hydrogenated derivatives are 2,3-dihydro- benzofuranyl, pyrrolinyl, pyrazolinyl, indolinyl, oxazolidinyl, oxazolinyl, oxazepinyl and the like.
The term heteroaryl-Ci.6-alkyl as used herein, denotes heteroaryl as defined above and Ci-6-alkyl as defined above.
The terms "aryl-Ci.6-alkyl" and "aryl-C2-6-alkenyl" as used herein denotes aryl as defined above and Ci-6-alkyl and C2-6-alkenyl, respectively, as defined above.
The term "acyl" as used herein denotes -(C=O)-Ci-6-alkyl wherein Ci_6-alkyl is as defined above.
DMT is:
Fmoc is:
Certain of the above defined terms may occur more than once in the structural formulae, and upon such occurrence each term shall be defined independently of the other. Furthermore, when combined terms are used for divalent or trivalent moities, the interpretation of such combined terms into chemical structures is done by reading the combined terms from left to right or vice versa. Hence, a term like a divalent aminoalkyl moiety also covers an alkylamino moiety. Herein, the term moiety is preferaly used in connection with divalent and trivalent radicals. To be more specific, here follows the names of some divalent moieties consisting of a combination of different terms each divalent moiety followed by an alternative definition in square paranthesis and optionally one or more specific examples of such divalent moieties: alkyleneaminocarbonylalkylamino [-alkylene-NH-CO-alkylene-NH-, for example, -NH-- CH2CH2-CO-NH-CH2CH2CH2CH2-], alkylenecarbonylamino(polyalkoxy)alkylamino [-alkylene- CO-NH-(polyalkoxy)-alkylene-NH-], alkyleneoxyalkyl [-alkylene-O-alkylene-, for example, - CH2CH2-O-CH2CH2-], carbonylalkylamino [-CO-alkylene-NH-, for example, -NHCH2CH2C(O)- ], carbonylalkylcarbonylamino(polyalkoxy)alkylamino [-CO-alkylene-CO-NH-(polyalkoxy)- alkylene-NH-], carbonylalkoxyalkylamino [-CO-alkoxy-alkylene-NH-], carbonylalkoxyalkylcarbonylamino(polyalkoxy)alkylamino [-CO-alkoxy-alkylene-CO-NH- (polyalkoxy)-alkylene-NH-], carbonyl(polyalkoxy)alkylamino [-CO-(polyalkoxy)-alkylene-NH-], (polyalkoxy)alkyl [-(polyalkoxy)-alkylene-, for example, -CH2OCH2CH2OCH2CH2O-CH2- and - CH2CH2OCH2CH2OCH2CH2O-CH2-], (polyalkoxy)alkylcarbonyl [-(polyalkoxy)-alkylene-CO-]. In the formulae in the square brackets, the bonds between the different moieties are indicated by "-".
The term "optionally substituted" as used herein means that the groups in question are either unsubstituted or substituted with one or more of the substituents specified. When the groups in question are substituted with more than one substituent, the substituents may be the same or different.
The term "treatment" as used herein means the prevention, management and care of a patient for the purpose of combating a disease, disorder or condition. The term is intended to include the prevention of the disease, delaying of the progression of the disease, disorder or condition, the alleviation or relief of symptoms and complications, and/or the cure or elimination of the disease, disorder or condition. In an embodiment the patient to be treated is a mammal, in particular a human being.
The term "excipient" as used herein means the chemical compounds which are normally added to pharmaceutical compositions, for example, buffers, tonicity agents, preservatives and the like.
The term "effective amount" as used herein means a dosage which is sufficient to be effective for the treatment of the patient compared with no treatment.
The term "pharmaceutical composition" as used herein means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative, and optionally a tonicity modifier and/or a stabilizer. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation.
The present invention relates to branched polymers attached to GLP-1 which branched polymers are made up of a precise number of monomer building blocks. The monomer building blocks may be oligomerised either on solid support or in solution using suitable monomer protection and activation strategies. A branched polymer attached to GLP- 1 is herein also designated a conjugated GLP-1 or an GLP-1 conjugate. Using the methods described below, it is possible to prepare a conjugated GLP-1 wherein the branched polymer is structural well defined. Hence, the compounds of this invention are monodisperse. Using the process described herein, it is possible to prepare compounds of the general formula I below having a purity above 50%. In an embodiment it is above 75%. In an embodiment it is above 90%. In an embodiment it is above 95%. In an embodiment it is above 99% (weight/weight). In an embodiment, this invention relates to a product containing a single, specific compound of formula I in such a high purity. An embodiment of this invention provides an GLP-1 conjugate as described above, which is represented by the general formula I:
ITA-L4-(L3)m-Y1 (Y2(Y3(Y4(Y5(Y6)r)q)p)s)n (I)
wherein ITA represents an insulinotropic agent from which a hydrogen has been removed from an alpha-amino group present in the insulinotropic agent, or from an epsilon amino group present in lysine at any position in the insulinotropic agent, for the 1 st generation of bifurcated compounds,
Y1 is Yb; Y2 is Z; r, q, p, and s are all zero; and n is 2; for the 2nd generation of bifurcated compounds,
Y1 and Y2 are Yb; Y3 is Z; r, q, and p are all zero; s is 4; and n is 2; for the 3rd generation of bifurcated compounds,
Y1 , Y2, and Y3 are all Yb; Y4 is Z; r and q are zero; p is 8; s is 4; and n is 2; for the 4th generation of bifurcated compounds, Y1 , Y2, Y3, and Y4 are all Yb; Y5 is Z; r is zero; q is 16; p is 8; s is 4; and n is 2; and for the 5th generation of bifurcated compounds,
Y1 , Y2, Y3, Y4, and Y5 are all Yb; Y6 is Z; r is 32; q is 16, p is 8; s is 4; and n is 2; wherein
Yb is -A-LrX3
L-B-
for the 1 st generation of trifurcated compounds, Y1 is Yt; Y2 is Z; r, q, p, and s are all zero; and n is 3; for the 2nd generation of trifurcated compounds, Y1 and Y2 are Yt; Y3 is Z; r, q, and p are all zero; s is 9; and n is 3; for the 3rd generation of trifurcated compounds,
Y1 , Y2, and Y3 are all Yt; Y4 is Z; r and q are zero; p is 27; s is 9; and n is 3; and for the 4th generation of trifurcated compounds,
Y1 , Y2, Y3, and Y4 are all Yt; Y5 is Z; r is zero; q is 81 ; p is 27; s is 9; and n is 3; wherein
wherein A is -CO-, -C(O)O-, -P(=O)(OR)- or -P(=S)(OR)-, wherein R is hydrogen, alkyl or optionally substituted aryl; and B is -NH- or -0-; with the proviso that when B is -NH-, then A is -CO- or -C(O)O-, and when B is -0-, then A is -P(=O)(OR)- or -P(=S)(OR)-; and wherein the group B of one monomer layer (generation) (exemplified by Y1 , Y2, and Y3) is connected to the group A of the adjacent, following layer where Y has the following number as suffix (exemplified by Y2; Y3, and Y4, respectively) or is connected to Z;
X3 is a nitrogen atom, alkantriyl, arenetriyl, alkantrioxy, an aminocarbonyl moiety of the formula -CO-N<, an acetamido moiety of the formula -CH2CO-N< or a moiety of the formula: -CO-NH-Q-NH-CO- I wherein Q is alkantriyl;
X4 is alkantetrayl or arenetetrayl;
In embodiments of the invention L1 is a valence bond, oxy, alkylene, alkyleneoxyalkyl, polyalkoxydiyl, (polyalkoxy)alkylcarbonyl, oxyalkyl or (polyalkoxy)alkyl wherein the terminal alkyl moiety of the last 3 moieties is connected to A ; In embodiments of the invention Li is connected to A in the oxy- part of the last three moieties.
In embodiments of the invention L2 is a valence bond, oxy, alkylene, alkyleneoxyalkyl, polyalkoxydiyl, (polyalkoxy)alkylcarbonyl, oxyalkyl or (polyalkoxy)alkyl wherein the terminal alkyl moiety of the last 3 moieties is connected to B;
In embodiments of the invention L2 is connected to B in the other end of the divalent radicals.
In embodiments of the invention L3 represents a valence bond, alkylene, oxy, polyalkoxydiyl, oxyalkyl, alkylamino, carbonylalkylamino, alkylaminocarbonylalkylamino, carbonylalkyl- carbonylamino(polyalkoxy)alkylamino, carbonylalkoxyalkylcarbonylamino(polyalkoxy)- alkylamino, alkylcarbonylamino(polyalkoxy)alkylamino, carbonyl(polyalkoxy)alkylamino or carbonylalkoxyalkylamino wherein the terminal carbonyl, alkyl and oxy moiety of the last 10 moieties, is connected to the ITA group, optionally via the L4 moiety; In an embodiments of the invention the moieties are connected in the other end of the divalent radical.
m is zero, 1 , 2 or 3;
In embodiments of the invention L4 is selected among a valence bond and a moiety of the formula -CO-L5-CH=N-O-, wherein L5 is a valence bond, alkylene or arylene, and wherein the terminal carbonyl moiety in said L4 moiety, is connected to the ITA moiety; In embodiments of the invention the moieties are connected in the other end of the divalent radical.
and
Z is hydrogen, alkyl, alkoxy, hydroxyalkyl, polyalkoxy, oxyalkyl, acyl, polyalkoxyalkyl, or polyalkoxyalkylcarbonyl.
As defined above, L1, L2, L3 and L4 all shall be interpreted as divalent radicals, X3 is a trivalent radical and X4 is a tetravalent radical.
In the definition of formula I above and elsewhere herein, the three terms bifurcated, trifurcated and generation are used in an attempt to facilitate the understanding hereof and they should not in any way result in a restricted interpretation.
Hence, the 1st generation of bifurcated compounds can be illustrated by the formula Ia:
ITA-U-(U)m-Y1 (Y2)2 (la)
which also can be illustrated by formula Ia'
ITA-L4-(L3)m-Yb-Z
I (Ia')
Z wherein ITA, Y1 , Y2, L3, L4, m, Yb and Z each are as defined above. Furthermore, the 2nd generation bifurcated compounds can be illustrated by the formula Ib ITA-l_4-(L3)m-Y1 (Y2(Y3)4)2 (Ib)
which also can be illustrated by the formula Ib'
Z-Yb-Z
ITA-L4-(L3)m-Yb (Ib')
Z-Yb-Z wherein ITA, Y1 , Y2, Y3, L3, L4, m, Yb and Z each are as defined above.
Alternatively, this invention can be illustrated by drawing the formula of, for example, the 4th generation bifurcated compounds as in the following formula Ic:
wherein all the symbols are as mentioned above (and the perpendicular lines are not a part of the formula, but illustrates the different levels). Formula Ic is only given in an attempt to illustrate this invention and is not to be used to limit the scope of protection.
In an embodiment of this invention, r is zero. In another embodiment of this invention, r and q are each zero. In another embodiment of this invention, n is 2 (for bifurcated compounds) or 3 (for trifurcated compounds). In another embodiment of this invention, s is 4 (for bifurcated compounds) or 9 (for trifurcated compounds). In another embodiment of this invention, s is 4, and p is 8 (for bifurcated compounds) or s is 9, and p is 27 (for trifurcated compounds). As mentioned above, compounds of formula I contains one or more Yb moieties. If a compound of formula I contains more than one Yb moiety, those moieties may be the same or different. In an embodiment of this invention, all Yb moieties are identical. In another embodiment of this invention, the Yb moieties from the same level are identical, but the Yb moieties (or moiety) in one level are (is) different from the Yb moieties (or moiety) in another level, each level being identified by the suffixes n, s, p, q and r, respectively, in formula I. In a specific moiety Yb, the two B moieties are the same or different. In an embodiment such two B moieties are the same. Furthermore, in an embodiment the specific moiety Yb, the two L2 moieties are the same or different. In an embodiment such two L2 moieties are the same. This, similarly, applies for the Yt moieties and the B and L2 moieties present therein. In an embodiment of this invention, it relates to bifurcated compounds, in another embodiment, it relate to trifurcated compounds.
The two or three L2 moieties present in any Yb or Yt moiety, respectively, may be the same or different. In an embodiment of this invention, the two or three L2 moieties present in any Yb or Yt moiety, respectively, are the same.
At least for illustrative purposes but also to some extent in practice, the major part of the non insulinotropic agent of compounds of formula I can be build from compounds of formula IVb or IVc having the following formula:
A'-L-X^ A-L1-X4-L^-B' formula IVb: 1^6' , formula IVc: L?B' wherein L1, L2, X3 and X4 are as defined above, and wherein -A' and -B' are groups which can react to form the moiety -A-B-; wherein A and B are as defined above. The nature of the covalent bond formed by reaction between the groups A' and B' depends upon the selection of A' and B', and include, as indicated above, amide bonds, carbamate bonds, phosphate ester bonds, thiophosphate ester bonds, and phosphit bonds.
In an embodiment, A' is selected from the group consisting of -COOH, -COOR, - OCOOR, -OP(NR2)OR, -(O=)P(OR)2, -(S=)P(OR)(OR'), -(S=)P(SR)(OR'), -(S=)P(SR)(SR'), - COCI, -COBr, -OCOBr, -P(OR)3, p-nitrophenyl carbonate (-OC(=O)OC6H4NO2), succinimidyl carbonate (-OC(=O)-Nhs, where Nhs is N-hydroxysuccinimid), carbonylimidazole (-C(=0)-lm, where Im is imidazol), oxycarbonylimidazole (-0C(=0)-lm, where Im is imidazol), carbonylchlorides (-C(=O)CI), chloroformiate (-OC(=O)CI), isocyanate (-N=C=O) and isothiocyanates (-N=C=S), wherein R and R' represents Ci-6-alkyl, aryl or substituted aryl.
In another embodiment, B' is selected from the group consisting of -NH2, -OH, -N3, - NHR' and -OR'; where R' is a protection group, that facilitates stepwise monomer oligomerization as used in, for example, peptide chemistry and oligonucleotide chemistry. Non-limiting examples of protecting groups includes 9-fluorenylmethoxycarbonyl (designated Fmoc), terf-butoxycarbonyl (designated Boc), phthaloyl, triphenylmethyl, and substituted triphenylmethyl, trihaloacetyl such as trifluoroacetyl or trichloroacetyl, pixyl, trimethylsilyl, terf-butyldimethylsilyl, and terf-butyldiphenylsilyl. Other examples of appropriate protection groups are known to the skilled person, and suggestions can be found in Green & Wuts "Protection groups in organic synthesis", 3rd edition, Wiley-interscience. X3 may be a branched, trivalent organic radical (linker). In an embodiment X3 is of hydrophilic nature. In an embodiment, it includes a multiply-functionalised alkyl group containing up to 18. In an embodiment it contains from 1 to about 10 carbon atoms. In another embodiment, X3 is a single nitrogen atom. In another embodiment, X3 is alkantriyl. In another embodiment, X3 is propan-1 ,2,3-triyl. In another embodiment X3 is an alkantrioxy. In another embodiment, X3 is alkantriyl, alkantrioxy or a moiety of the formula: -CO-NH-Q-NH- CO-, wherein Q is alkantriyl; and, furthermore, X3 can be an aminocarbonyl moiety of the formula -CO-N< or an acetamido moiety of the formula -CH2CO-N< and, in another embodiment X3 has one of the following formulas: the two last moieties being (R) and (S)-1 ,5-bis(aminocarbonyl)pentyl.
In an embodiment of this invention, X4 is symmetrical. In an embodiment X4 is benzen-1 ,3,4,5-tetrayl. In another embodiment of this invention, X3 or X4 is symmetrically.
Examples of L1 and L2 are alkylene and -((CH2)mO)n-, where m' is 2, 3, 4, 5, or 6, and n' is an integer from O to 10. In an embodiment, L1 and L2 are of hydrophilic nature. In another embodiment of this invention, L1, L2 or both are valence bonds.
In another embodiment of this invention, L1 is -CH2(OCH2CH2)n-OCH2C(O)-, where n" is an integer from O to 10.
In another embodiment of this invention, L1 and L2 are independently selected from water soluble organic divalent radicals. In another embodiment of this invention, either L1 or L2 or both are divalent organic radicals containing about 1 to 5 PEG (-CH2CH2O-) groups. In another embodiment of this invention, L1 and L2 are each, independently of each other, a tri, tetra or pentaethylenglycol moiety, i.e., (-CH2CH2O)3, (-CH2CH2O)4 or (-CH2CH2O)5. In another embodiment of this invention, L1 is oxy (-0-) or oxymethyl (-OCH2-), and L2 is a
moiety of the formula (-CH2CH2O-)2, also having the formula: ° .
In an embodiment of this invention, L1 is a valence bond, oxy, alkyleneoxyalkyl, oxyalkyl or
(polyalkoxy)alkyl. In an embodiment L1 is a valence bond, -O- or one of the following three moieties: -OCH2-, -CH2OCH2CH2OCH2CH2OCH2- and -CH2OCH2-. In another embodiment of this invention, L1 is a valence bond, oxy, alkylene, polyalkoxydiyl or oxyalkyl. In another embodiment, L1 is (polyalkoxy)alkylcarbonyl, oxyalkyl or (polyalkoxy)alkyl wherein the terminal alkyl moiety, preferably, is connected to A . In an embodiment the moieties are connected in the other end of the divalent radical. In an embodiment of this invention, L2 is alkylene, alkyleneoxyalkyl, polyalkoxydiyl, or
(polyalkoxy)alkyl. In an embodiment L2 is one of the following four moieties: moieties: -CH2- CH2OCH2CH2O-, -CH2CH2OCH2CH2OCH2CH2OCH2-, -CH2CH2OCH2CH2- or -CH2CH2-. In another embodiment of this invention, L2 is a valence bond, oxy, alkylene, polyalkoxydiyl or oxyalkyl. In another embodiment, L2 is (polyalkoxy)alkylcarbonyl, oxyalkyl or (polyalkoxy)alkyl wherein the terminal alkyl moiety, preferably, is connected to B. In an embodiment the moieties are connected in the other end of the divalent radical.
In an embodiment of this invention, X3 is a nitrogen atom, alkantriyl, arenetriyl or a moiety of the formula: -CO-NH-Q-NH-CO-
wherein Q is alkantriyl.
In an embodiment of this invention, Q is 1 ,1 ,5-pentatriyl. In an embodiment of this invention, m is an integer or 1 , 2 or 3. In another embodiment of this invention, m is an integer. In another embodiment of this invention, m is 1 , 2 or 3.
In an embodiment of this invention, L3 is alkylene, polyalkoxydiyl, oxy, oxyalkyl, and a valence bond, L4 is a bond, and a part of L3 may contain one of the following moieties: oxyiminoarylcarbonyl (-0-N=CH-Ar-CO-) in both isomeric (syn and anti) forms and oxyimino- carbonyl (-0-N=CH-CO-) in both isomeric (syn and anti) forms, in which case, L3 as a hole is aminoalkenyl or aminopolyalkoxydiyl derivatives such as aminoalkyloxyiminoarylcarbonyl or aminopolyalkoxyiminocarbonyl. In another embodiment of this invention, L3 is alkylene, polyalkoxydiyl, oxy, oxyalkyl, and a valence bond, L4 is a bond, and a part of L3 may contain one of the following moieties: oxyiminomethylarylcarbonyl (-0-N=CH-Ar-CO-) in both isomeric (syn and anti) forms and oxyiminoacetyl (-0-N=CH-CO-) in both isomeric (syn and anti) forms, in which case, L3 as a hole is aminoalkenyl or aminopolyalkoxydiyl derivatives such as aminoalkyloxyiminomethylarylcarbonyl or aminopolyalkoxyiminoacetyl. In another embodiment of this invention, L3 is alkylamino, carbonylalkylamino or alkylaminocarbonyl- alkylamino. In an embodiment L3 is one of the following three moieties: -C(O)CH2CH2NH-, - CH2CH2CH2CH2NHC(O)CH2CH2NH- or -CH2CH2CH2CH2NH- .
In an embodiment L3 is a valence bond or a divalent linker radical such as those illustrated by the following six formulae:
wherein each end of the divalent radicals can be attached to the ITA group. In an embodiment the ITA group is attached via the carbonyl group. In an embodiment L4 and the adjacent L3 is a divalent linker radical such as those illustrated by the following eight formulae:
wherein each ends of the divalent radicals can be connected to the ITA group. In embodiments the carbonyl group is attached to ITA group.
In an embodiment of this invention, L4 is an oxyiminoalkylcarbonyl moiety, such as oxyiminoacetyl (-0-N=CH-CO-) in both isomeric (syn and anti) forms. In another embodiment of this invention, L4 is an oxyiminoalkylarylcarbonyl moiety, such as oxyiminoalkylaryl- carbonyl (-0-N=CH-Ar-CO-) in both isomeric (syn and anti) forms. In another embodiment of this invention, L4 is a valence bond. In another embodiment of this invention, L4 is syn or anti forms of one of the moieties of the formulae:
In another embodiment of this invention, L4 is syn and anti forms of the moieties of the formulae:
In an embodiment of this invention, A is -CO-, -C(O)O-, -P(=O)(OR)- or -P(=S)(OR)-, wherein R is hydrogen. In an embodiment A is -CO-, -C(O)O-, -P(O)O - or -P(S)O -.
In an embodiment of this invention, B is -NH- or -0-.
In an embodiment of this invention, Z is hydrogen, alkyl, acyl or polyalkoxyalkyl- carbonyl and in an embodiment Z is H-, CH3OCH2CH2OCH2CH2OCH2C(O)-, CH3- or C6H5C(O)-. In an embodiment Z is hydrogen or one of the three groups: CH3OCH2CH2OCH2- CH2OCH2C(O)-, CH3- and C6H5C(O)-.
In an embodiment of this invention, A' is a carboxyl group, and B' is a protected amino group which after deprotection may be coupled to a new monomer of same structure via its carboxy group to form an amide. Larger polymers can be assembled in a repetitive manner as is known from standard oligopeptide synthesis. In another embodiment of this invention, A' is p-nitrophenylcarbonate (-C(O)-O-pC4H6NO2), carbonylimidazol (-C(O)-lm), - COOH or -C(O)CI. In another embodiment of this invention, B' is N3-, FmocNH- or a group of one of the formulae:
In an embodiment L3 is aminoalkyl, the carbon atom thereof is connected to the part of formula I containing the ITA-L4 moiety. In an embodiment L3 is oxyalkyl, the carbon atom thereof is connected to the part of formula I containing the ITA-L4 moiety. In an embodiment L1 is polyalkoxydiyl, polyalkoxyalkyl or oxyalkyl, the terminal alkylene moiety thereof is, connected to the A moiety. In an embodiment L2 is polyalkoxydiyl, or oxyalkyl, the oxy part thereof is connected to the X3 and X4 moiety.
In another embodiment of this invention, A' is a phosphoramidite and B' is a hydroxyl group suitable protected, which upon deprotection can be coupled to another monomer of the same type to form a phosphite triester which subsequently is oxidised to form a stable phosphate triester or thiophosphate triester. Thus, larger polymers can be assembled in a repetitive manner as is known from standard oligonucleotide synthesis.
In another embodiment, A' is a reactive carbonate such as nitrophenyl carbonate, and B' is an amino group. In an embodiment this is in its protected form. Thus, larger polymers can be assembled in a repetitive manner as is known from standard oligocarbamate synthesis.
In another embodiment, A' is an acyl halide such as -COCI or -COBr, and B' is an amino group. In an embodiment this is in its protected form. In an embodiment A is one of the three moieties: -CO-, -P(O)O- and -P(S)O-. In an embodiment B is oxy or the moiety -NH-.
In an embodiment, the branched polymer of the compounds of this invention has a molecular weight of above about 500 Da. In an embodiment it is above about 3 kDa. In an embodiment it is above about 5 KDa.
In an embodiment, the branched polymer of the compounds of this invention has a molecular weight of below about 10 kDa. In an embodiment it is below about 7 kDa.
In an embodiment, the compounds of this invention have an isoelectric point between about 3 and about 7.
In an embodiment, the compounds of this invention have a net negative charge under physiological conditions. In a further embodiment, the monomer building blocks of the formula
A'-LrX3-(L2-B')2 is
In another embodiment, the monomer building blocks of the formula A'-Li-X3-(L2-B')2 is
In another embodiment, the monomer building blocks of the formula A'-Li-X3-(L2-B')2 is
In an embodiment, L3 is a divalent linker radical such as the following three formula:
In an embodiment, Z is a capping agent that can react with a terminal amino group or hydroxy group. In embodiments Z include the following three examples: o
0 ^O' ^-"Me
In an embodiment, for bifurcated compounds, the major part of the non ITA part of the compounds of formula I is build from monomers of the formula A'-LrX3-(L2-B')2:
In an embodiment of this invention, for trifurcated compounds, the major part of the non ITA part of the compounds of formula I is build from monomers of the formula A'-LrX4- (U-BV
n = 5 n = 5
In one embodiment, ITA is GLP-1 from any natural species and salts thereof, active derivatives of GLP-1 , or GLP-1 analogues, from which a hydrogen atom has been removed as mentioned above. In a still further embodiment, ITA is any of the GLP-1 molecules mentioned specifically in the example below, from which a hydrogen atom has been removed as mentioned above.
In another embodiment of the invention the insulinotropic agent is derived from a peptide having a length between 27 and 45 amino acid residues in which 22 out of the first 28 amino acid residues are identical to those found in corresponding positions in GLP-1 (7-37) (SEQ ID No. 1 ) or in corresponding positions in Exendin-4(1 -39) (SEQ ID No. 2).
In another embodiment of the invention the insulinotropic agent is derived from a peptide having a length between 28 and 45 amino acid residues in which 22 out of the first 28 amino acid residues are identical to those found in corresponding positions in GLP-1 (7- 37) or in corresponding positions in Exendin-4(1 -39). In another embodiment of the invention the insulinotropic agent is selected from a peptide comprising the amino acid sequence of the formula (II):
Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaai6-Ser-Xaai8-Xaai9-Xaa20-Glu-Xaa22-Xaa23-Ala- Xaa25-Xaa26-Xaa27-Phe-lle-Xaa3o-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa4o- Xaa4i"Xaa42-Xaa43-Xaa44"Xaa45-Xaa46
Formula (II) (SEQ ID No: 3)
wherein Xaa7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy- histidine, homohistidine, Nα-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
Xaa8 is Ala, D-AIa, GIy, VaI, Leu, He, Lys, Aib, (1 -aminocyclopropyl) carboxylic acid, (1 - aminocyclobutyl) carboxylic acid, (1 -aminocyclopentyl) carboxylic acid, (1 -aminocyclohexyl) carboxylic acid, (1 -aminocycloheptyl) carboxylic acid, or (1 -aminocyclooctyl) carboxylic acid; Xaaie is VaI or Leu;
Xaais is Ser, Lys or Arg;
Xaaig is Tyr or Gln;
Xaa2o is Leu or Met;
Xaa22 is GIy, GIu or Aib; Xaa23 is GIn, GIu, Lys or Arg;
Xaa25 is Ala or VaI;
Xaa26 is Lys, GIu or Arg;
Xaa30 is Ala, GIu or Arg; Xaa33 is VaI or Lys;
Xaa34 is Lys, GIu, Asn or Arg;
Xaa35 is GIy or Aib;
Xaa36 is Arg, GIy or Lys;
Xaa37 is GIy, Ala, GIu, Pro, Lys, amide or is absent; Xaa38 is Lys, Ser, amide or is absent.
Xaa39 is Ser, Lys, amide or is absent;
Xaa40 is GIy, amide or is absent;
Xaa4i is Ala, amide or is absent;
Xaa42 is Pro, amide or is absent; Xaa43 is Pro, amide or is absent; Xaa44 is Pro, amide or is absent; Xaa45 is Ser, amide or is absent; Xaa46 is amide or is absent ; provided that if Xaa3s, Xaa3g, Xaa4o, Xaa4i, Xaa42, Xaa43, Xaa44, Xaa45 or Xaa46 is absent then each amino acid residue downstream is also absent.
In another embodiment of the invention the insulinotropic agent is a peptide comprising the amino acid sequence of formula (III):
Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaai8-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala- Xaa26-Glu-Phe-lle-Xaa3o-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38
Formula (III) (SEQ ID No: 4) wherein Xaa7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy- histidine, homohistidine, Nα-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; Xaa8 is Ala, D-AIa, GIy, VaI, Leu, He, Lys, Aib, (1 -aminocyclopropyl) carboxylic acid, (1 - aminocyclobutyl) carboxylic acid, (1 -aminocyclopentyl) carboxylic acid, (1 -aminocyclohexyl) carboxylic acid, (1 -aminocycloheptyl) carboxylic acid, or (1 -aminocyclooctyl) carboxylic acid;
Xaais is Ser, Lys or Arg;
Xaa22 is GIy, GIu or Aib; Xaa23 is GIn, GIu, Lys or Arg;
Xaa26 is Lys, GIu or Arg;
Xaa30 is Ala, GIu or Arg;
Xaa34 is Lys, GIu or Arg;
Xaa35 is GIy or Aib; Xaa36 is Arg or Lys;
Xaa37 is GIy, Ala, GIu or Lys;
Xaa38 is Lys, amide or is absent.
In another embodiment of the invention the insulinotropic agent is selected from
GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-36)-amide, GLP-1 (7-37), GLP-1 (7-38), GLP-1 (7-39), GLP-1 (7-40), GLP-1 (7-41 ) or an analogue thereof.
In another embodiment of the invention the insulinotropic agent comprises no more than fifteen amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ), or no more than ten amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ). In another embodiment of the invention the insulinotropic agent comprises no more than six amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ).
In another embodiment of the invention the insulinotropic agent comprises no more than 4 amino acid residues which are not encoded by the genetic code.
In another embodiment of the invention the insulinotropic agent comprises an Aib residue as the second amino acid residue from the N-terminal.
In another embodiment of the invention the N-terminal amino acid residue (position 7 in formulae Il and III) of said insulinotropic agent is selected from the group consisting of D- histidine, desamino-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, Nα- acetyl-histidine , α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2- pyridylalanine and 4-pyridylalanine.
In another embodiment of the invention the insulinotropic agent is selected from the group consisting of [Arg34]GLP-1 (7-37), [Arg26'34]GLP-1 (7-37)l_ys, [Lys36Arg26'34]GLP-1 (7-36), [Aib8'22'35]GLP-1 (7-37),
[Aib8'35]GLP-1 (7-37), [Aib8'22]GLP-1 (7-37), [Aib8'22'35 Arg26'34]GLP-1 (7-37)Lys, [Aib8'35 Arg26'34]GLP-1 (7-37)Lys, [Aib8'22 Arg26'34]GLP-1 (7-37)Lys, [Aib 8,22,35 Arg 26,34 ]GLp.1 (7_37)Lys> [Aib8'35 Arg26'34]GLP-1 (7-37)Lys,
[Aib8'22'35 Arg26]GLP-1 (7-37)Lys, [Aib8'35 Arg26]GLP-1 (7-37)Lys, [Aib8'22 Arg26]GLP-1 (7-37)Lys, [Aib8'22'35 Arg34]GLP-1 (7-37)Lys, [Aib8'35Arg34]GLP-1 (7-37)Lys, [Aib8'22Arg34]GLP-1 (7-37)Lys, [Aib8'22'35Ala37]GLP-1 (7-37)Lys, [Aib8'35Ala37]GLP-1 (7-37)Lys, [Aib8'22Ala37]GLP-1 (7-37)Lys, [Aib8'22'35 Lys37]GLP-1 (7-37), [Aib8'35l_ys37]GLP-1 (7-37), [Aib8'22Lys37]GLP-1 (7-37) or derivatives thereof which has been amidated on the C-terminal.
In another embodiment of the invention the insulinotropic agent comprises at least one Aib residue.
In another embodiment of the invention the insulinotropic agent contains two Aib residues.
In another embodiment of the invention the insulinotropic agent comprises a serine residue at position 18 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 12 relative to Exendin-4(1 -39).
In another embodiment of the invention the insulinotropic agent comprises a tyrosine residue at position 19 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 13 relative to Exendin-4(1 -39). In another embodiment of the invention the insulinotropic agent comprises a glycine residue at position 22 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 16 relative to Exendin-4(1 -39).
In another embodiment of the invention the insulinotropic agent comprises a glutamine residue at position 23 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 17 relative to Exendin-4(1 -39).
In another embodiment of the invention the insulinotropic agent comprises a lysine residue at position 26 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 20 relative to Exendin-4(1 -39). In another embodiment of the invention the insulinotropic agent comprises a glutamate residue at position 27 relative to GLP-1 (7-37) (SEQ ID. No. 1 ), corresponding to position 21 relative to Exendin-4(1 -39).
In another embodiment of the invention the insulinotropic agent is exendin-4(1 -39). In another embodiment of the invention the insulinotropic agent is ZP-10, i.e. [Ser38Lys39]Exendin-4(1 -39)LysLysLysLysLys-amide (SEQ ID No. 5).
In another embodiment of the invention the insulinotropic agent is attached the branched polymer via the amino acid residue in position 25 to 45 relative to the amino acid sequence SEQ ID No 1 .
In another embodiment of the invention the insulinotropic agent is attached to the branched polymer via an amino acid residue selected from one of the 10 C-terminal amino acid residues.
In another embodiment of the invention the insulinotropic agent is attached to the branched polymer via the amino acid residue in position 23, 26, 34, 36 or 38 relative to the amino acid sequence SEQ ID No: 1 . In another embodiment of the invention the insulinotropic agent is attached to the branched polymer via the amino acid residue in position 17, 20, 28, 30 or 32 relative to the amino acid sequence SEQ ID No: 2.
In another embodiment of the invention the insulinotropic agent is attached to the branched polymer via the C-terminal amino acid residue. In another embodiment of the invention the insulinotropic agent is attached to the branched polymer via a carboxyl group, an amino group, a keto group, a hydroxyl group, a thiol group or a hydrazide group.
In another embodiment of the invention the insulinotropic agent is attached to the branched polymer via a the epsilon-amino group on a lysine residue. In another embodiment of the invention the insulinotropic agent comprises only one lysine residue.
In another embodiment of the invention the insulinotropic agent comprises only one lysine residue which is the C-terminal amino acid residue of said insulinotropic agent. In another embodiment the compound according to the present invention has an
EC50 of less than 1000 pM, less than 500 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM or less than 10 pM as determined by the functional receptor assay disclosed herein.
In another embodiment the compound according to the present invention is selected from the group consisting of
The GLP-1 analogs can be produced by classical peptide synthesis, for example, solid phase peptide synthesis using t-Boc or F-Moc chemistry or other well established techniques., see the examples and for example, Houben-Weyl, Methods of organic Chemistry, Volume E 22a, E 22b and E 22 c; Green and Wuts, "Protecting Groups in Organic Synthesis", Jogn Wiley & Sons, 1999. These methods are preferred when the insulinotropic agent is a peptide comprising non-natural amino acid residues.
When the insulinotropic agent is a polypeptide comprising only amino acid residues encoded by the genetic code, the polypeptides can also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide, after which the resulting peptide is recovered from the culture and then derivatized to the compound of formula (I).
The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (for example, in catalogues of the American Type Culture Collection). The peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration. For extracellular products the proteinaceous components of the supernatant are isolated by filtration, column chromatography or precipitation, for example, microfiltation, ultrafiltration, isoelectric precipitation, purification by a variety of chromatographic procedures, for example, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question. For intracellular or periplasmic products the cells isolated from the culture medium are disintegrated or permeabilised and extracted to recover the product polypeptide or precursor thereof.
The DNA sequence encoding the therapeutic polypeptide may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the peptide by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the polypeptide may also be prepared synthetically by established standard methods, for example, the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981 ), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801 - 805. The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
The DNA sequence may be inserted into any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, for example, a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
In an embodiment the vector is an expression vector in which the DNA sequence encoding the polypeptide is operably linked to additional segments required for transcription of the DNA, such as a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the peptide of the invention in a variety of host cells are well known in the art, cf. for instance Sambrook et al., supra.
The DNA sequence encoding the polypeptide may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
The vector may also comprise a selectable marker, for example, a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, for example, ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. In embodiments of large scale manufacture the selectable marker is not antibiotic resistance, for example, antibiotic resistance genes in the vector are excised when the vector is used for large scale manufacture. Methods for eliminating antibiotic resistance genes from vectors are known in the art, see for example, US 6,358,705 which is incorporated herein by reference.
To direct a parent peptide of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the peptide. The secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.
The procedures used to ligate the DNA sequences coding for the present peptide, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook etal.., supra).
The host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
Obviously, by combining one or more of the embodiments of this invention described herein including the claims below, new embodiments are obtained.
Branched polymers can in general be assembled from the monomer building blocks described above using one of two fundamentally different oligomerisation strategies called the divergent approach and the convergent approach.
Divergent assembly of branched polymers: In one embodiment, the branched polymers are assembled by an iterative process of synthesis cycles, where each cycle use suitable activated, reactive bi or trifurcated monomer building blocks, them self containing functional end groups - allowing for further elongation (i.e. polymer "growth"). The functional end groups usually needs to be protected in order to prevent self polymerisation and a deprotection step will in such cases be needed in order to generate a functional end group necessary for further elongation. One such cycle of adding an activated (reactive) monomer building block and subsequent deprotection in the iterative process completes a generation. The divergent approach is illustrated in reaction scheme 4 using solution phase chemistry and in reaction scheme 3 using solid phase chemistry.
Convergent assembly of branched polymers:
However, when higher generation materials are reached in such an iterative process, a high packing density of functional end groups will frequently appear which prevents further regular growth leading to incomplete generations. In fact, with all systems in which growth requires the reaction of large numbers of surface functional groups, it is difficult to ensure that all will react at each growth step. Since unreacted functional end groups may lead to failure sequences (truncation) or spurious reactivity at later stages of the stepwise growth sequence, this poses a significant problem in the synthesis of regular monodispersed and highly organised branched structures.
In an embodiment, the branched polymer therefore is assembled by the convergent approach described in US patent 5,041 ,516. The convergent approach to build macromolecules involves building the final molecule by beginning at its periphery, rather than at its core as in the divergent approach. This avoids problems, such as incomplete formation of covalent bonds, typically associated with the reaction at progressively larger numbers of sites. The convergent approach for assembly 2nd generation branched polymer is illustrated in reaction scheme 1 and reaction scheme 2 using a specific example involving one of the monomer building blocks.
Rigidity of the branched polymer can be controlled by the design of the particular monomer, for example by using a rigid core structure (X3 or X4) or by using rigid linker moieties (L1 and L2). In another embodiment, adjustment of the rigity is obtained by using the rigid monomer in one or more specific layers intermixed with monomers of more flexible nature. In another embodiment, the overall hydrophilic nature of the polymer is controllable. This is achieved by choosing monomers with more hydrophobic core structure (X3 or X4) or more hydrophobic linker moieties (L1 and L2), in one or more of the dendritic layers. In another embodiment, a different monomer is used in the outer terminal layer (Z) of the branched polymer, which in the final GLP-1 conjugate will be exposed to the surrounding environment. Some of the monomers described here have protected amine functions as terminal end groups (B'), which after a deprotection step, and under physiological conditions, i.e. neutral physiological buffered to a pH value around 7.4, will be protonated, causing the overall structure to be polycationically charged. Alternatively, neutral structures can be made by capping with various acylating reagents. One example as depicted in reaction scheme 5 uses CH3(OCH2CH2)2CH2COOH for capping the final layer (Z) of a dendritic structure, that otherwise would be terminated in amines.
In another embodiment, branched polymers is provided which imitates the natural occurring glycopeptides, which commonly has multiple anionic charged sialic acids as termination groups on the antenna structure of their N-glycans. By a proper choice of monomer used to create the final layer (Z), such glycans can be imitated with respect to their poly anionic nature. One such example is depicted in reaction scheme 6, where the branched polymer is capped with succinic acid mono terf-butyl esters which upon deprotection with acids render a polymer surface that is negatively charged under physiological conditions.
The assembly of monomers into polymers may for example be conducted either on solid support as described by NJ. Wells, A. Basso and M. Bradley in Biopolymers 47, 381 - 396 (1998), or in an appropriate organic solvent by classical solution phase chemistry, for example, as described by Frechet et al. in U.S. patent 5,041 ,516.
Thus in an embodiment, the branched polymer is assembled on a solid support derivatised with a suitable linkage in an iterative divergent process as described above and illustrated in reaction scheme 3. For monomers designed with Fmoc or Boc protected amino groups (B'), and reactive functional acylating moieties (A'), solid phase protocols useful for conventional peptide synthesis can conveniently be adapted. Applicably standard solid phase techniques such as those described in the literature (see Fields, editor, Solid phase peptide synthesis, in Meth Enzymol 289) can be conducted either by use of suitable programmable instruments (for example, ABI 430A) or similar home build machines, or manually using standard filtration techniques for separation and washing of support. For monomers with, for example, DMT protected alcohol groups (B') and, for example, reactive phosphor amidites (A'), solid phase equipment used for standard oligonucleotide synthesis such as Applied Biosystems Expidite 8909, and conditions such as those recently described by M. Dubber and J.M.J. Frechet in Bioconjugate chem. 2003, 14, 239-246, can conveniently be applied. Solid phase synthesis of such phosphate diesters according to the conventional phosphoramidite methodology usually requiers that an intermediate phosphite triester is oxidised to a phosphate triester. This type of solid support oxidation is typically achieved with iodine/water or peroxides such as but not limited to tert- butyl hydrogenperoxid and 3-chloroperbenzoic acid and requires that the monomers with or without protection resist oxidation condition. The phosphor amidite methodology also allows for convenient synthesis of thiophosphates by simple replacement of the iodine with elementary sulfur in pyridine or organic thiolation reagents such as 3H-1 ,2-benzodithiole-3- one-1 ,1 -dioxide (see, for example, M. Dubber and J.M.J. Frechet in Bioconjugate chem. 2003, 14, 239-246).
The resin attached branched polymer, when complete, can then be cleaved from the resin under suitable conditions. It is important, that the cleavable linker between the growing polymer and the solid support is selected in such way that it will stay intact during the oligomerisation process of the individual monomers, including any deprotection steps, oxidation or reduction steps used in the individual synthesis cycle, but when desired under appropriate conditions can be cleaved leaving the final branched polymer intact. The skilled person will be able to make suitable choices of linker and support, as well as reaction conditions for the oligomerisation process, the deprotection process, and optionally oxidation process, depending on the monomers in question.
Resins derivatised with appropriate functional groups, that allows for attachment of monomer units and later act as cleavable moieties, are commercial available (see, for example, the catalogue of Bachem and NovoBiochem).
In another embodiment, the branched polymer is synthesised on a resin with a suitable linker, which upon cleavage generates a branched polymer product furnished with a functional group that directly can act as an attachment group in a subsequent solution phase conjugation process to the insulionotropic agent (ITA) as described below or, alternatively, by appropriate chemical means can be converted into such an attachment group.
In another embodiment, the dendritic branched polymers of a certain size and compositions is synthesised using classical solution phase techniques.
In this embodiment, the branched polymer is assembled in an appropriate solvent, by sequential addition of suitable activated monomers to the growing polymer. After each addition, a deprotection step may be needed before construction of the next generation can be initiated. It may be desirable to use excess of monomer in order to reach complete reactions. In an embodiment, the removal of excess monomer takes advantages of the fact that hydrophilic polymers have low solubility in diethyl ether or similar types of solvents. The growing polymer can thus be precipitated leaving the excess of monomers, coupling reagents, by-products etc. in solution. Phase separation can then be performed by simple decantation. In embodiments by centrifugation followed by decantation. Polymers can also be separated from by-products by conventional chromatographic techniques on, for example, silica gel, or by the use of HPLC or MPLC systems under either normal or reverse phase conditions as described by P. R. Ashton in et al. in J.Org.Chem. 1998, 63, 3429-3437. Alternatively, the considerably larger polymer can be separated from low molecular components, such as excess monomers and by-products, using size exclusion chromatography, optionally in combination with dialysis as described by E.R.Gillies and J.M.J. Frechet in J. Am. Chem. Soc. 2002, 124, 14137-14146.
In another embodiment, a convergent solution phase synthesis is used. In contrast to solid phase techniques, solution phase also makes it possible to use the convergent approach for assembly of branched polymers as described above and further reviewed by S.M.Grayson and J. M. J. Frechet in Chem. Rev. 2001 , IQl, 3819-3867. In this approach, it is desirable to initiate the synthesis with monomers, where the protected functional end groups (B') initially are converted into moieties that eventually will be present on the outer surface of the final branched polymer. Therefore, the functional moiety (A') of general formula I in most cases will need suitable protection that allows for stepwise chemical manipulation of the end groups (B'). The choice of protection groups for the functional moiety (A') depends on the actually functional group. For example, if A' in general formula IVb or IVc is a carboxyl group, a terf-butyl ester derivate that can be removed by TFA would be an appropriate choice. Suitable protection groups are known to the skilled person, and other examples can be found in Green & Wuts "Protection groups in organic synthesis", 3rd edition, Wiley-interscience. The convergent assembly of branched polymers is illustrated in reaction scheme 1 and reaction scheme 2. The rection schemes can be found below. In step (i) of reaction scheme 1 , a tert- butyl ester functionality (A') is prepared by reaction of a suitable precurser with terf-butyl α- bromoacetate. In step (ii), the terminal end groups (B') are manipulated in such way that they allow for the acylation of step (iii) with a carboxylic acid that is converted into an acyl halid in step (iv). In step (v), the terf-butyl ester functionality (A') is removed creating an end (B') capped monomer. This end capped monomer serves as starting material for preparing the second generation product in reaction scheme 2, where two equivalents are used in an acylation reaction with the product of step (ii) in reaction scheme 1. The product of this reaction is a new terf-butyl ester, which after deprotection can re-enter in the initial step of reaction scheme 2 in an iterative manner creating higher generation materials.
To effect covalent attachment of the branched polymer molecule(s) to the insulinotropic agent (ITA), either in solution or on solid support, the branched polymer must be provided with a reactive handle, i.e., furnished with a reactive functional group, examples of which include carboxylic acids, primary amino groups, hydrazides, O-alkylated hydroxylamines, thiols, succinates, succinimidyl succinates, succimidyl proprionates, succimidyl carboxymethylates, hydrazides arylcarbonates and aryl carbamates such as nitrophenylcarbamates and nitrophenyl carbonates, chlorocarbonates, isothiocyanates, isocyanates, malemides, and activated esters such as:
The conjugation of the branched polymer to ITA is conducted by conventional methods, known to the skilled artisan. The skilled person will be aware that the activation method and/or conjugation chemistry (for example, choice of reaction groups ect.) to be use depends on the attachment group(s) selected on the ITA (for example, amino groups, hydroxyl groups, thiol groups ect.) and the branched polymer (for example, succimidyl proprionates, nitrophenylcarbonates, malimides, vinylsulfones, haloacetates ect.). In another embodiment, suitable attachment moieties on the branched polymer, such as those mentioned above, are created after the branched polymer has been assembled using conventional solution phase chemistry. Embodiments of this invention, illustrating different ways to create nucleophilic attachment moieties on a branched polymer containing a carboxylic acid group are listed in reaction scheme 7.
As an alternative to direct acylation of ε-amino group on lysine with branched polymer derivatives, insulinotropic agent (ITA) may initially be acylated with formyl derivated carboxyl acids, for example, using activation such as N-hydroxysuccinimide esters, 1 - hydroxybenzotriazol esters and the like. The resulting ITA carrying an aldehyde functionality may then in turn be condensed with mono-, oligo- or polymeric building blocks of the invention suitable derivatized as, for example, O-substituted hydroxylamines, hydrazines or hydrazides, by mixing the two components in an aqueous media, optionally containing organic co-solvents at neutral, acid or alkaline pH. In this case L4 is a valence bond, and the divalent radical L3 in the general formula I contains an oxime group. Representative non limiting examples of the moiety L4 plus the adjacent L3 include (as syn and anti forms):
O
In embodiments L3 is a divalent radical according to the definitions, and L4 is selected among a valence bond and a moiety of the formula -CO-L5-CH=N-O-, wherein L5 is a valence bond, alkylene or arylene, and wherein the terminal carbonyl moiety in said L4 moiety is connected to the ITA moiety. Representative non limiting examples of L4 includes (as syn and anti forms):
Alternatively insulinotropic agent (ITA) may be derivatized with a moiety that after a chemical reaction, such as, for example, a periodate oxidation, may generate an ITA molecule containing an aldehyde functionality. The ITA carrying an aldehyde functionality may then as above be condensed with mono-, oligo- or polymeric building blocks of the invention similarly derivatized as, for example, O-substituted hydroxylamines, hydrazines or hydrazides, by mixing the two components in an aqueous media, optionally containing organic co-solvents at neutral, acid or alkaline pH.
A particular example is an initial acylation of ε-amino group on lysine with serine, followed by a periodate cleavage to generate glyoxyl derived ITA. In this case, representative non limiting examples of the divalent L4 moiety plus the adjacent L3 moiety include (as syn and anti forms):
L3 may also be a divalent radical according to the definitions, and L4 may be an oxyiminoalkylcarbonyl group. Representative non limiting examples includes (as syn and anti forms):
The biologically active ITA is reacted with the activated branched polymers in an aqueous reaction medium which is optionally buffered, depending upon the pH requirements of the ITA. The optimum pH value for the reaction is generally between about 6.5 and about 8. In an embodiment about 7.4 for most ITA analogues.
The optimum reaction conditions for the insulinotropic agent (ITA) stability, reaction efficiency, etc. is within level of ordinary skill in the art. In an embodiment the temperature range is from about 40C to about 370C. The temperature of the reaction medium cannot exceed the temperature at which the insulinotropic agent (ITA) may denature or decompose. In an embodiment the insulinotropic agent (ITA) is reacted with an excess of the activated branched polymer. Following the reaction, the conjugate is recovered and purified such as by diafiltration, column chromatography including size exclusion chromatography, ion-exchange chromatograph, affinity chromatography, electrophoreses, or combinations thereof, or the like.
In an embodiment, the method of conjugation is based upon standard chemistry, which is performed in the following manner. The branched polymer has an aminooxyacetyl group attached during synthesis, for example, by acylation of diaminoalkyl linked aminooxyacetic acid as depicted in reaction scheme 7. The ITA has a terminal serine or threonine residue, which is oxidised to a glyoxylyl group under mild conditions with periodate according to Rose in J. Am. Chem. Soc. 1994, V\ 6, 30-33, and European patent 0243929. Alternatively, an aldehyde function may be introduced by acylating an exposed amino group such as an epsilon amino group of a lysin residue with an acylating moiety containing an aldehyde or a temporarily protected aldehyd group. The aminooxy component of the branched polymer and the aldehyde component of the ITA are mixed in approximately equal proportions at a concentration in the range from about 1 to about 10 mM in aqueous solution at mildly acid conditions, for example at a pH value in the range from about 2 to about 5, especially at around room temperature, and the conjugation reaction (in this case oximation) is followed by reversed phase high pressure liquid chromatography (HPLC) and electrospray ionisation mass spectrometry (ES-MS). The reaction speed depends on concentrations, pH value, and steric factors but is normally at equilibrium within a few hours, and the equilibrium is greatly in favour of conjugate (Rose, et al., Bioconjugate Chemistry 1996, 7, 552-556). A slight excess (up to about five fold) of one component forces the conjugation reaction towards completion. Products are isolated and characterised as previously described for oximes. ITA analogues are purified, for example, by reversed phase HPLC (Rose, J Am. Chem.Soc, supra and Rose, et al., Bioconjugate Chemistry, supra).
In another embodiment, the method of conjugation is performed in the following manner: The branched polymer is synthesised on the Sasrin or Wang resin (Bachem) as depicted in reaction scheme 3. Using the procedure recommended by the resin manufacturer (Bachem), the branched polymer is cleaved from the resin by repeated treatment with TFA in dichloromethane and the solution of cleaved polymer is neutralised with pyridine in methanol. After evaporation of solvents at room temperature (no heat is applied) and purification of the cleaved polymer as if it was GLP-1 , the carboxyl group which was connected to the resin is activated (for example, with HBTU, TSTU or HATU) and coupled to a nucleophilic group (such as an amino group, i.e., an epsilon amino group on the side chain of lysin) on the insulinotropic agent (ITA) by standard techniques of peptide chemistry. If desired, the modified target molecule or material can be purified from the reaction mixture by one of numerous purification methods that are well known to those of ordinary skill in the art such as size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, preparative isoelectric focusing, etc. General methods and principles for macromolecule purification, particularly peptide purification, can be found, for example, in "Protein Purification: Principles and Practice" by Seeres, 2nd edition, Springer-Verlag, New York, NY, (1987), which is incorporated herein by reference.
Many of the parent GLP-1 analogues or GLP-1 derivatives used for preparing the compounds of this invention are known (see L. B. Knudsen et al., J. Med. Chem. 2000, 43, 1664-1669, for a series of non-limited examples) and other can be prepared analogously with the preparation of the known compounds or by other methods which will be obvious for the skilled art worker.
The foregoing is illustrative of the insulinotropic agent (ITA) includes GLP-1 analogues, which are suitable for conjugation with the branched polymers. It is to be understood that insulinotropic agent and analogues not specifically mentioned but having suitable properties are also intended and are within the scope of the present invention.
In another embodiment, water soluble polymers are provided. These are important as agents for enhancing the properties of the GLP-1 analogues. For example, by conjugating water soluble polymers to GLP-1 analogues to increased solubility. The attachment of a branched polymer to GLP-1 analogues, that have inherent immunogenic properties provides conjugates with decreased immune response compared to the immune response generated by the non conjugated GLP-1 analogues, or an increased pharmacokinetic profile, an increased shelf-life, and an increased biological half-life. This invention provides GLP-1 analogues which are modified by the attachment of the hydrophilic water soluble branched polymers without substantially reducing or interfering with the biologic activity of the non modified GLP-1 analogues.
This invention GLP-1 analogues modified by the structurally well defined polymers, which are essentially homogeneous compounds, wherein the number of generations of the branched polymer is well defined.
This invention provides conjugates which have maintained the biological activity of the non conjugated GLP-1 . In another embodiment of this invention, the conjugated GLP-1 has improved characteristics compared to the non-conjugated GLP-1.
In another embodiment of this invention, the branched polymers conjugated to certain parts of GLP-1 reduce the bioavailability, the potency, and the efficacy or the activity of GLP- 1 . Such reduction can be desirable in drug delivery systems based on the sustain release principle. In another embodiment, a sustain release principle in which the branched polymer is used in connection with a linker that can be cleaved under physiological conditions, thereby releasing the bio-active GLP-1 slowly from the branched polymer, is contemplated. In this case, the GLP-1 may not be biological active before the branched polymer is removed. In a specific embodiment, the cleavable linker is a small peptide that can function as a substrate for, for example, proteases present in the blood serum.
It will be understood that the polymer conjugation is designed so as to produce the optimal molecule with respect to the number of polymer molecules attached, the size, and composition (for example, number of generations and particular monomer used in each generation), and the attachment site(s) on GLP-1 . The particular molecular weight of the branched polymer to be used may, for example, be chosen on the basis of the desired effect to be achieved. For instance, if the primary purpose of the conjugate is to achieve a conjugate having a high molecular weight (for example, to reduce renal clearance), it is usually desirable to conjugate as few high molecular branched polymer molecules as possible to obtain the desirable molecular weight. In other cases, protection against specific or unspecific proteolytical cleavage or shielding of an immunogenic epitope on the GLP-1 can be desirable, and a branched polymer with a specific low molecular weight may be the optimal choice. Thus, by this invention, polymer derivatised GLP-1 analogues (conjugates) with a fine-tuned predefined mass is obtained.
In still another embodiment of this invention, a branched polymer prepared as described herein, is conjugated to GLP-1 . In another embodiment of this invention, this produces a conjugate with increased pulmonal bioavailability. In another embodiment of this invention, this produce a conjugate with increased pulmonary duration of action.
In a related embodiment, a branched polymer as described herein is used to shield immunogenic epitopes on biopharmaceutical GLP-1 obtained from non-human sources.
In yet another embodiment, a branched water soluble polymer is conjugated to GLP-1 that in its unmodified state and under physiological conditions has a low solubility.
In another embodiment, the in vivo half life of certain GLP-1 conjugates of this invention is improved by more than 10%. In an embodiment, the in vivo half life of certain GLP-1 conjugates is improved by more than 25%. In an embodiment, the in vivo half life of GLP-1 conjugates is improved by more than 50%. In an embodiment, the in vivo half life of certain GLP-1 conjugates is improved by more than 75%. In an embodiment, the in vivo half life of certain GLP-1 conjugates is improved by more than 100%. In another embodiment, the in vivo half life of certain GLP-1 increased 250 % upon conjugation of a branched polymer.
In another embodiment, the functional in vivo half life of certain GLP-1 conjugates of this invention is improved by more than 10%. In another embodiment, the functional in vivo half life of certain GLP-1 conjugates is improved by more than 25%. In another embodiment, the functional in vivo half life of certain GLP-1 conjugates is improved by more than 50%. In another embodiment, the functional in vivo half life of certain GLP-1 conjugates is improved by more than 75%. In another embodiment, the functional in vivo half life of certain GLP-1 conjugates is improved by more than 100%. In another embodiment, the functional half life of certain GLP-1 is increased 250 % upon conjugation of a branched polymer.
Generally, the stability of GLP-1 analogues in solution is very poor. Therefore, in one embodiment of this invention, well defined water soluble branched polymers as described herein can conjugate GLP-1 analogues and stabilize the GLP-1 by minimizing structural transformations such as refolding and maintain GLP-1 activity. In a related embodiment, the shelf-half life of GLP-1 is improved upon conjugation to a branched polymer as described herein.
In another embodiment of the invention the insulinotropic agent is a DPPIV protected peptide.
In another embodiment of the invention the insulinotropic agent has an EC50 of less than 1 nM as determined by the functional receptor assay disclosed herein. In another embodiment of the invention the insulinotropic agent has an EC50 of less than 300 pM, less than 200 pM or less than 100 pM as determined by the functional receptor assay disclosed herein.
Reaction scheme 1 - Convergent synthesis in solution - Capped - first generation
Reaction scheme 2: Second generation with protected focal point
Reaction scheme 3: Solid phase synthesis of a second generation branched polymer
R=BoC
R=H
Reaction scheme 4: Divergent synthesis of a second generation material in solution
Reaction scheme 5: illustration of end capping of a second generation polymer using a Me(PEG)2CH2COOH acid.
DIC, HOBt
Reaction scheme 6: illustration of end capping of a second generation polymer attatched to a solid support or an insulinotropic agent (R) using succinic acid mono tert butyl ester to create a poly anionic glyco mimic polymer.
Reaction scheme 7: Formation of suitable reactive handles for polymer conjugation to ITA molecules. Illustrated for a second generation polymer material.
PHARMACEUTICAL ADMINISTRATION
The conjugated GLP-1 analogues of this invention of formula I can, for example, be administered subcutaneously, orally, or pulmonary. For subcutaneous administration, the compounds of formula I are formulated analogously with the formulation of known GLP-1 analogues. Furthermore, for subcutaneous administration, the compounds of formula I are administered analogously with the administration of known GLP-1 analogues and, generally, the physicians are familiar with this procedure. For oral administration, the compounds of formula I are formulated analogously with the formulation of other medicaments which are to be administered orally. Furthermore, for oral administration, the compounds of formula I are administered analogously with the administration of known oral medicaments and, principally, the physicians are familiar with such procedure. For pulmonary products, the following details are given:
The conjugated GLP-1 analogues of this invention may be administered by inhalation in a dose effective manner to increase circulating GLP-1 levels and/or to lower circulating glucose levels. Such administration can be effective for treating disorders such as diabetes or hyperglycemia. Achieving effective doses of GLP-1 requires administration of an inhaled dose of more than about 0.5 μg/kg to about 50 μg/kg of conjugated GLP-1 of this invention. A therapeutically effective amount can be determined by a knowledgeable practitioner, who will take into account factors including GLP-1 level, blood glucose levels, the physical condition of the patient, the patient's pulmonary status, or the like. According to the invention, conjugated GLP-1 of this invention may be delivered by inhalation to achieve rapid absorption thereof. Administration by inhalation can result in pharmacokinetics comparable to subcutaneous administration of GLP-1 analogues. Inhalation of a conjugated GLP-1 of this invention leads to a rapid rise in the level of circulating GLP-1 followed by a rapid fall in blood glucose levels. Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.
According to the invention, conjugated GLP-1 of this invention may be delivered by any of a variety of inhalation devices known in the art for administration of a therapeutic agent by inhalation. These devices include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. In an embodiment conjugated GLP-1 of this invention is delivered by a dry powder inhaler or a sprayer. There are a several desirable features of an inhalation device for administering conjugated GLP-1 of this invention. For example, delivery by the inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device should deliver small particles, for example, less than about 10 μm, for example about 1 -5 μm, for good respirability. Some specific examples of commercially available inhalation devices suitable for the practice of this invention are Turbohaler™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed by Inhale Therapeutics, AEFtx™ (Aradigm), the Ultravent® nebulizer (Mallinckrodt), the Acorn II® nebulizer (Marquest Medical Products), the Ventolin® metered dose inhaler (Glaxo), the Spinhaler® powder inhaler (Fisons), or the like.
As those skilled in the art will recognize, the formulation of conjugated GLP-1 of this invention, the quantity of the formulation delivered, and the duration of administration of a single dose depend on the type of inhalation device employed. For some aerosol delivery systems, such as nebulizers, the frequency of administration and length of time for which the system is activated will depend mainly on the concentration of GLP-1 conjugate in the aerosol. For example, shorter periods of administration can be used at higher concentrations of GLP-1 conjugate in the nebulizer solution. Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods to deliver the desired amount of GLP-1 conjugate. Devices such as powder inhalers deliver active agent until a given charge of agent is expelled from the device. In this type of inhaler, the amount of conjugated GLP-1 of this invention in a given quantity of the powder determines the dose delivered in a single administration.
The particle size of conjugated GLP-1 of this invention in the formulation delivered by the inhalation device is critical with respect to the ability of GLP-1 to make it into the lungs. In an embodiment into the lower airways or alveoli. In an embodiment the conjugated GLP-1 of this invention is formulated so that at least about 10% of the GLP-1 conjugate delivered is deposited in the lung. In an embodiment about 10 to about 20%, or more. It is known that the maximum efficiency of pulmonary deposition for mouth breathing humans is obtained with particle sizes of about 2 μm to about 3 μm. When particle sizes are above about 5 mμ, pulmonary deposition decreases substantially. Particle sizes below about 1 μm cause pulmonary deposition to decrease, and it becomes difficult to deliver particles with sufficient mass to be therapeutically effective. Thus, in embodiments of the invention particles of GLP- 1 conjugate delivered by inhalation have a particle size less than about 10 μm. In an embodiments in the range of about 1 μm to about 5 μm. The formulation of GLP-1 conjugate is selected to yield the desired particle size in the chosen inhalation device.
In an embodiment for administration as a dry powder, conjugated GLP-1 of this invention is prepared in a particulate form with a particle size of less than about 10 μm. In an embodiment about 1 to about 5 μm. In an embodiment the particle size is effective for delivery to the alveoli of the patient's lung. In an embodiment the dry powder is largely composed of particles produced so that a majority of the particles have a size in the desired range. In an embodiment at least about 50% of the dry powder is made of particles having a diameter less than about 10 μm. Such formulations can be achieved by spray drying, milling, or critical point condensation of a solution containing GLP-1 conjugate and other desired ingredients. Other methods also suitable for generating particles useful in the current invention are known in the art.
The particles are usually separated from a dry powder formulation in a container and then transported into the lung of a patient via a carrier air stream. Typically, in current dry powder inhalers, the force for breaking up the solid is provided solely by the patient's inhalation. One suitable dry powder inhaler is the Turbohaler™ manufactured by Astra (Sødertalje, Sweden). In another type of inhaler, air flow generated by the patient's inhalation activates an impeller motor which deagglomerates the monomeric GLP-1 analogue particles. The Dura Spiros™ inhaler is such a device.
Formulations of conjugated GLP-1 of this invention for administration from a dry powder inhaler typically include a finely divided dry powder containing GLP-1 conjugate, but the powder can also include a bulking agent, carrier, excipient, another additive, or the like. Additives can be included in a dry powder formulation of GLP-1 conjugate, for example, to dilute the powder as required for delivery from the particular powder inhaler, to facilitate processing of the formulation, to provide advantageous powder properties to the formulation, to facilitate dispersion of the powder from the inhalation device, to stabilize the formulation (for example, antioxidants or buffers), to provide taste to the formulation, or the like.
Advantageously, the additive does not adversely affect the patient's airways. The GLP-1 conjugate can be mixed with an additive at a molecular level or the solid formulation can include particles of the GLP-1 conjugate mixed with or coated on particles of the additive. Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like. Typically an additive, such as a bulking agent, is present in an amount effective for a purpose described above, often at about 50% to about 90% by weight of the formulation. Additional agents known in the art for formulation of a protein such as GLP-1 analogue protein can also be included in the formulation.
A spray including conjugated GLP-1 of this invention can be produced by forcing a suspension or solution of GLP-1 conjugate through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed. In an embodiment particles of GLP- 1 conjugate delivered by a sprayer have a particle size less than about 10 μm. In an embodiment in the range of about 1 μm to about 5 μm.
Formulations of conjugated GLP-1 of this invention suitable for use with a sprayer typically include GLP-1 conjugate in an aqueous solution at a concentration of about 1 mg to about 20 mg of GLP-1 conjugate per ml of solution. The formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant. In an embodiment the formulations contain zinc. The formulation can also include an excipient or agent for stabilization of the GLP-1 conjugate, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating GLP-1 conjugates include albumin, protamine, or the like. Typical carbohydrates useful in formulating GLP-1 conjugates include sucrose, mannitol, lactose, trehalose, glucose, or the like. The GLP-1 conjugate formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the GLP-1 conjugate caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation. In an embodiment the surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as GLP-1 analogue protein can also be included in the formulation. Conjugated GLP-1 of this invention can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source is used to create a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-pressure region is created, which draws a solution of GLP-1 conjugate through a capillary tube connected to a liquid reservoir. The liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol. A range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer. In an ultrasonic nebulizer, high- frequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer. This energy is transmitted to the formulation of GLP-1 conjugate either directly or through a coupling fluid, creating an aerosol including the GLP-1 conjugate. Advantageously, particles of GLP-1 conjugate delivered by a nebulizer have a particle size less than about 10 μm. In an embodiment in the range of about 1 μm to about 5 μm.
Formulations of GLP-1 conjugate suitable for use with a nebulizer, either jet or ultrasonic, typically include GLP-1 conjugate in an aqueous solution at a concentration of about 1 mg to about 20 mg of GLP-1 conjugate per ml of solution. The formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant. The formulation can also include an excipient or agent for stabilization of the GLP-1 conjugate, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating GLP-1 conjugates include albumin, protamine, or the like. Typical carbohydrates useful in formulating GLP-1 conjugates include sucrose, mannitol, lactose, trehalose, glucose, or the like. The GLP-1 conjugate formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the GLP-1 conjugate of this invention caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation In an embodiment the surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as GLP-1 analogue protein can also be included in the formulation.
In a metered dose inhaler (MDI), a propellant, an GLP-1 conjugate of this invention, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol. In an embodiment containing particles in the size range of less than about 10 μm. In an embodiment about 1 μm to about 5 μm. The desired aerosol particle size can be obtained by employing a formulation of GLP-1 conjugate of this invention produced by various methods known to those of skill in the art, including jet-milling, spray drying, critical point condensation, or the like. In an embodiment the metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant. Formulations of a GLP-1 conjugate of this invention for use with a metered-dose inhaler device will generally include a finely divided powder containing GLP-1 conjugate of this invention as a suspension in a non aqueous medium, for example, suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1 ,1 ,1 ,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like. In an embodiment the propellant is a hydrofluorocarbon. The surfactant can be chosen to stabilize the GLP-1 conjugate of this invention as a suspension in the propellant, to protect the active agent against chemical degradation, and the like. Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. In some embodiments solution aerosols are using solvents such as ethanol. Additional agents known in the art for formulation of a protein such as GLP-1 analogue protein can also be included in the formulation.
One of ordinary skill in the art will recognize that the methods of the current invention may be achieved by pulmonary administration of conjugated GLP-1 of this invention via devices not described herein.
The present invention also relates to a pharmaceutical composition or formulation including an GLP-1 conjugate of this invention and suitable for administration by inhalation. According to the invention, an GLP-1 conjugate of this invention can be used for manufacturing a formulation or medicament suitable for administration by inhalation. The invention also relates to methods for manufacturing formulations including an GLP-1 conjugate of this invention in a form that is suitable for administration by inhalation. For example, a dry powder formulation can be manufactured in several ways, using conventional techniques. Particles in the size range appropriate for maximal deposition in the lower respiratory tract can be made by micronizing, milling, spray drying, or the like. And a liquid formulation can be manufactured by dissolving an GLP-1 conjugate of this invention in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients.
Hence, in an embodiment, this invention relates to a method of administering a conjugated insulin of formula Il comprising administering an effective amount of the conjugated insulin of formula Il to a patient in need thereof by pulmonary means; In an embodiment said conjugated insulin of formula Il is inhaled through the mouth of said patient.
To be more precise, this invention also relates to the following embodiments: a) The method as described herein, wherein the conjugated GLP1 analogue of formula I is delivered to a lower airway of the patient. b) The method as described herein, wherein the conjugated GLP1 analogue of formula I is deposited in the alveoli. c) The method as described herein, wherein the conjugated GLP1 analogue of formula I is administered as a pharmaceutical formulation comprising the conjugated GLP1 analogue of formula I in a pharmaceutically acceptable carrier. d) The method as described herein, wherein the formulation is selected from the group consisting of a solution in an aqueous medium and a suspension in a non-aqueous medium. e) The method as described herein, wherein the formulation is administered as an aerosol. f) The method as described herein, wherein the formulation is in the form of a dry powder. g) The method as described herein, wherein the conjugated GLP1 analogue of formula I has a particle size of less than about 10 microns. h) The method as described herein, wherein the conjugated GLP1 analogue of formula I has a particle size of about 1 to about 5 microns. i) The method as described herein, wherein the conjugated GLP1 analogue of formula I has a particle size of about 2 to about 3 microns. j) The method as described herein, wherein at least about 10% of the conjugated GLP1 analogue of formula I delivered is deposited in the lung. k) The method as described herein, wherein the conjugated GLP1 analogue of formula I is delivered from an inhalation device suitable for pulmonary administration and capable of depositing the insulin analog in the lungs of the patient.
I) The method as described herein, wherein the device is selected from the group consisting of a nebulizer, a metered-dose inhaler, a dry powder inhaler, and a sprayer, m) The method as described herein, wherein the device is a dry powder inhaler, n) The method as described herein, wherein the device is a nebulizer, o) The method as described herein, wherein the device is a metered-dose inhaler, p) The method as described herein, wherein the device is a sprayer. q) The method as described herein, wherein actuation of the device administers about 3 μg/kg to about 20 μg/kg of said conjugated GLP1 analogue of formula I. In embodiments about 7 μg/kg to about 14 μg/kg of said conjugated GLP1 analogue of formula I. r) The method as described herein, wherein said conjugated GLP1 analogue of formula I is any of the compounds mentioned specifically in any of the above examples. s) A method as described herein for treating diabetes comprising administering an effective dose of said conjugated GLP1 analogue of formula I to a patient in need thereof by pulmonary means. t) The method as described herein, wherein the conjugated GLP1 analogue of formula I is administered as a pharmaceutical formulation comprising the conjugated GLP1 analogue of formula I in a pharmaceutically acceptable carrier.
The method as described herein, wherein the GLP1 conjugate is any of the specific compounds of formula Il specifically mentioned herein, especially in the specific examples herein. Even though the above embodiments are here described specifically in relation to a method, they apply analogously for the product or formulation to be used.
One object of the present invention is to provide a pharmaceutical formulation comprising a compound according to the present invention which is present in a concentration from about 0.1 mg/ml to about 25 mg/ml, and wherein said formulation has a pH from 2.0 to 10.0. The formulation may further comprise a buffer system, preservative(s), isotonicity agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment of the invention the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water. Such formulation is typically a solution or a suspension. In a further embodiment of the invention the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50 %w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 %w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 %w/w water.
In another embodiment the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use. In another embodiment the pharmaceutical formulation is a dried formulation (for example, freeze-dried or spray-dried) ready for use without any prior dissolution.
In a further aspect the invention relates to a pharmaceutical formulation comprising an aqueous solution of a compound according to the present invention, and a buffer, wherein said compound is present in a concentration from 0.1 mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0.
In another embodiment of the invention the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention the pH of the formulation is from about 3.0 to about 7.0. In another embodiment of the invention the pH of the formulation is from about 5.0 to about 7.5. In another embodiment of the invention the pH of the formulation is from about 7.5 to about 9.0. In another embodiment of the invention the pH of the formulation is from about 7.5 to about 8.5. In another embodiment of the invention the pH of the formulation is from about 6.0 to about 7.5. In another embodiment of the invention the pH of the formulation is from about 6.0 to about 7.0.
In another embodiment of the invention the pH of the formulation is from about 3.0 to about 9.0, and said pH is at least 2.0 pH units from the isoelectric pH of compound of the present invention.
In a further embodiment of the invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginin, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.
In a further embodiment of the invention the formulation further comprises a pharmaceutically acceptable preservative. In a further embodiment of the invention the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p- hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p- hydroxybenzoate, benzethonium chloride, chlorphenesine (3p-chlorphenoxypropane-1 ,2-diol) or mixtures thereof. In a further embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In a further embodiment of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995. In a further embodiment of the invention the formulation further comprises an isotonic agent. In a further embodiment of the invention the isotonic agent is selected from the group consisting of a salt (for example, sodium chloride), a sugar or sugar alcohol, an amino acid (for example, L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (for example, glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3- propanediol, 1 ,3-butanediol) polyethyleneglycol (for example, PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one embodiment the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galacititol, dulcitol, xylitol, and arabitol. In one embodiment the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of the invention. In one embodiment, the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In a further embodiment of the invention the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
In a further embodiment of the invention the formulation further comprises a chelating agent. In a further embodiment of the invention the chelating agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In a further embodiment of the invention the chelating agent is present in a concentration from 0.1 mg/ml to 5mg/ml. In a further embodiment of the invention the chelating agent is present in a concentration from 0.1 mg/ml to 2mg/ml. In a further embodiment of the invention the chelating agent is present in a concentration from 2mg/ml to 5mg/ml. Each one of these specific chelating agents constitutes an alternative embodiment of the invention. The use of a chelating agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
In a further embodiment of the invention the formulation further comprises a stabiliser. The use of a stabilizer in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
More particularly, compositions of the invention are stabilized liquid pharmaceutical compositions whose therapeutically active components include a polypeptide that possibly exhibits aggregate formation during storage in liquid pharmaceutical formulations. By
"aggregate formation" is intended a physical interaction between the polypeptide molecules that results in formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution. By "during storage" is intended a liquid pharmaceutical composition or formulation once prepared, is not immediately administered to a subject. Rather, following preparation, it is packaged for storage, either in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject. By "dried form" is intended the liquid pharmaceutical composition or formulation is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and PoIIi (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991 ) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491 - 676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1 169-1206; and Mumenthaler et al. (1994) Pharm. Res. 1 1 :12-20), or air drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991 ) Biopharm. 4:47-53). Aggregate formation by a polypeptide during storage of a liquid pharmaceutical composition can adversely affect biological activity of that polypeptide, resulting in loss of therapeutic efficacy of the pharmaceutical composition. Furthermore, aggregate formation may cause other problems such as blockage of tubing, membranes, or pumps when the polypeptide-containing pharmaceutical composition is administered using an infusion system.
The pharmaceutical compositions of the invention may further comprise an amount of an amino acid base sufficient to decrease aggregate formation by the polypeptide during storage of the composition. By "amino acid base" is intended an amino acid or a combination of amino acids, where any given amino acid is present either in its free base form or in its salt form. Where a combination of amino acids is used, all of the amino acids may be present in their free base forms, all may be present in their salt forms, or some may be present in their free base forms while others are present in their salt forms. In one embodiment, amino acids to use in preparing the compositions of the invention are those carrying a charged side chain, such as arginine, lysine, aspartic acid, and glutamic acid. Any stereoisomer (i.e., L, D, or DL isomer) of a particular amino acid (for example, glycine, methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof) or combinations of these stereoisomers, may be present in the pharmaceutical compositions of the invention so long as the particular amino acid is present either in its free base form or its salt form. In one embodiment the L-stereoisomer is used. Compositions of the invention may also be formulated with analogues of these amino acids. By "amino acid analogue" is intended a derivative of the naturally occurring amino acid that brings about the desired effect of decreasing aggregate formation by the polypeptide during storage of the liquid pharmaceutical compositions of the invention. Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable methionine analogues include S-ethyl homocysteine and S-butyl homocysteine and suitable cystein analogues include S-methyl-L cystein. As with the other amino acids, the amino acid analogues are incorporated into the compositions in either their free base form or their salt form. In a further embodiment of the invention the amino acids or amino acid analogues are used in a concentration, which is sufficient to prevent or delay aggregation of the protein. In a further embodiment of the invention methionine (or other sulphur containing amino acids or amino acid analogous) may be added to inhibit oxidation of methionine residues to methionine sulfoxide when the polypeptide acting as the therapeutic agent is a polypeptide comprising at least one methionine residue susceptible to such oxidation. By "inhibit" is intended minimal accumulation of methionine oxidized species over time. Inhibiting methionine oxidation results in greater retention of the polypeptide in its proper molecular form. Any stereoisomer of methionine (L, D, or DL isomer) or combinations thereof can be used. The amount to be added should be an amount sufficient to inhibit oxidation of the methionine residues such that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxide. Generally, this can be achieved by adding methionine such that the ratio of methionine added to methionine residues ranges from about 1 :1 to about 1000:1 , such as 10:1 to about 100:1 .
In a further embodiment of the invention the formulation further comprises a stabiliser selected from the group of high molecular weight polymers or low molecular compounds. In a further embodiment of the invention the stabilizer is selected from polyethylene glycol (for example, PEG 3350), polyvinylalcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates thereof (for example, HPC, HPC-SL, HPC-L and
HPMC), cyclodextrins, sulphur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and different salts (for example, sodium chloride). Each one of these specific stabilizers constitutes an alternative embodiment of the invention.
The pharmaceutical compositions may also comprise additional stabilizing agents, which further enhance stability of a therapeutically active polypeptide therein. Stabilizing agents of particular interest to the present invention include, but are not limited to, methionine and EDTA, which protect the polypeptide against methionine oxidation, and a nonionic surfactant, which protects the polypeptide against aggregation associated with freeze-thawing or mechanical shearing. In a further embodiment of the invention the formulation further comprises a surfactant. In a further embodiment of the invention the surfactant is selected from a detergent, ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (for example, poloxamers such as Pluronic® F68, poloxamer 188 and 407, Triton X-100 ), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives such as alkylated and alkoxylated derivatives (tweens, for example, Tween-20, Tween-40, Tween-80 and Brij- 35), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lecitins and phospholipids (for example, phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, diphosphatidyl glycerol and sphingomyelin), derivates of phospholipids (for example, dipalmitoyl phosphatidic acid) and lysophospholipids (for example, palmitoyl lysophosphatidyl-L-serine and 1 -acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine or threonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkyl ether) derivatives of lysophosphatidyl and phosphatidylcholines, for example, lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, that is cholines, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (for example, cephalins), glyceroglycolipids (for example, galactopyransoide), sphingoglycolipids (for example, ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives- (for example, sodium tauro-dihydrofusidate etc.), long-chain fatty acids and salts thereof C6-C12 (for example, oleic acid and caprylic acid), acylcarnitines and derivatives, Nα- acylated derivatives of lysine, arginine or histidine, or side-chain acylated derivatives of lysine or arginine, Nα-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, Nα-acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CAS registry no [577-11 -7]), docusate calcium, CAS registry no [128-49- 4]), docusate potassium, CAS registry no [7491 -09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate), sodium caprylate, cholic acid or derivatives thereof, bile acids and salts thereof and glycine or taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate, N-hexadecyl-N,N-dimethyl- 3-ammonio-1 -propanesulfonate, anionic (alkyl-aryl-sulphonates) monovalent surfactants, zwitterionic surfactants (for example, N-alkyl-N,N-dimethylammonio-1 -propanesulfonates, 3- cholamido-1 -propyldimethylammonio-1 -propanesulfonate, cationic surfactants (quarternary ammonium bases) (for example, cetyl-trimethylammonium bromide, cetylpyridinium chloride), non-ionic surfactants (for example, dodecyl β-D-glucopyranoside), poloxamines (for example, Tetronic's), which are tetrafunctional block copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine, or the surfactant may be selected from the group of imidazoline derivatives, or mixtures thereof. Each one of these specific surfactants constitutes an alternative embodiment of the invention.
The use of a surfactant in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
It is possible that other ingredients may be present in the peptide pharmaceutical formulation of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (for example human serum albumin, gelatin or proteins) and a zwitterion (for example an amino acid such as betaine, taurine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention. Pharmaceutical compositions containing a compound according to the present invention may be administered to a patient in need of such treatment at several sites, for example, at topical sites, for example, skin and mucosal sites, at sites which bypass absorption, for example, administration in an artery, in a vein, in the heart, and at sites which involve absorption, for example, administration in the skin, under the skin, in a muscle or in the abdomen.
Administration of pharmaceutical compositions according to the invention may be through several routes of administration, for example, lingual, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary, for example, through the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ocular, for examples through the conjunctiva, uretal, and parenteral to patients in need of such a treatment.
In one aspect the present invention relates to a pharmaceutical composition comprising a compound according to Formula (I), and a pharmaceutically acceptable excipient. In one embodiment the pharmaceutical composition is suited for pulmonary administration.
In another aspect the present invention relates to the use of a compound of formula (I) for the preparation of a pulmonary medicament.
Compositions of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example, hard gelatine capsules and soft gelatine capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, infusion solution, and implants.
Compositions of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the compound, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof. Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to, polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, polyvinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and block co-polymers thereof, polyethylene glycols, carrier proteins, for example albumin, gels, for example, thermogelling systems, for example block co-polymeric systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticulates, liquid crystals and dispersions thereof, L2 phase and dispersions there of, well known to those skilled in the art of phase behaviour in lipid-water systems, polymeric micelles, multiple emulsions, self- emulsifying, self-microemulsifying, cyclodextrins and derivatives thereof, and dendrimers.
Compositions of the current invention are useful in the formulation of solids, semisolids, powder and solutions for pulmonary administration of the compound, using, for example a metered dose inhaler, dry powder inhaler and a nebulizer, all being devices well known to those skilled in the art.
Compositions of the current invention are specifically useful in the formulation of controlled, sustained, protracting, retarded, and slow release drug delivery systems. More specifically, but not limited to, compositions are useful in formulation of parenteral controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), well known to those skilled in the art. I In an embodiment controlled release and sustained release systems are administered subcutaneous. Without limiting the scope of the invention, examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles, Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co- cystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenization, encapsulation, spray drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D.L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, EJ. , ed. Marcel Dekker, New York, 2000).
Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a solution or suspension for the administration of the compound according to the present invention in the form of a nasal or pulmonal spray. As a still further option, the pharmaceutical compositions containing the compound of the invention can also be adapted to transdermal administration, for example, by needle-free injection or from a patch, optionally an iontophoretic patch, or transmucosal, for example, buccal, administration.
The term "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability. The term "physical stability" of the protein formulation as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. Physical stability of the aqueous protein formulations is evaluated by means of visual inspection and/or turbidity measurements after exposing the formulation filled in suitable containers (for example, cartridges or vials) to mechanical/physical stress (for example, agitation) at different temperatures for various time periods. Visual inspection of the formulations is performed in a sharp focused light with a dark background. The turbidity of the formulation is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a formulation showing no turbidity corresponds to a visual score 0, and a formulation showing visual turbidity in daylight corresponds to visual score 3). A formulation is classified physical unstable with respect to protein aggregation, when it shows visual turbidity in daylight. Alternatively, the turbidity of the formulation can be evaluated by simple turbidity measurements well-known to the skilled person. Physical stability of the aqueous protein formulations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. In an embodiment the probe is a small molecule. In an embodiment it binds to a non-native conformer of the protein. One example of a small molecular spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths.
Other small molecules can be used as probes of the changes in protein structure from native to non-native states. For instance the "hydrophobic patch" probes that bind to exposed hydrophobic patches of a protein. The hydrophobic patches are generally buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature. Examples of these small molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthroline or the like. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.
The term "chemical stability" of the protein formulation as used herein refers to chemical covalent changes in the protein structure leading to formation of chemical degradation products with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure. Various chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation can most probably not be completely avoided and increasing amounts of chemical degradation products is often seen during storage and use of the protein formulation as well- known by the person skilled in the art. Most proteins are prone to deamidation, a process in which the side chain amide group in glutaminyl or asparaginyl residues is hydrolysed to form a free carboxylic acid. Other degradations pathways involves formation of high molecular weight transformation products where two or more protein molecules are covalently bound to each other through transamidation and/or disulfide interactions leading to formation of covalently bound dimer, oligomer and polymer degradation products (Stability of Protein Pharmaceuticals, Ahem. T.J. & Manning M. C, Plenum Press, New York 1992). Oxidation (of for instance methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein formulation can be evaluated by measuring the amount of the chemical degradation products at various time-points after exposure to different environmental conditions (the formation of degradation products can often be accelerated by for instance increasing temperature). The amount of each individual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatography techniques (for example, SEC-HPLC and/or RP-HPLC).
Hence, as outlined above, a "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a formulation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached. In one embodiment of the invention the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 6 weeks of usage and for more than 3 years of storage.
In another embodiment of the invention the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 4 weeks of usage and for more than 3 years of storage. In a further embodiment of the invention the pharmaceutical formulation comprising the compound according to the present invention is stable for more than 4 weeks of usage and for more than two years of storage.
In an even further embodiment of the invention the pharmaceutical formulation comprising the compound is stable for more than 2 weeks of usage and for more than two years of storage.
In another aspect the present invention relates to the use of a compound according to the invention for the preparation of a medicament.
In one embodiment a compound according to the invention is used for the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers.
In another embodiment a compound according to the invention is used for the preparation of a medicament for delaying or preventing disease progression in type 2 diabetes.
In another embodiment a compound according to the invention is used for the preparation of a medicament for decreasing food intake, decreasing β-cell apoptosis, increasing β-cell function and β-cell mass, and/or for restoring glucose sensitivity to β-cells.
The treatment with a compound according to the present invention may also be combined with combined with a second or more pharmacologically active substances, for example, selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity. Examples of these pharmacologically active substances are: Insulin, sulphonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenosis, glucose uptake modulators, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the β- cells; Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, repaglinide; β-blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, alatriopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and α-blockers such as doxazosin, urapidil, prazosin and terazosin; CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin agonists, β3 agonists, MSH (melanocyte- stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists, serotonin re-uptake inhibitors, serotonin and noradrenaline reuptake inhibitors, mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists, bombesin agonists, galanin antagonists, growth hormone, growth hormone releasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR (retinoid X receptor) modulators, TR β agonists; histamine H3 antagonists.
It should be understood that any suitable combination of the compounds according to the invention with one or more of the above-mentioned compounds and optionally one or more further pharmacologically active substances are considered to be within the scope of the present invention.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).
All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.
The use of any and all examples, or exemplary language (for example "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. The mentioning herein of references is no admission that they constitute prior art.
Herein, the word "comprise" is to be interpreted broadly meaning "include", "contain" or "comprehend".
This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. The present invention is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof.
EXAMPLES
The following examples and general procedures refer to intermediate compounds and final products identified in the structural specification and in the synthesis schemes. The preparation of the compounds of the present invention is described in detail using the following examples, but the chemical reactions described are disclosed in terms of their general applicability to the preparation of selected branched polymers of the invention. Occasionally, the reaction may not be applicable as described to each compound included within the disclosed scope of the invention. The compounds for which this occurs will be readily recognised by those skilled in the art. In these cases the reactions can be successfully performed by conventional modifications known to those skilled in the art, that is, by appropriate protection of interfering groups, by changing to other conventional reagents, or by routine modification of reaction conditions. Alternatively, other reactions disclosed herein or otherwise conventional will be applicable to the preparation of the corresponding compounds of the invention. In all preparative methods, all starting materials are known or may easily be prepared from known starting materials. All temperatures are set forth in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight when referring to yields and all parts are by volume when referring to solvents and eluents. All reagents were of standard grade as supplied from Aldrich, Sigma, ect. Proton, carbon and phosphor nuclear magnetic resonance (1 H-, 13C- and 31 P-NMR) were recorded on a Bruker NMR apparatus, with chemical shift (δ) reported down field from tetramethylsilane or phosphoric acid. LC-MS mass spectra were obtained using apparatus and setup conditions as follows:
GLP-1 conjugates were generally analysed using following HPLC systems:
HPLC (Method A): The RP-analysis was performed using a Waters 2690 systems fitted with a Waters 996 diode array detector. UV detections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6 mm x 250 mm 5μ C-18 silica column (The Seperations Group, Hesperia), which was eluted at 1 ml/min at 420C. The column was equilibrated with 10% of a 0,5 M ammonium sulfate, which was adjusted to pH 2.5 with 4M sulfuric acid. After injection, the sample was eluted by a gradient of 0% to 60% acetonitrile in the same aqueous buffer during 50 min.
LC-MS (Method A) analysis (electrospray) was performed on HP1 100 MSD equipped with binary pump, column compartment, diode array detector and a single quadropole massspectrometer detector. The analysis was performed at 40 C on Waters Xterra MS C-18 X 3 mm column with a linear gradient from 10% aqueous acetonitrile - 100% acetonitrile containing 0,01 %TFA running over 7.5 min. UV detection at 210nm and MS scanning range from 100-1000 amu.
LC-MS (Method B): was performed on a setup consisting of Hewlett Packard series 1100 G1312A Bin Pump, Hewlett Packard series 1100 Column compartment, Hewlett Packard series 1 100 G1315A DAD diode array detector, Hewlett Packard series 1100 MSD and Sedere 75 Evaporative Light Scattering detectorcontrolled by HP Chemstation software. The HPLC pump was connected to two eluent reservoirs containing: (A) 1 OmM NH4OH in water and (B) 10mM NH4OH in 90% acetonitrile. The analysis was performed at 23° C by injecting an appropriate volume of the sample (preferably 20 μl) onto the column which is eluted with a gradient of A and B. The HPLC conditions, detector settings and mass spectrometer settings used was as follows: Column Waters Xterra MS C-18 X 3 mm id 5 urn Gradient 5% - 100% acetonitrile linear during 6.5 min at 1 .5ml/min Detection 210 nm (analogue output from DAD) ELS (analogue output from ELS) MS ionisation mode API-ES. Scan 100-1000 amu step 0.1 amu
Some of the NMR data shown in the following examples are only selected data.
In the examples the following terms are intended to have the following, general meanings:
Abbreviations
AEEAc Aminoethoxyethoxyacetyl AcOEt: Ethyl acetate
Ala: Alanine tBu: tert butyl
Boc: terf-Butoxycarbonyl
CDI: Carbonyldiimidazole CH.CN: Acetonitrile DBU: 1 ,8-Diazabicyclo[5,4,0]undec-7-ene
DCM: Dichloromethane, methylenechloride
Dde: 1 -(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl
DIC: Diisopropylcarbodiimide
DIPEA: Λ/,Λ/-Diisopropylethylamine
DhbtOH: 3-Hydroxy-1 ,2,3-benzotriazin-4(3H)-one
DMAP: 4-Dimethylaminopyridine
DMF: Λ/,Λ/-dimethylformamide
DMSO: Dimethyl sulphoxide
DTT: Dithiothreitol
Et: Ethyl
Et2O: Diethylether
EtOH: Ethanol
Fmoc: 9-Fluorenylmethyloxycarbonyl
H2O: water
HBTU: 2-(1 H-Benzotriazol-1 -yl-)-1 ,1 ,3,3 tetramethyluroniumhexafluorophosphate
HCI: Hydrochloric acid
HOBt: 1 -Hydroxybenzotriazole ivDde: 1 -(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl
MeCN: Acetonitrile
MeOH: Methanol
Mtt: 4-methyltrityl
Mmt: 4-methoxytrityl
NMP: Λ/-methyl-2-pyrrolidinone
NEt3: Triethylamine
OtBu: tert butyl ester
Pmc: 2,2,5,7,8-Pentamethyl-chroman-6-sulfonyl
PhMe: Toluene
Rf: Retention factor
Rt: Retention time r.t: Room temperature
SiO2: Silica gel
THF: Tetrahydrofuran
TFA: Trifluoroacetic acid
TIS: triisopropylsilane TLC: Thin Layer Chromatography
Trt: triphenylmethyl
TSTU: 2-Succinimido-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate
The following non limiting examples illustrate the synthesis of monomers and polymerisation technique using solid phase synthesis or solution phase synthesis.
General synthetics methods and procedures
Synthesis of resin bound peptide.
The protected peptidyl resin was synthesized according to the Fmoc strategy on an Applied Biosystems 431 A peptide synthesizer in 0.25 mmol or 1 .0 mmol scale using the manufacturer supplied FastMoc UV protocols which employ HBTU (2-(1 H-benzotriazol-1 -yl)- 1 ,1 ,3,3 tetramethyluronium hexafluorophosphate) or HATU (O-(7-azabenzotriazol-1 -yl)- 1 ,1 ,3,3-tetra-methyluronium hexafluorophosphate) mediated couplings in NMP (N-methyl pyrrolidone), and UV monitoring of the deprotection of the Fmoc protection group. The starting resin used for the synthesis of the GLP-1 peptide amides was Rink-Amide resin and either Wang or chlorotrityl resin was used for GLP-1 peptides with a carboxy C-terminal. The protected amino acid derivatives used were standard Fmoc-amino acids (supplied from for example, Anaspec, or Novabiochem) supplied in preweighed cartridges suitable for the ABI433A synthesizer with the exception of unnatural aminoacids such as Fmoc-Aib-OH (Fmoc-aminoisobutyric acid). The N terminal amino acid was Boc protected at the alpha amino group (for example, Boc-His(Boc)OH was used for peptides with His at the N- terminal). The epsilon amino group of the lysine which has to be further derivatized was either protected with Mtt, Mmt, Dde, ivDde, or Boc, depending on the route for attachment of the albumin binding moiety and spacer.
The attachment of branched polymers and linkers to specific lysine residues on the crude resin bound protected peptide was carried out in a specific position by incorporation of Fmoc-Lys(Dde)-OH, Lys(ivDde)-OH, Lys(Mtt)-OH, or Lys(Mmt)-OH during automated synthesis followed by selective deprotection with hydrazine or TFA.
Procedure for removal of ivDde or Dde-protection. The resin (0.25 mmol) was placed in a manual shaker/filtration apparatus and treated with 2% hydrazine in N-methyl pyrrolidone (20 ml, 2x12 min) to remove the DDE group and wash with N-methyl pyrrolidone (4x20 ml). Procedure for removal of Mtt or Mmt-protection.
The resin (0.25 mmol) was placed in a manual shaker/filtration apparatus and treated with 2% TFA in DCM (20 ml, 5-10 min repeated 6-12 times) to remove the Mtt or Mmt group and wash with DCM (2x20 ml), 10%MeOH and 5% DIPEA in DCM (2x20ml) and N-methyl pyrrolidone (4x20 ml).
General procedure for on-resin attachment of branched polymers to lysine residues.
The branched polymers (4 molar equivalents relative to resin bound peptide) was dissolved in NMP/DCM (1 :1 ). Hydroxybenzotriazole (HOBt) (4 molar equivalents relative to resin bound peptide) and diisopropylcarbodiimide (4 molar equivalents relative to resin bound peptide) was added and the solution was stirred for 15 min. The solution was added to the resin and diisopropyethylamine (4 molar equivalents relative to resin bound peptide) was added. The resin was shaken 24 hours at room temperature. The resin was washed with twice with NMP, twice with NMP/DCM (1 :1 ) and twice with DCM.
Procedure for cleaving the peptide off the resin:
The peptide was cleaved from the resin by stirring for 180 min at room temperature with a mixture of trifluoroacetic acid, water and triisopropylsilane (95:2.5:2.5). The cleavage mixture was filtered and the filtrate was concentrated to an oil by a stream of nitrogen. The crude peptide was precipitated from this oil with 45 ml diethyl ether and washed 3 times with 45 ml diethyl ether.
The peptide products is purified using one of the protocol described below (unless described otherwise in the example):
Preparative HPLC protocol in a TFA (acid) based solvent system: The crude peptide was purified by semipreparative HPLC on a 20 mm x 250 mm column packed with 7μ C-18 silica. After drying the crude peptide was dissolved in 5 ml 50% acetic acid H2O and diluted to 20 ml with H2O and injected on the column which then was eluted with a gradient of 40-60 % CH3CN in 0.1 % TFA 10 ml/min during 50 min at 40 0C. The peptide containing fractions were collected. The purified peptide was lyophilized after dilution of the eluate with water.
Preparative HPLC protocol in a ammonium sulphate (basic) based solvent system: The crude peptide was purified by semipreparative HPLC on a 20 mm x 250 mm column packed with 7μ C-18 silica. The column was equilibrated with 40% CH3CN in 0.05M (NH4)2SO4, which was adjusted to pH 2.5 with concentrated H2SO4. After drying the crude peptide was dissolved in 5 ml 50% acetic acid H2O and diluted to 20 ml with H2O and injected on the column which then was eluted with a gradient of 40% - 60% CH3CN in 0.05M (NH4)2SO4, pH 2.5 at 10 ml/min during 50 min at 40 0C. The peptide containing fractions were collected and diluted with 3 volumes of H2O and passed through a Sep-Pak® C18 cartridge (Waters part. #:51910 ) which has been equilibrated with 0.1 % TFA. It was then eluted with 70% CH3CN containing 0.1 % TFA and the purified peptide was isolated by lyophilisation after dilution of the eluate with water.
The final product obtained was characterised by analytical RP-HPLC (retention time) and by LCMS. The RP-HPLC analysis was performed using UV detection at 214 nm and a Vydac 218TP54 4.6mm x 250mm 5μ C-18 silica column (The Separations Group, Hesperia,
USA) which was eluted at 1 ml/min at 42 0C. Two different elution conditions were used:
A1 : Equilibration of the column with in a buffer consisting of 0.1 M (NH4)2SO4, which was adjusted to pH 2.5 with concentrated H2SO4 and elution by a gradient of 0% to 60%
CH3CN in the same buffer during 50 min. B1 : Equilibration of the column with 0.1 % TFA / H2O and elution by a gradient of 0%
CH3CN / 0.1 % TFA / H2O to 60% CH3CN / 0.1 % TFA / H2O during 50 min. B6: Equilibration of the column with 0.1 % TFA / H2O and elution by a gradient of 0%
CH3CN / 0.1 % TFA / H2O to 90% CH3CN / 0.1 % TFA / H2O during 50 min.
SYNTHESIS OF MONOMER BUILDING BLOCKS AND LINKERS:
EXAMPLE 1
2-[2-(2-Chloroethoxy)ethoxymethyl]oxirane
2-(2-Chloroethoxy)ethanol (100.00 g; 0.802 mol) was dissolved in dichloromethane (100 ml) and a catalytic amount of boron trifluoride etherate (2.28 g; 16 mmol) was added. The clear solution was cooled to 0 5C, and epibromohydrin (104.46 g; 0.762 mol) was added dropwise maintaining the temperature at 0 5C. The clear solution was stirred for an additional 3h at 0 5C, then solvent was removed by rotary evaporation. The residual oil was evaporated once from acetonitrile, to give crude 1 -bromo-3-[2-(2-chloroethoxy)ethoxy]propan-2-ol, which was re-dissolved in THF (500 ml). Powdered potassium terf-butoxide (85.0 g; 0.765 mmol) was then added, and the mixture was heated to reflux for 30 min. Insoluble salts were removed by filtration, and the filtrate was concentrated, in vacuo, to give a clear yellow oil. The oil was further purified by vacuum distillation, to give 56.13 g (41 %) of pure title material. bp = 65-75°C (0.65 mbar). 1H-NMR (CDCI3): δ = 2.61 ppm (m, 1 H); 2.70 (m, 1 H); 3.17 (m, 1 H); 3.43 (dd, 1 H); 3.60-3.85 (m, 9H). 13C-NMR (CDCI3): δ = 42.73 ppm; 44.18; 50.80; 70.64 & 70,69 (may collapse); 71 .37; 72.65.
EXAMPLE 2
1 ,3-Bis[2-(2-chloroethoxy)ethoxy]propan-2-ol
2-[2-(2-Chloroethoxy)ethoxymethyl]oxirane (2.20 g; 12.2 mmol) was dissolved in DCM (20 ml), and 2-(2-chloroethoxy)ethanol (1 .52 g; 12.2 mol) was added. The mixture was cooled to 0 5C and a catalytical amount of boron trifluride etherate (0.2 ml; 1.5 mmol) was added. The mixture was stirred at 0 5C for 2h, then solvent was removed by rotary evaporation. Residual of boron trifluride etherate was removed by co-evaporating twice from acetonitrile. The oil thus obtained was purified by kuglerohr destilation. The title material was obtained as a clear viscous oil in 2.1 O g (45%) yield, bp. = 270 5C, 0.25 mbar. 1H-NMR (CDCI3): δ = 3.31 (bs, 1 H); 3.55 ppm (ddd, 4H); 3.65-3.72 (m, 12H); 3.75 (t, 4H); 3.90 (m, 1 H). 13C-NMR (CDCI3): δ = 43.12 ppm; 69.92; 70.95; 71 .11 ; 71 .69; 72.69.
EXAMPLE 3
1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-ol
1 ,3-Bis[2-(2-chloroethoxy)ethoxy]propan-2-ol (250 mg; 0.81 mmol) was dissolved in DMF (2.5 ml), and sodium azide (200 mg; 3.10 mmol) and sodium iodide (100 mg; 0.66 mmol) were added. The suspension was heated to 100 5C (internal temperature) over night. The mixture was then cooled and filtered. The filtrate was taken to dryness, and the semi crystalline oil re-suspended in DCM (5 ml). The non-soluble salts were removed by filtration; the filtrate was evaporated to dryness to give pure title material as a colourless oil. Yield: 210 mg (84%). 1H-NMR (CDCI3): δ = 3.48 ppm (t, 4H); 3.60-3.75 (m, 16H); 4.08 (m, 1 H). 13C- NMR (CDCI3): δ = 51 .05 ppm; 69.10; 70.24; 70.53; 70.78; 71.37. LC-MS: m/e = 319 (M+1 ) +; 341 (M+Na) +; 291 (M-N2) +. Rt = 2.78 min.
EXAMPLE 4
1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-yl-p-nitrophenylcarbonate
1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-ol (2.00 g; 6.6 mmol) was dissolved in THF (50 ml) and diisopropylethylamine (10 ml) was added. The clear yellow solution was then added 4- dimethylaminopyridine (1 .60 g; 13.1 mmol) and p-nitrophenylchloroformiate (2.64 g; 13.1 mmol) and stirred at ambient temperature. A precipitate rapidly formed. The suspension was stirred for 5 h at room temperature, then filtered and concentrated in vacuo. The residue was further purified by chromatography using ethyl acetate - heptane - triethylamine (40 / 60 / 2) as eluent. The product was obtained as a clear yellow oil in 500 mg (16%) yield. 1H-NMR (CDCI3): δ = 3.38 ppm (t, 4H); 3.60-3.72 (m, 12H); 3.76 (m, 4H); 5.12 (q, 1 H); 7.41 (d, 2H); 8.28 (d, 2H). LC-MS: m/e = 506 (M+Na)+; 456 (M-N2), Rt= 4.41 min.
EXAMPLE 5
1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-yl chloroformiate
Trichloroacetylchloride (1 ,42 g, 7.85 mmol) was dissolved in THF (10 ml), and the solution was cooled to 0 5C. A solution of 1 ,3-bis[2-(2-azidoethoxy)ethoxy]propan-2-ol (1 .00 g; 3.3 mmol) and triethylamine (0,32 g, 3.3 mmol) in THF (5 ml) was slowly added drop wise over 10 min. Cooling was removed, and the resulting suspension was stirred for 6h at ambient temperature. The mixture was filtered, and the filtrate was evaporated to give a light brown oil. The oil was treated twice with acetonitrile following evaporation, and the product was used without further purification.
1H-NMR (CDCI3): δ = 3.40 (t, 4H); 3,55-3,71 (m, 12H); 3,75 (d, 4H); 5.28 (m, 1 H).
EXAMPLE 6
2-(1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-yloxy)acetic acid
Sodium hydride (7.50 g; 80% oil suspension) was washed trice with heptanes, and then re- suspended in dry THF (100 ml). A solution of 1 ,3-bis[2-(2-azidoethoxy)ethoxy]propan-2-ol (10.00 g; 33.0 mmol) in dry THF (100 ml) was then slowly added over a period of 30 min at room temperature. Then a solution of bromoacetic acid (6.50 mg; 47 mmol) in THF (100 ml) was added drop wise over 20 min. -> slight heat evolution. A cream coloured suspension was formed. The mixture was stirred at ambient temperature over night. Excess sodium hydride was carefully destroyed by addition of water (20 ml) while cooling the mixture. The suspension was taken to dryness by rotary evaporation, and the residue partitioned between DCM and water. The water phase was extracted twice with DCM then acidified by addition of acetic acid (25 ml). The water phase was then extracted twice with DCM, and the combined organic phases were dried over sodium sulphate, and evaporated to dryness. The residual oil at this point contained the title material as well as bromoacetic acid. The later was removed by re-dissolving the oil in DCM (50 ml) containing piperidine (5 ml); stir for 30 min., and then wash of the organic solution trice with 1 N aquoeus HCI (3x). Pure title material was then obtained after drying (Na2SO4) and evaporation of the solvent. Yield: 7.54 g (63%). 1H-NMR (CDCI3): δ = 3.48 ppm (t, 4H); 3.55-3.80 (m, 16H); 4.28 (s, 2H); 4.30 (m, 1 H); 8.50 (bs, 1 H). 13C-NMR (CDCI3): δ = 51.04 ppm; 69.24; 70.50; 70.72; 71 .39; 71 .57; 80.76; 172.68. LC-MS: m/e = 399 (M+Na) +; 349 (M-N2). Rt = 2.34 min.
EXAMPLE 7 lmidazole-1 -carboxylic acid 1 ,3-bis(2-(2-azidoethoxy)ethoxy)propan-2-yl ester
1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-ol (1.00 g; 3.3 mmol) was dissolved in DCM (5 ml) and carbonyldiimidazole (1 .18 g , 6.3 mmol) was added. The mixture was stirred for 2h at room temperature. Solvent was removed and the residue was dissolved in methanol (20 ml) and stirred for 20 min. Solvent was removed and the clear oil, thus obtained was further purified by column chromatography on silica using 2 % MeOH in DCM as eluent. Yield: 372.4 mg (35%). 1H-NMR (CDCI3): δ =3.33 (t, 4H); 3,60-3,75 (m, 12H); 3,80 (d, 4H); 5.35 (m, 1 H); 7.06 (s, 1 H); 7.43 (s, 1 H); 8.16 (s, 1 H). LC-MS: m/e = 413 (M+1 ). Rt = 2.35 min.
EXAMPLE 8 terf-Butyl 2-(1 ,3-bis[2-(2-azidoethoxy)ethoxy]propan-2-yloxy)acetate
2-(1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-yloxy)acetic acid (5.0 g; 13.28 mmol) was dissolved in toluene (20 ml), and the reaction mixture was heated to reflux under an inert atmosphere. N,N-dimethylformamid-di-tert-butylacetal (13 ml; 54.21 mmol) was then added dropwise over 30 min. Reflux was continued for 24h. The dark brown solution was then filtered through Celite. Solvent was removed under vacuum, and the oily residue was purified by flash chromatography on silica, using 3% methanol dichloromethane as eluent. Pure fractions were pooled and evaporated to dryness. The title material was obtained as a yellow clear oil. Yield: 5.07 g (88%). 1H-NMR (CDCI3): δ = 1 .42 ppm (s, 9H); 3.35 (t, 4H); 3.54-3.69 (m, 16H); 3.75-3.85 (m, 1 H); 4.16 (s, 2H). 13C-NMR (CDCI3, selected peaks): δ = 30.35 ppm.; 52.93; 70.65; 72.25; 73.12; 73.90; 80.44; 83.55; 172.28. TLC: Rf = 0.33 in ethyl acetate - heptane (1 :1 ).
EXAMPLE 9 terf-Butyl 2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)acetate tert-Buty\ 2-(1 ,3-bis[2-(2-azidoethoxy)ethoxy]propan-2-yloxy)acetate (5.97 g, 1 1 .7 mmol) was dissolved in ethanol-water (25 ml; 2:1 ), and acetic acid (5 ml) was added, followed by a aqueous suspension of Raney-Nickel (5 ml). The mixture was then hydrogenated at 3 atm., for 16 h using a Parr apparatus. The catalyst was then removed by filtration, and the reaction mixture was taken to dryness by rotary evaporation. The oily residue was dissolved in water and freeze dried to give a quantitative yield of title material. 1H-NMR (CDCI3): δ = 1 .45 ppm (s, 9H); 3.15 (bs, 4H); 3.48-3.89 (broad m, 17H); 4.15 (s, 2H). 13C-NMR (CDCI3, selected peaks): δ = 28.44 ppm.; 39.81 ; 68.17; 70.58; 70.79; 70.99; 78.81 ; 82.31 ; 170.59.
EXAMPLE 10
2-(1 ,3-Bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)acetic acid
2-(1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-yloxy)acetic acid (1 .00 g; 2.65 mmol) was dissolved in 1 N aqueous hydrochloric acid (10 ml) and a 50% aqueous suspension of 5 % palladium on carbon (1 ml) was added. The mixture was hydrogenated at 3.5 atm using a Parr apparatus. After one hour the reaction was stopped, and the catalyst removed by filtration. The solvent was removed by rotary evaporation, and the residue was evaporated twice from acetonitrile. Yield: 930 mg (88 %). 1H-NMR (D2O): δ = 3.1 1 ppm (t, 4H); 3.53-3.68 (m, 16H); 3.80 (m, 1 H); 4.25 (s, 2H). 13C-NMR (D2O): δ = 38.18 ppm.; 65.43; 66.09; 68.55: 69.13; 69.23; 77.18; 173.42.
EXAMPLE 1 1
2-(1 ,3-Bis[2-(2-{9-fluorenylmethyloxycarbonylamino}ethoxy)ethoxy]propan-2-yloxy)acetic acid
2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)acetic acid (9.35 g; 28.8 mmol) was added DIPEA (10 ml; 57 mmol). The reaction mixture was cooled on an ice bath, and chlorotrimethylsilane (15 ml; 118 mmol) dissolved in DCM (50 ml) was added dropwise, followed by DIPEA (11 ml; 62.7 mmol). To the almost clear solution was added dropwise a solution of Fmoc-CI (15.0 g; 57 mmol) in DCM (50 ml). The reaction mixture was stirred overnight, then diluted with DCM (500 ml) and added to 0.01 N aqueous HCI solution (500 ml). The organic layer was separated; washed with water (3x 200 ml) and dried over anhydrous sodium sulfate. Solvent was removed by rotary evaporation. The crude product was purified by flash chromatography on silica using ethylacetate-heptane (1 :1 ) as eluent. Pure fractions were collected and taken to dryness to give 9.20 g (42%) of title material. 1H-NMR (D2O): δ = 3.34 ppm (t, 4H); 3.45-3.65 (m, 16H); 3.69 (bs, 1 H); 4.20 (t, 2H); 4.26 (s, 2H); 4.38 (d, 4H); 5.60 (t, 2H); 7.30 (t, 4H); 3.35 (t, 4H); 7.58 (d, 4H); 7.72 (d, 4H). 13C-NMR (D2O; selected peaks): δ = 21 .20 ppm.; 30.75; 34.64; 67.66; 68.90; 70.38; 70.51 ; 80.02; 120.37; 125.54; 127.48; 128.09; 128.67; 136.27; 141 .69; 173.63; 176.80.
EXAMPLE 12 2-[2-(2-azidoethoxy)ethoxy]ethanol
A slurry of 2-(2-(-2-chloroethoxy)ethoxy)ethanol (25.Og, 148 mmol) and sodiumazide (14.5g, 222mmol) in dimethylformamide (250ml) was standing at 100 0C night over. The reaction mixture was cooled on an ice bath, filtered and the organic solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (200ml), washed with water (75ml), the water- phase was extracted with additional dichloromethane (75ml) and the combined organic phases were dried with magnesium sulphate (MgSO4), filtered and evaporated in vacuo giving an oil which was used without further purification. Yield: 30.0 g (100%). 13C-NMR (CDCI3): δ = 72.53; 70.66-70.05; 61.74; 50.65
EXAMPLE 13 (2-[2-(2-Azidoethoxy)ethoxy]ethoxy)acetic acid
The above 2-[2-(2-azidoethoxy)ethoxy]ethanol (26g,148mmol) was dissolved in tetrahydrofuran (100ml) and under an nitrogen atmosphere slowly added to an ice cooled slurry of sodium hydride (24 g, 593 mmol, 60% in oil)) (which in advance had been washed with heptane (2x10OmI)) in tetrahydrofuran (250ml). The reaction mixture was standing for 40 min. then cooled on a ice bath followed by slowly addition of bromoacetic acid (31 g, 223mmol) dissolved in tetrahydrofuran (150ml) and then standing about 3 hours at RT. The organic solvent was evaporated in vacuo. The residue was suspended in dichloromethane (400ml). Water (100ml) was slowly added, where after the mixture was standing for 30 min. under mechanical stirring. The water phase was separated, acidified with hydrochloride (4N) and extracted with dichloromethane (2x75ml). All the combined organic phases were evaporated in vacuo giving a yellow oil. To the oil was slowly added a solution of piperidine (37 ml, 371 mmol) in dichloromethane (250ml), the mixture was standing under mechanical stirring for 1 hour. The clear solution was diluted with dichloromethane (100ml) and washed with hydrochloride (4N, 2x100ml). The water phase was extracted with additional dichloromethane (2x75ml) and the combined organic phases were evaporated in vacuo, giving an yellow oil which was used without further purification. Yield: 27.0 g (66%). 13C-NMR (CDCI3): δ = 173.30; 71 .36; 70.66-70.05; 68.65; 50.65
EXAMPLE 14
(S)-2,6-Bis-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}acetylamino)hexanoic acid methyl ester
The above (2-[2-(2-azidoethoxy)ethoxy]ethoxy)acetic acid (13g, 46.9mol) was dissolved in dichloromethane (100ml). N-Hydroxysuccinimide (6.5g, 56.3mmol) and 1 -ethyl-3-(3- dimethylaminopropylcarbodiimide hydrochloride (10.8g, 56.3mmol) was added and the reaction mixture was standing for 1 hour. Diisopropylethylamine (39ml, 234mmol) and L- lysine methyl ester dihydrochloride (6.Og, 25.8mmol) were added and the reaction mixture was standing for 16 hours. The reaction mixture was diluted with dichloromethane (300ml), extracted with water (100ml), hydrochloride (2N, 2x100ml), water (100ml), 50% saturated sodiumhydrogencarbonate (100ml) and water (2x100ml). The organic phase was dried with magnesium sulphate, filtered and evaporated in vacuo, giving an oil, which was used without further purification. Yield: 1 1 g (73 %). LCMS: m/z = 591 . 13C-NMR (CDCI3): (selected) δ = 172.48; 169.87; 169.84; 71 .093-70.02; 53.51 ; 52.34; 51 .35; 50.64; 38.48; 36.48; 31 .99; 31 .40; 29.13; 22.82.
EXAMPLE 15 (S)-2,6-Bis-(2-{2-[2-(2-t-butyloxycarbonylaminoethoxy)ethoxy]ethoxy}acetylamino)hexanoic acid methyl ester
To a solution of the above (S)-2,6-bis-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy} acetylamino)hexanoic acid methyl ester (1 .Og, 1 .7mmol) in ethylacetate (15ml) was added di- tert-butyl dicarbonat (0.9g, 4.24mmol) and 10% Pd/C (0.35g). Hydrogen was then constantly bubbled through the solution for 3 hours. The reaction mixture was filtered and the organic solvent was removed in vacuo. The residue was purified by flash chromatography using ethylacetate/methanol 9:1 as the eluent. Frations containing product were pooled and the organic solvent was removed in vacuo giving an oil. Yield: 0.6Og (50%). LC-MS: m/z = 739 (M+1 ).
EXAMPLE 16
(S)-2,6-Bis-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}acetylamino)hexanoic acid methyl ester
The above (S)-2,6-bis-(2-{2-[2-(2-t-butyloxycarbonylaminoethoxy)ethoxy]ethoxy}acetylamino) hexanoic acid methyl ester (0.6g, 0.81 mmol) was dissolved in dichloromethane (5ml). Trifluoroacetic acid (5ml) was added and the reaction mixture was standing about 1 hour. The reaction mixture was evaporated, in vacuo, giving an oil, which was used without further purification. Yield: 0.437 g (100%). LC-MS m/z = 539 (M+1 )
EXAMPLE 17 (S)-2,6-Bis-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}acetylamino)hexanoic acid
To a solution of (S)-2,6-bis-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}acetylamino) hexanoic acid methyl ester (2.Og, 3.47mmol) in methanol (10ml) was added sodium hydroxide (4N,1 .8ml, 6.94mmol) and the reaction mixture was standing for 2 hours. The organic solvent was evaporated in vacuo, and the residue was dissolved in water (45ml) and acidified with hydrochloric acid (4N). The mixture was extracted with dichloromethane (150ml) which was washed with saturated aqueous sodium chloride (2x25ml). The organic phase was dried over magnesium sulphate, filtered and evaporated, in vacuo, giving an oil. LC-MS m/z = 577 (M+1 ).
EXAMPLE 18
N-(tert-Butyloxycarbonylaminoxybutyl)phthalimide
To a stirred mixture of N-(4-bromobutyl)phthalimide (18.9 g, 67.0 mmol), MeCN (14 ml), and N-Boc-hydroxylamine (12.7 g, 95.4 mmol) was added DBU (15.0 ml, 101 mmol) in portions. The resulting mixture was stirred at 50 0C for 24 h. Water (300 ml) and 12 M HCI (10 ml) were added, and the product was extracted three times with AcOEt. The combined extracts were washed with brine, dried (MgSO4), and concentrated under reduced pressure. The resulting oil (28 g) was purified by chromatography (140 g SiO2, gradient elution with heptane/AcOEt). 17.9 g (80%) of the title compound was obtained as an oil. 1H NMR (DMSO-Cf6) δ = 1 .36 (s, 9H), 1 .50 (m, 2H), 1 .67 (m, 2H), 3.58 (t, J = 7 Hz, 2H), 3.68 (t, J = 7 Hz, 2H), 7.85 (m, 4H), 9.90 (s, 1 H).
EXAMPLE 19 4-(tert-Butyloxycarbonylaminoxy)butylamine
To a solution of N-(tert-butyloxycarbonylaminoxybutyl)phthalimide (8.35 g, 25.0 mmol) in EtOH (10 ml) was added hydrazine hydrate (20 ml), and the mixture was stirred at 80 0C for 38 h. The mixture was concentrated and the residue co-evaporated with EtOH and PhMe. To the residue was added EtOH (50 ml), and the precipitated phthalhydrazide was filtered off and washed with EtOH (50 ml). Concentration of the combined filtrates yielded 5.08 g of an oil. This oil was mixed with a solution of K2CO3 (10 g) in water (20 ml), and the product was extracted with DCM. Drying (MgSO4) and concentration yielded 2.28 g (45%) of the title compound as an oil, which was used without further purification. 1H NMR (DMSO-CZ6): δ = 1 .38 (m, 2H), 1.39 (s, 9H), 1 .51 (m, 2H), 2.51 (t, J = 7 Hz, 2H), 3.66 (t, J = 7 Hz, 2H).
EXAMPLE 20
2-(1 ,3-Bis[2-(2-hydroxyethoxy)ethoxy]propan-2-yloxy) acetic acid tert-butyl ester
1 ,3-Bis[2-(2-trityloxyethoxy)ethoxy]propan-2-ol (0.3 g, 0.40 mmol) was evaporated once from dry pyridine and once from dry acetonitrile. The residual was dissolved in dry DMF (2 mL), under nitrogen, 60% NaH - oil suspension (24 mg, 0.6 mmol) was added. The mixture was stirred at room temperature for 15 minutes. terf-Butylbromoacetate (0.07 mL, 0.48 mmol) was added and the mixture was stirred for an additional 60 minutes. The reaction was quenched with ice, then partitioned between diethyl ether (100 ml_) and water (100 ml_). The organic phase was collected, dried (Na2SO4), and solvent removed in vacuo to afford an oil which was eluted on silical gel column with EtOAc/Heptane/Et3N (49:50:1 ). Fraction containing main product was collected. The solvent was removed in vacuo and the residue was dissolved in 80 % aqueous acetic acid (5 ml_) and stirred at room temperature overnight. Solvent was removed in vacuo and the crude material dissolved in diethyl ether (25 ml_), and washed with water (2 x 5ml_). The water phases were collected and the water removed on rotorvap to yield 63 mg of the title compound. 1H NMR (CDCI3): δ = 4.19 (s, 2H), 3.78-3.55 (m, 21 H), 1 .49 (s, 9H).
EXAMPLE 21 Λ/,Λ/-Bis(2-(2-phthalimidoethoxy)ethyl)-0-tert-butylcarbamate
Λ/,Λ/-Bis(2-hydroxyethyl)-0-tert-butylcarbamate is dissolved in a polar, non-protic solvent such as THF or DMF. Sodium hydride (60 % suspension in mineral oil) is added slowly to the solution. The mixture is stirred for 3 hours. Λ/-(2-Bromoethyl)phthalimide is added. The mixture is stirred until the reaction is complete. The reaction is quenched by slow addition of methanol. Ethylacetate is added. The solution is washed with aqueous sodium hydrogencarbonate. The organic phase is dried, filtered, and subsequently concentrated under vacuum as much as possible. The crude compound is purified by standard column chromatography.
EXAMPLE 22 N,N-Bis(2-(2-aminoethoxy)ethyl)-O-tert-butylcarbamate
N,N-Bis(2-(2-phthalimidoethoxy)ethyl)-O-tert-butylcarbamate is dissolved in a polar solvent such as ethanol. Hydrazine (or another agent known to remove the phthaloyl protecting group) is added. The mixture is stirred at room temperature (or if necessary elevated temperature) until the reaction is complete. The mixture is concentrated under vacuum as much as possible. The crude compound is purified by standard column chromatography or if possible by vacuum destination.
EXAMPLE 23 N,N-Bis(2-(2-benzyloxycarbonylaminoethoxy)ethyl)-O-tert-butylcarbamate
N,N-Bis(2-(2-aminoethoxy)ethyl)-O-tert-butylcarbamate is dissolved in a mixture of aqueous sodium hydroxide and THF or in a mixture of aqueous sodium hydroxide and acetonitrile. Benzyloxychloroformate is added. The mixture is stirred at room temperature until the reaction is complete. If necessary, the volume is reduced in vacuo. Ethyl acetate is added. The organic phase is washed with brine. The organic phase is dried, filtered, and subsequently concentrated in vacuo as much as possible. The crude compound is purified by standard column chromatography.
EXAMPLE 24
Bis(2-(2-phthalimidoethoxy)ethyl)amine
Bis(2-(2-phthalimidoethoxy)ethyl)-tert-butylcarbamate is dissolved in trifluoroacetic acid. The mixture is stirred at room temperature until the reaction is complete. The mixture is concentrated in vacuo as much as possible. The crude compound is purified by standard column chromatography.
EXAMPLE 25
1 1 -Oxo-17-phthalimido-12-(2-(2-phthalimidoethoxy)ethyl)-3,6,9,15-tetraoxa-12-azahepta- decanoic acid
3,6,9-Trioxaundecanoic acid is dissolved in dichloromethane. A carbodiimide (for example NjN-dicyclohexylcarbodiimide or N,N-diisopropylcarbodiimide) is added. The solution is stirred over night at room temperature. The mixture is filtered. The filtrate can be concentrated in vacuo if necessary. The acylation of amines with the formed intramolecular anhydride is known from literature (for example Cook, R. M.; Adams, J. H.; Hudson, D. Tetrahedron Lett., 1994, 35, 6777-6780 or Stora, T.; Dienes, Z.; Vogel, H.; Duschl, C. Langmuir 2000, 16, 5471 -5478). The anhydride is mixed with a solution of bis(2-(2- phthalimidoethoxy)ethyl)amine in a non-protic solvent such as dichloromethane or N, N- dimethylformamide. The mixture is stirred until the reaction is complete. The crude compound is purified by extraction and subsequently standard column chromatography.
EXAMPLE 26
5-Oxo-11 -phthalimido-6-(2-(2-phthalimidoethoxy)ethyl)-3,9-dioxa-6-azaundecanoic acid
A solution of diglycolic anhydride in a non-protic solvent such as dichloromethane or N, N- dimethylformamide is added dropwise to a solution of bis(2-(2-phthalimidoethoxy)ethyl)amine in a non-protic solvent such as dichloromethane or N,N-dimethylformamide. The mixture is stirred until the reaction is complete. The crude compound is purified by extraction and subsequently standard column chromatography.
EXAMPLE 27
Bis-[2-(1 ,3-dioxo-1 ,3-dihydroisoindol-2-yl)ethyl]ammonium acetate
Diethylenetriamine (15.8 ml, 145.4 mmol) was added slowly to glacial acetic acid (175 ml) while it was cooled on icebath. Phthalic anhydride (43.1 g, 290.8 mmol) was added. The resulting mixture was refluxed for 20 h. The solution was cooled. The mixture was concentrated in vacuo. The resulting viscous oil was co-evaporated from acetonitrile 3 times.
The oil was mixed with acetonitrile (250 ml) and the mixture was heated to reflux briefly. The solution was kept in fridge over night. The formed crystals were isolated by filtration. The isolated bright yellow crystals were dried in vacuum oven.
Yield: 33.5 g, 63 %
Additional material could be obtained by crystallisation of the filtrate after concentration (in vacuo).
1H-NMR (Gf-DMSO) δ: 7.81 -7.74 (m, 8H), 3.60 (t, J = 6.32 Hz, 4H), 3.70-3.20 (b, 15H), 2.76
(t, J = 6.32 Hz, 4H), 1 .91 (s, 5H presumably residual acetic acid is present).
13C-NMR (Cf-DMSO) O: 168.3, 146.0, 134.5, 132.0, 123.2, 46.5, 37.5 - minor peaks from acetonitrile and acetic acid also observed.
LC-MS (ES-positive mode), m/z: 364
EXAMPLE 28
2-[2-({Bis-[2-(1 ,3-dioxo-1 ,3-dihydroisoindol-2-yl)ethyl]carbamoyl}methoxy)ethoxy]- ethoxyjacetic acid
3,6,9-Trioxaununcandioic acid (2.67 g, 12 mmol) and fy/V'-dicyclohexylcarbodiimide (2.48 g, 12 mmol) were mixed in dichloromethane (90 ml). The resulting mixture was stirred for 30 minutes. The mixture was filtered and subsequently concentrated in vacuo. The formed compound was dissolved in dichloromethane (250 ml). Bis-[2-(1 ,3-dioxo-1 ,3-dihydro-isoindol- 2-yl)-ethyl]-ammonium acetate (4.0 g, 9.45 mmol) and Λ/,Λ/,Λ/',Λ/'-tetramethylguanidine (1 .15 g, 10.0 mmol) were added to solution. The resulting mixture was stirred for at least 20 h. The mixture was concentrated in vacuo. Ethyl acetate (150 ml) and aqueous sodium hydrogencarbonate (5 % w/w, 150 ml) were added. The phases were separated. Concentrated hydrochloric acid was added to the aqueous phase until pH was 1 -2. The solution was extracted with dichloromethane (4 x100 ml). The combined organic extracts were dried over magnesium sulphate, filtered, and concentrated in vacuo to yield bright yellow syrup. Yield: 2.55 g, 48 %
1H-NMR (CDCI3) δ: 7.86-7.69 (m, 8H), 4.17 (s, 2H), 4.09 (s, 2H), 3.94-3.51 (several multiplets, 16H).
13C- NMR (CDCI3) δ: 172.2, 170.4, 168.3, 168.0, 134.4, 134.0, 132.0, 131 .7, 123.6, 123.4, 71 .3, 70.5, 70.4, 70.3, 69.2, 69.0, 44.6, 44.0, 35.7, 35.4 LC-MS (ES-positive mode), m/z: 568 [M+H]+ and 591 [M+Na]+
EXAMPLE 29
({Bis-[2-(1 ,3-dioxo-1 ,3-dihydroisoindol-2-yl)ethyl]carbamoyl}methoxy)acetic acid
Bis-[2-(1 ,3-dioxo-1 ,3-dihydroisoindol-2-yl)ethyl]ammonium acetate (25.8 g, 60.9 mmol) was suspended in DCM (100 ml). Λ/,Λ/,Λ/',Λ/'-Tetramethylguanidine (7.00 g, 60.9 mmol) was added upon which massive precipitation occurred. Additional dichloromethane (50 ml) was added. Diglycolic anhydride (8.48 g, 73.0 mmol) was added in 1 portion. The mixture was stirred for at least 20 h. The mixture was concentrated in vacuo. The resulting syrup was dissolved in a mixture of ethyl acetate (750 ml) and saturated aqueous sodium hydrogencarbonate (750 ml). The organic phase was extracted with saturated aqueous sodium hydrogencarbonate (2x200 ml). The combined aqueous phases were acidified with concentrated hydrochloric acid (pH 1 -2) - massive precipitation of white solid. The combined aqueous phases were extracted with dichloromethane (400 and 2 x 200 ml). The organic phase was dried over magnesium sulphate and filtered. The organic phase was concentrated in vacuo to about 200 ml after which it was filtered again. Further precipitation occurred during filtration and concentration. The filtrate was evaporated to yield a white solid. Yield: 6.04 g
1H-NMR (af-DMSO) δ: 12.65 (b, 1 H), 7.89-7.80 (m, 8H), 4.08 (s, 2H), 3.81 -3.75 (m, 6H), 3.59-3.50 (m, 4H)
13C- NMR (CDCI3) δ: 171 .4, 169.4, 168.3, 168.1 , 134.9, 134.7, 131 .9, 131 .8, 123.5, 123.3, 68.4, 67.4, 44.3, 43.5, 35.9, 35.5 LC-MS (ES-positive mode), m/z: 480 [M+H]+
EXAMPLE 30
Benzyl phenyl carbonate
According to: Pittelkow, M.; Lewinsky, R.; and Christensen, J. B. Synthesis 2002, 15, 2195- 2202.
Phenyl chloroformate (54.1 g, 500 mmol) was added dropwise to a mixture of benzyl alcohol (78.3 g, 500 mmol), dichloromethane (90 ml) and pyridine (50 ml) in a 1 l-f lask with condenser and addition funnel. The mixture was stirred for 1 h. Water (125 ml) was added. The phases were separated. The organic phase was washed with dilute sulfuric acid (2 M, 2x125 ml). Brine had to be added in the final wash in order to obtain good separation. The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. The crude compound was vacuum destilled to yield a colourless liquid. Yield:104.3 g, 91 %
1H-NMR (CDCI3) δ: 7.46-7.17 (2 multiplets, 10H), 5.27 (s, 2H)
13C- NMR (CDCI3) δ: 152.5, 149.9, 133.5, 128.3, 127.6, 127.5, 127.3, 124.8, 119.8, 69.1
EXAMPLE 31 Bis-(2-benzyloxycarbonylaminoethyl)ammonium chloride cf
According to: Pittelkow, M.; Lewinsky, R.; and Christensen, J. B. Synthesis 2002, 15, 2195-
2202.
Benzyl phenylcarbonate (25,1 g, 1 10 mmol) was added dropwise to a solution of diethylenetriamine (5,16 g, 50 mmol) in dichloromethane (100 ml). The mixture was stirred for at least 20 h. The organic phase was washed with phosphate buffer (0.025 M K2HPO4,
0.025 M NaH2PO4, 2000 ml, pH adjusted to 3 with 2 M sulfuric acid). The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo.
Yield: 25.2 g A portion (5 g) of the crude oil was mixed with hydrochloric acid (2 M, 15 ml). The mixture was stirred for 15 minutes. The mixture was filtered. The isolated solid was mixed with abs. ethanol (600 ml). The mixture was brought to reflux. The boiling mixture was decanted in order to remove insoluble impurities. The compound crystallized over night at 5 °C.
Yield: 2.84 g (white crystals) 1H-NMR (af-DMSO) δ: 8.96 (b, 2H), 7.51 (t, J = 5.56 Hz, 2H), 7.40-7.30 (b, 10H), 5,04 (s,
4H), 3.33 (q, J = 6.06 Hz, 4H), 3.00 (b, 4H)
13C- NMR (Gf-DMSO) δ: 156.6, 137.2, 128.7, 128.3, 128.2, 66.0, 46.8, 37.1
LC-MS (ES-positive mode), m/z: 372.5 [M+H]+
EXAMPLE 32
[2-(2-{[Bis-(2-benzyloxycarbonylaminoethyl)carbamoyl]methoxy}ethoxy)ethoxy]acetic acid
3,6,9-Trioxaundecandioic acid (1 .83 g, 8.3 mmol) and fy/V'-dicyclohexylcarbodiimide (1 .70 g, 8.3 mmol) were mixed in dichloromethane (10 ml). The resulting mixture was stirred for 30 minutes. The mixture was filtered and subsequently concentrated in vacuo. The formed compound was mixed with bis-(2-benzyloxycarbonylaminoethyl)ammonium chloride (2.8 g, 6.87 mmol) and Λ/,Λ/,Λ/',Λ/'-tetramethylguanidine (791 mg, 6.87mmol)(250 ml) in Λ/,Λ/-di- methylformamide (27 ml). The resulting mixture was stirred for 20 h. The mixture was concentrated in vacuo. Ethyl acetate (150 ml) and aqueous sodium hydrogencarbonate (5 % w/w, 150 ml) were added. The phases were separated. The organic phase was extracted with aqueous sodium hydrogencarbonate (5 % w/w, 2 x 100 ml). The combined aqueous extracts were mixed with ethyl acetate (200 ml). Concentrated hydrochloric acid was added to the mixture until pH was 2-3. The phases were separated immidiately. The aqueous phase was extracted with ethyl acetate (2 x 200 ml). The combined organic extracts were dried with magnesium sulphate, filtered, and concentrated in vacuo to yield colourless syrup. Yield: 2.17 g, 55 %
1H-NMR (CDCI3) δ: 10.2 (b, 1 H), 7.31 (b, 10H), 6.10 (b, 1 H), 5.84 (b, 1 H), 5.06 (s, 2H), 5.04 (s, 2H), 4.17-4.09 (m, 4H), 3.72-3.22 (several multiplets, 16H)
13C- NMR (CDCI3) δ: 172.9, 171 .2, 157.3, 137.0, 128.9-128.4 (several signals), 71 .3, 70.8, 70.7, 70.3, 68.9, 67.1 , 67.0, 47.5, 46.0, 39.6 LC-MS (ES-positive mode), m/z: 576 [M+H]+
EXAMPLE 33 [2-(2-{[Bis-(2-aminoethyl)carbamoyl]methoxy}ethoxy)ethoxy]acetic acid H2N NH2
[2-(2-{[Bis-(2-benzyloxycarbonylaminoethyl)carbamoyl]methoxy}ethoxy)ethoxy]acetic acid (725 mg, 1 .26 mmol) is dissolved in methanol (50 ml). Palladium on activated carbon (150 mg, 5 % Pd, wet, Degussa catalyst type E101 NO/W) was added. The mixture was stirred in an atmosphere of hydrogen gas for 20 h. The mixture was filtered. The filtrate was concentrated in vacuo. LC-MS (ES-positive mode), m/z: 309 [M+H]+, 291 [M-H2O]+
EXAMPLE 34 [2-(2-{[Bis-(2-benzyloxycarbonylaminoethyl)carbamoyl]methoxy}ethoxy)ethoxy]acetic acid 2,5-dioxopyrrolidin-1 -yl ester
[2-(2-{[Bis-(2-benzyloxycarbonylaminoethyl)carbamoyl]methoxy}ethoxy)ethoxy]acetic acid (1 .45 g, 2.52 mmol) was mixed with Λ/-hydroxysuccinic imide (291 mg, 2.53 mmol), 1 -ethyl-3- (3'-dimethylaminopropyl)carbodiimide hydrochloride (485 mg, 2.53 mmol), and N,N,N',N'- tetramethylguanidine (291 mg, 2.53 mmol). The mixture was stirred for 20 h. Aqueous sodium hydrogensulfate (5 % w/w, 150 ml) and dichloromethane (100 ml) were added. The phases were separated. The aqueous phase was extracted with dichloromethane (2 x 100 and 2 x 50 ml). The combined organic phases were dried over solid sodium sulfate, filtered, and concentrated in vacuo. LC-MS (ES-positive mode), m/z: 674 [M+H]+, 577 (unreacted starting material).
EXAMPLE 35
1 ,2,3-Benzotriazin-4(3H)-one-3-yl 2-[2-(2-methoxyethoxy)ethoxy]acetate
3-Hydroxy-1 ,2,3-benzotriazin-4(3H)-one (10.0 g; 61 .3 mmol) and 2-[2-(2- methoxyethoxy)ethoxy]acetic acid (10.9 g; 61.3 mmol) was suspended in DCM (125 ml) and DIC (7,7 g; 61 .3 mmol) was added. The mixture was stirred under a dry atmosphere at ambient temperature over night. A precipitate of diisopropyl urea was formed, which was filtered off. The organic solution was washed extensively with aqueous saturated sodium hydrogen carbonate solution, then dried (Na2SO4) and evaporated in vacuo, to give the title product as a clear yellow oil. Yield was 16.15 g (81 %). 1H-NMR (CDCI3): δ = 3.39 ppm (s, 3H); 3.58 (t, 2H); 3.68 (t, 2H); 3.76 (t, 2H); 3.89 (t, 2H); 4.70 (s, 2H); 7.87 (t, 1 H); 8.03 (t, 1 H); 8.23 (d, 1 H); 8.37 (d, 1 H). 13C-NMR (CDCI3, selected peaks): δ = 57.16 ppm; 64.96; 68.71 ; 68.79; 69.59; 69.99; 120.32; 123.87; 127.17; 130.96; 133.63; 142.40; 148.22; 164.97.
OLIGOMERIC PRODUCTS:
Solid Phase Oligomerisation: The reactions described below are all performed on polystyrene functionalised with the Wang linker. The reactions will in general also work on other types of solid supports, as well as with other types of functionalised linkers.
Solid phase azide reduction: The reaction is known (Schneider, S. E. et al. Tetrahedron, 1998, 54(50) 15063-15086) and can be performed by treating the support bound azide with excess of triphenyl phosphine in a mixture of THF and water for 12-24 hours at room temperature. Alternatively, trimethylphosphine in aqueous THF as described by Chan, T. Y. et al Tetrahedron Lett. 1997, 38(16), 2821 -2824 can be used. Reduction of azides can also be performed on solid phase using sulfides such as dithiothreitol (Meldal, M. et al. Tetrahedron Lett. 1997, 38(14), 2531 - 2534) 1 ,2-dimercaptoethan and 1 ,3-dimercaptopropan (Meinjohanns, E. et al. J. Chem. Soc, Perkin Trans 1 , 1997,6, 871 -884) or tin(ll) salts such as tin(ll)chloride (Kim, J. M. et al. Tetrahedron Lett, 1996, 37(30), 5305-5308).
Solid phase carbamate formation:
The reaction is known and is usually performed by reacting an activated carbonate, or a halo formiate derivative with an amine, preferable in the presence of a base.
EXAMPLE 36 3-(1 ,3-Bis{2-[2-([benzoylamino]ethoxy)ethoxy}propan-2-yloxycarbonyl)amino)propanoicacid
This example uses the 1 ,3-bis[2-(2-azidoethoxy)ethoxy]propan-2-yl-p-nitrophenylcarbonate monomer building block prepared in example 4 in the synthesis of a second generation carbamate based branched polymer capped with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid. The coupling chemistry is based on standard solid phase carbamate chemistry, and the protection methodology is based on a solid phase azide reduction step as described above.
Step 1 : Fmoc-β-Ala-Wang resin (100 mg; loading 0.31 mmol/g BACHEM) was suspended in dichloromethane for 30 min, and then washed twice with DMF. A solution of 20% piperidine in DMF was added, and the mixture was shaken for 15 min at ambient temperature. This step was repeated, and the resin was washed with DMF (3x) and DCM (3x).
Step 2: Coupling of monomer building blocks: A solution of 1 ,3-bis[azidoethoxyethyl]propan- 2-yl-p-nitrophenylcarbamate (527 mg; 1 ,4 mmol, 4x) was added to the resin together with DIPEA (240 μl; 1 ,4 mmol, 4x). The resin was shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
Step 3: Capping with acetic anhydride: The resin was then treated with a solution of acetic anhydride, DIPEA, DMF (12:4:48) for 10 min. at ambient temperature. Solvent was removed and the resin was washed with DMF (3x) and DCM (3x). Step 4: Deprotection (reduction of azido groups): The resin was treated with a solution of DTT (2M) and DIPEA (1 M) in DMF at 50 5C for 1 hour. The resin was then washed with DMF (3x) and DCM (3x). A small amount of resin was withdrawn and treated with a solution of benzoylchloride (0.5 M) and DIPEA (1 M) in DMF for 1 h. The resin was cleaved with 50% TFA/DCM and the dibenzoylated product analysed with NMR and LC-MS. 1H-NMR (CDCI3): δ = 3.50-3.75 (m, 20H); 3.85 (s, 1 H); 4.25 (d, 2H); 6.95 (t, 1 H); 7.40-7.50 (m, 6H); 7.75 (m, 4H). LC-MS: m/z = 576 (M+1 ); R1 = 2.63 min.
EXAMPLE 37 3-[2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetylamino}ethoxy)ethoxy]propan-2-yl- oxy)acetylamino]propanoic acid
Step 1 : Fmoc-β-Ala linked Wang resin (A22608, Nova Biochem, 3.00 g; with loading 0.83 mmol/g) was swelled in DCM for 20 min. then washed with DCM (2x20 ml) and NMP (2x20 ml). The resin was then treated twice with 20% piperidine in NMP (2x15 min). The resin was washed with NMP (3x20 ml) and DCM (3x20 ml).
Step 2: 2-(1 ,3-Bis[2-(2-azidoethoxy)ethoxy]propan-2-yloxy)acetic acid (3.70 g; 10 mmol) was dissolved in NMP (30 ml) and DhbtOH (1 .60 g; 10 mmol) and DIC (1 .55 ml; 10 mmol) was added. The mixture was stirred at ambient temperature for 30 min, and then added to the resin obtained in step 1 together with DIPEA (1.71 ml; 10 mmol). The reaction mixture was shaken for 1 .5 h, then drained and washed with NMP (5x20 ml) and DCM (3x20 ml).
Step 3: A solution of SnCI2.2H2O (1 1.2 g; 49.8 mmol) in NMP (15 ml) and DCM (15 ml) was then added. The reaction mixture was shaken for 1 h. The resin was drained and washed with NMP:MeOH (5x20 ml; 1 :1 ). The resin was then dried in vacuo. Step 4: A solution of 2-[2-(2-methoxyethyl)ethoxy]acetic acid (1 .20 g; 6.64 mmol), DhbtOH (1 .06 g; 6.60 mmol) and DIC (1 .05 ml; 6.60 mmol) in NMP (10 ml) was mixed for 10 min, at room temperature, and then added to the 3-[2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2- yloxy)acetylamino]propanoic acid tethered wang resin (1.0 g; 0.83 mmol/g) obtained in step 3. DIPEA (1 .15 ml, 6.60 mmol) was added, and the reaction mixture was shaken for 2.5 h. Solvent was removed, and the resin was washed with NMP (5x20 ml) and DCM (10x20 ml).
Step 5: The resin product of step 4 was treated with TFA:DCM (10 ml, 1 :1 ) for 1 hour. The resin was filtered and washed once with TFA:DCM (10 ml, 1 :1 ). The combined filtrate and washing was then taken dryness, to give a yellow oil (711 mg). The oil was dissolved in 10% acetonitril-water (20 ml), and purified over two runs on a preparative HPLC apparatus using a C18 column, and a gradient of 15-40% acetonitril-water. Fractions were subsequently analysed by LC-MS. Fractions containing product were pooled and taken to dryness. Yield: 222 mg (37%). LC-MS: m/z = 716 (M+1 ), Rt = 1 .97 min. 1H-NMR (CDCI3): δ = 2.56 ppm (t, 2H); 3.36 (s, 6H); 3.46-3.66 (m, 39H); 4.03 (s, 4H); 4.16 (s, 2H); 7.55 (t, 2H); 8.05 (t, 1 H). 13C-NMR (CDCI3, selected peaks): δ = 33.71 ppm; 34.90; 58.89; 68.94; 69.40; 69.98; 70.09; 70.33; 70.74; 70.91 ; 71 .07; 71.74; 79.07; 171 .62; 171 .97; 173.63.
EXAMPLE 38 3-(1 ,3-Bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetamino}ethoxy)ethoxy]- propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)acetylamino)propanoic acid
This material was prepared from 3-[2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)- acetylamino]propanoic acid tethered wang resin (1 .0 g; 0.83 mmol/g), obtained in step 3 of example by repeating step 2-5, doubling the amount of reagents used. Yield: 460 mg (33%). MALDI-MS (α-cyano-4-hydroxycinnamic acid): m/z = 1670 (M+Na). 1H-NMR (CDCI3): δ = 2.57 ppm (t, 2H); 3.38 (s, 12H); 3.50-3.73 (m, 85 H); 4.05 (s, 8H); 4.17 (s, 2H); 4.19 (s, 4H); 7.48 (m, 4H); 7.97 (m, 3H). 13C-NMR (CDCI3, selected peaks): δ = 38.81 ppm; 58.92; 69.46; 69.92; 70.05; 70.05; 70.13; 70.40; 70.73; 70.97; 71 .11 ; 71.88; 76.74; 77.06; 77.38; 171.33; 172.02.
Alternative mode of preparation:
This example uses the 2-(1 ,3-bis[azidoethoxyethyl]propan-2-yloxy)acetic acid monomer building block prepared in example 6 in the synthesis of a second generation amide based branched polymer capped with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid. The coupling chemistry is based on standard solid phase peptide chemistry, and the protection methodology is based on a solid phase azide reduction step as described above.
Step 1 : Fmoc-β-Ala-Wang resin (100 mg; loading 0.31 mmol/g BACHEM) was suspended in dichloromethane for 30 min, and then washed twice with DMF. A solution of 20% piperidine in DMF was added, and the mixture was shaken for 15 min at ambient temperature. This step was repeated, and the resin was washed with DMF (3x) and DCM (3x).
Step 2: Coupling of monomer building blocks: A solution of 2-(1 ,3-bis[azidoethoxyethyl]- propan-2-yloxy)acetic acid (527 mg; 1 ,4 mmol, 4x) and DhbtOH (225 mg; 1 ,4 mmol, 4x) were dissolved in DMF (5 ml) and DIC (216 μl, 1 ,4 mmol, 4x) was added. The mixture was left for 10 min (pre-activation) then added to the resin together with DIPEA (240 ul; 1 ,4 mmol, 4x). The resin was shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
Step 3: Capping with acetic anhydride: The resin was then treated with a solution of acetic anhydride, DIPEA, DMF (12:4:48) for 10 min. at ambient temperature. Solvent was removed and the resin was washed with DMF (3x) and DCM (3x).
Step 4: Deprotection (reduction of azido groups): The resin was treated with a solution of DTT (2M) and DIPEA (1 M) in DMF at 50 5C for 1 hour. The resin was then washed with DMF (3x) and DCM (3x). A small amount of resin was withdrawn and treated with a solution of benzoylchloride (0.5 M) and DIPEA (1 M) in DMF for 1 h. The resin was cleaved with 50% TFA/DCM and the dibenzoylated product analysed with NMR and LC-MS. 1H-NMR (CDCI3): δ = 3.50-3.75 (m, 20H); 3.85 (s, 1 H); 4.25 (d, 2H); 6.95 (t, 1 H); 7.40-7.50 (m, 6H); 7.75 (m, 4H). LC-MS: m/e = 576 (M+1 ); Rt = 2.63 min. Step 5-7 was performed as step 2-4 using a double molar amount of reagents but same amount of solvent.
Step 8: Capping with 2-[2-(2-methoxyethoxy)ethoxy]acetic acid: A solution of 2-[2-(2- methoxyethoxy)ethoxy]acetic acid (997 mg; 5.6 mmol, 16x with respect to resin loading) and DhbtOH (900 mg; 5.6 mmol, 16x) were dissolved in DMF (5 ml) and DIC (864 ul, 5.6 mmol, 16x) was added. The mixture was left for 10 min (pre-activation) then added to the resin together with DIPEA (960 ul; 5.6 mmol, 16x). The resin was shaken for 90 min, then drained and washed with DMF (3x) and DCM (3x).
Step 9: Cleavage from resin: The resin was treated with a 50% TFA - DCM solution at ambient temperature for 30 min. The solvent was collected and the resin was washed an additional time with 50% TFA - DCM. The combined filtrates were evaporated to dryness, and the residue was purified by chromatography.
EXAMPLE 39
3-(1 ,3-Bis{2-(2-[2-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetamino}- ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)acetylamino)- ethoxy)ethoxy}propan-2-yloxy)acetylamino)propanoic acid
This material was prepared from 3-[2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)- acetylamino]propanoic acid tethered wang resin (1 .0 g; 0.83 mmol/g), obtained in step 3 of example by repeating step 2-3 with 2x the amount of reagents used, then repeating step 2-5 with 4x the amount of reagent used. Yield: 84 mg (4%). LC-MS: (m/2)+1 = 1758; (m/3)+1 = 1 172; (m/4)+1 = 879; (m/5)+1 = 704. Rt = 2.72 min. 1H-NMR (CDCI3): δ = 2.51 ppm (t, 2H); 3.33 (s, 24H); 3.44-3.70 (m, 213H); 3.93 (s, 16H); 4.08 (s, 14H); 7.25 (m, 8H); 7.69 (m, 7H). 13C-NMR (CDCI3, selected peaks): δ = 38.94 ppm; 59.33; 69.78; 70.08; 70.37; 70.44; 70.56; 70.82; 71 .10; 71 .26; 71 .51 ; 72.17; 79.24; 170.60; 171 .22.
EXAMPLE 40
N-Hydroxysuccinimidyl 3-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetylamino}- ethoxy)ethoxy]propan-2-yloxy)acetylamino]propanoate
3-[2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetylamino}ethoxy)ethoxy]propan-2-yl- oxy)acetylamino]propanoic acid (67 mg; 82 μmol) was dissolved in THF (5 ml). The reaction mixture was cooled on an ice bath. DIPEA (20 μl; 120 μmol) and TSTU (34 mg; 120 μmol) was added. The mixture was stirred at ambient temperature overnight at which time, the reaction was complete according to LC-MS. LC-MS: m/z = 813 (M+H); Rt = 2.22 min.
EXAMPLE 41 N-Hydroxysuccinimidyl 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acet- amino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)acetyl- amino)propanoate
Prepared from 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetamino}- ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)- acetylamino)propanoic acid and TSTU similarly as as described in example 40. LC-MS: (m/2)+1 = 873, Rt = 2.55 min. EXAMPLE 42
N-Hydroxysuccimidyl 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)- ethoxy]acetamino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)- acetylamino)ethoxy)ethoxy}propan-2-yloxy)acetylamino)propanoate
Prepared from N-hydroxysuccinimidyl 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2- methoxyethoxy)ethoxy]acetamino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}- propan-2-yloxy)acetylamino)ethoxy)ethoxy}propan-2-yloxy)acetylamino)propanoic acid and TSTU as described in example 40. LC-MS: (m/4)+1 = 903, Rt = 2.69 min.
EXAMPLE 43
N-(4-tert-Butoxycarbonylaminoxybutyl) 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxy- ethoxy)ethoxy]acetamino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-
2-yloxy)acetylamino)propanamide
N-Hydroxysuccinimidyl 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acet- amino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)acetyl- amino)propanoate (105 mg; 0.06 mmol) was dissolved in DCM (2 ml). Then a solution of 4- (tert-butyloxycarbonylaminoxy)butylamine (49 mg; 0.24 mmol) was added followed by DIPEA (13 μl; 0.07 mmol). The mixture was stirred at ambient temperature for one hour, then concentrated under reduced pressure. The residue was dissolved in 20% acetonitril-water (4 ml), and purified on a preparative HPLC apparatus using a C18 column, and a step gradient of 0, 10, 20, 30, and 40% (10 ml elutions each) of acetonitril-water. Fractions containing pure product was concentrated and dried for 16 hours in a vacuum oven to give a yellow oil. Yield: 57 mg (51 %). LC-MS: (m/2)+1 = 918, Rt = 2.75 min. 1H-NMR (CDCI3): δ = 1.42 ppm (s, 9H); 2.40 (t, 2H); 3.21 (dd, 2H); 3.33 (s, 12H); 3.38-3.72 (m, 99H); 3.80 (m, 2H); 3.95 (s, 8H); 4.08 (s, 6H); 6.99 (m, 1 H); 7.23 (m, 4H); 7.69 (m, 2H); 7.85 (m, 1 H); 8.00 (m, 1 H). 13C-NMR (CDCI3, selected peaks): δ = 28.27 ppm; 38.58; 58.97; 69.42; 69.72; 70.01 ; 70.08; 70.20; 70.41 ; 70.46; 70.73; 70.91 ; 71.16; 71.22; 71 .81 ; 78.89; 81.33; 170.27; 170.89.
EXAMPLE 44
N-(4-Aminoxybutyl) 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acet- amino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)acetyl- amino)propanamide
N-(4-tert-Butoxycarbonylaminoxybutyl) 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxy- ethoxy)ethoxy]acetamino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan- 2-yloxy)acetylamino)propanamide (19 mg; 10 μmol) was dissolved in 50% TFA/DCM (10 ml), and the clear solution was stirred at ambient temperature for 30 min. The solvent was removed by rotary evaporation, and the residue was stripped twice from DCM, to give a quantitative yield (19 mg) of the title product. LC-MS: (m/2)+1 = 868, (m/3)+1 = 579, R1 = 2,35 min.
EXAMPLE 45 terf-Butyl 2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetamino}ethoxy)ethoxy]propan-2- yloxy)acetate terf-Butyl 2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)acetate (1 .74 g; 4.5 mmol, example 9) and 1 ,2,3-benzotriazin-4(3H)-one-3-yl 2-[2-(2-methoxyethoxy)ethoxy]acetate (2.94 g; 9 mmol, example 35) were dissolved in DCM (100 ml). DIPEA (3.85 ml; 22.3 mmol) was added and the clear mixture was stirred for 90 min at room temperature. Solvent was removed in vacuo, and the residue was purified by chromatography on silica, using MeOH - DCM (1 :16) as eluent. Pure fractions were pooled and taken to dryness to give the title material as a clear oil. Yield was 1 .13 g (36 %). 1H-NMR (CDCI3): δ = 1 .46 ppm (s, 9H); 3.38 (s, 6H); 3.49-3.69 (m, 37H); 4.01 (s, 4H); 4.18 (s, 2H); 7.20 (bs, 2H).
EXAMPLE 46
2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetamino}ethoxy)ethoxy]propan-2-yloxy)- acetic acid:
terf-Butyl 2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)acetate (470 mg; 0.73 mmol) was dissolved in DCM-TFA (25 ml, 1 :1 ) and the mixture was stirred for 30 min at ambient temperature. The solvent was removed, in vacuo, and the residue was stripped twice from DCM. LC-MS: (M+1 ) = 645, R1 = 2,26 min. 1H-NMR (CDCI3): δ = 3.45 ppm (s, 6H); 3.54-3.72 (m, 37H); 4.15 (s, 4H); 4.36 (s, 2H).
EXAMPLE 47
N-Hydroxysuccimidyl 2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetamino}ethoxy)- ethoxy]propan-2-yloxy)acetate
2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetamino}ethoxy)ethoxy]propan-2-yloxy)- acetic acid (115 mg; 0.18 mmol) was dissolved in THF (5 ml). The reaction mixture was placed on an ice bath. TSTU (65 mg, 0.21 mmol) and DIPEA (37 μl; 0.21 mmol) was added and the reaction mixture was stirred at 0 5C for 30 min, then at room temperature overnight. The reaction was then taken to dryness, to give 130 mg of the title material as an clear oil. LC-MS: (m+1 ) = 743, (m/2)+1 = 372, Rt = 2,27 min.
EXAMPLE 48 t-Butyl 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acet- amino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)acetyl- amino)ethoxy)ethoxy}propan-2-yloxy)acetate
The material was prepared from two equivalents of N-hydroxysuccimidyl 2-(1 ,3-bis[2-(2-{2-[2- (2-methoxyethoxy)ethoxy]acetamino}ethoxy)ethoxy]propan-2-yloxy)acetate and one equivalent of terf-butyl 2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan-2-yloxy)acetate, using the protocol and purification method described in example 45.
Further dendritic growth may be acchieved by removing the terf-butyl group as described in example 46 and subsequent N-hydroxysuccimidyl ester formation as described in example 47 followed by coupling to terf-butyl 2-(1 ,3-bis[2-(2-aminoethoxy)ethoxy]propan- 2-yloxy)acetate as described in this example.
EXAMPLE 49
(S)-2,6-Bis(2-[2-(2-[2-(2,6-bis-[2-(2-[2-(2-azidoethoxy)ethoxy]ethoxy)acetylamino]hexanoyl- amino)ethoxy]ethoxy)ethoxy]acetylamino)hexanoic acid methyl ester
(S)-2,6-Bis(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}acetylamino)hexanoic acid (1.8g, 3.10mmol)) was dissolved in a mixture of dimethylformamide/dichloromethane 1 :3 (10ml), pH was adjusted to basic reaction using diisopropylethylamine, N-hydroxybenzotriazole and N- ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride were added and the reaction mixture was standing for 30 min. Then this reaction mixture was added to a solution of (S)- 2,6-bis-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}acetylamino)hexanoic acid methyl ester (0.37g, 0.70mmol in dichloromethane) and the reaction mixture was standing the night over. The reaction mixture was diluted with dichloromethane (150ml), washed with water (2x40ml), 50% saturated sodium hydrogen carbonate (2x30ml) and water (3x40ml). The organic phase was dried over magnesium sulphate, filtered and evaporated in vacuo giving an oil. Yield: 1 .6g (89%). LC-MS: m/z = 1656 (M+1 ), 828.8 (M/2)+1 and 553 (M/3)+1 .
EXAMPLE 50 (S)-2,6-Bis(2-[2-(2-[2-((S)-2,6-bis[2-(2-[2-(2-tert-butoxycarbonylaminoethoxy)ethoxy]ethoxy)- acetylamino]hexanoylamino)ethoxy]ethoxy)ethoxy]acetylamino)hexanoic acid methyl ester
To a solution of the above (S)-2,6-Bis(2-[2-(2-[2-((S)-2,6-bis[2-(2-[2-(2-azidoethoxy)ethoxy]- ethoxy)acetylamino]hexanoylamino)ethoxy]ethoxy)ethoxy]acetylamino)hexanoic acid methyl ester (1 .6g, 0.97mmol) in ethylacetate (60ml), was added di-terf-butyl dicarbonate (1.Og, 4.8mmol) and Pd/C (10%, 1 .1 g). Hydrogen was constantly bubbled through the reaction mixture for 2 hours. The reaction mixture was filtered and the organic solvent was removed in vacuo giving an oil which was used without further purification. Yield: 1 .8 g (98%). LC-MS: m/z = 1953 (M+1 ), 977 (M/2)+1 .
EXAMPLE 51
(S)-2,6-Bis(2-[2-(2-[2-((S)-2,6-bis[2-(2-[2(2aminoethoxy)ethoxy]ethoxy)acetylamino]- hexanoylamino)ethoxy]ethoxy)ethoxy]acetylamino)hexanoic acid methyl ester
The above (S)-2,6-bis(2-[2-(2-[2-((S)-2,6-bis[2-(2-[2-(2-tert-butoxycarbonylaminoethoxy)- ethoxy]ethoxy)acetylamino]hexanoylamino)ethoxy]ethoxy)ethoxy]acetylamino)hexanoic acid methyl ester was dissolved in dichloromethane (20ml) and trifluoroacetic acid (20ml) was added. The reaction mixture was standing for 2 hours. The organic solvent was evaporated in vacuo, giving an oil.
Yield: 1 .4g (100%). LC-MS: m/z = 1552 (M+1 ); 777.3 (M/2)+1 ; 518.5 (M/3)+1 and 389.1
(M/4)+1 .
EXAMPLE 52
(S)-2,6-Bis-(2-[2-(2-[2-((S)-2,6-bis-[2-(2-[2-(2-(2-(2-(2-methoxyethoxy)ethoxy)acetylamino)- ethoxy)ethoxy]ethoxy)acetylamino]hexanoylamino)ethoxy]ethoxy)ethoxy]acetylamino)- hexanoic acid methyl ester
To a solution of 2-(2-(methoxyethoxy)ethoxy)acetic acid (1 .3 g, 7.32 mmol) in a mixture of dichloromethane and dimethylformamide 3:1 (20 ml) was added N-hydroxysuccinimide (0.8g, 7.32 mmol) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.4g, 7.32mmol). The reaction mixture was standing for 1 hour, where after the mixture was added to a solution of (S)-2,6-bis(2-[2-(2-[2-((S)-2,6-bis[2-(2-[2(2aminoethoxy)ethoxy]ethoxy)acetyl- amino]hexanoylamino)ethoxy]ethoxy)ethoxy]acetylamino)hexanoic acid methyl ester (1 .42g, 0.92mmol) and diisopropylethylamine (2.4ml, 14.64 mmol) in dichloromethane (10ml). The reaction mixture was standing night over. The reaction mixture was diluted with dichloromethane (100ml) and extracted with water (3x25 ml). The combine water-phases were extracted with additional dichloromethane (2x75 ml). The combined organic phases were dried over magnesium sulphate, filtered and evaporated in vacuo. The residue was purified by flash chromatography using 500 ml ethyl acetate, followed by 500ml ethyl acetate / methanol 9:1 and finally methanol as the eluent. Fractions containing product were evaporated in vacuo giving an oil. Yield: 0.75 g (38%). LC-MS: m/z = 1097 (M/2)+1 ; 732 (M/3)+1 and 549 (M/4)+1 .
The (S)-2,6-Bis-(2-[2-(2-[2-((S)-2,6-bis-[2-(2-[2-(2-(2-(2-(2-methoxyethoxy)ethoxy)acetyl- amino)ethoxy)ethoxy]ethoxy)acetylamino]hexanoylamino)ethoxy]ethoxy)ethoxy]acetyl- amino)hexanoic acid methyl ester can be saponified to the free acid and attached to a free amino group of ITA for example, on either ε amino lysin residues or on the terminal α-amino group using an activated ester. The activated ester may be produced and coupled to the amino group of the ITA peptide by standard coupling methods known in the art such as diisopropylethylamine and N-hydroxybenzotriazole or other activating conditions.
Alternatively, the tertbutyl protected carboxylic acids intermediate above, may be deprotected and subsequemtly activated as OSu esters (for example, as described in example 40) for attachment to the ITA peptide.
EXAMPLE 53 [2-(2-{[Bis-(2-{2-[2-(2-{[bis-(2-benzyloxycarbonylaminoethyl)carbamoyl]methoxy}ethoxy)- ethoxy]acetylamino}ethyl)carbamoyl]methoxy}ethoxy)ethoxy]acetic acid: A solution of [2-(2-{[bis-(2-benzyloxycarbonylaminoethyl)carbamoyl]methoxy}ethoxy)ethoxy]- acetic acid 2,5-dioxopyrrolidin-1 -yl ester (2.53 mmol - from previous experiment, mass not determined) in tetrahydrofuran (50 ml) and a solution of [2-(2-{[bis-(2-aminoethyl)carbamoyl]- methoxy}ethoxy)ethoxy]acetic acid (1.26 mmol - from previous experiment, mass not determined) in aqueous sodium hydrogencarbonate (5 % w/w, 50 ml) were mixed. The mixture was stirred for at least 20 h. Solid sodium hydrogensulfate was added until pH was 2-3. The phases were separated. The aqueous phase was extracted with dichloromethane (3 x 50 ml). The combined organic phases were washed with aqueous sodium hydrogensulfate (5 % w/w, 50 ml). The aqueous phase was extracted with dichloromethane (2 x 50 ml). The combined organic phases were dried over magnesium sulfate, filtered, and concentrated in vacuo.
Yield: 989 mg LC-MS (ES-positive mode), m/z: 1423 [M+H]+
General procedure for synthesis of dendrimers with charged phosphate backbones.
Alternatively, the tertbutyl protected carboxylic acids intermediate above, may be deprotected and subsequemtly activated as OSu esters (for example as described in example 47) for attachment to insulin.
EXAMPLE 54
2-(2-Trityloxyethoxy)ethanol:
Triphenyl chloromethane (1 Og, 35.8 mmol) was dissolved in dry pyridine, diethyleneglycol (3.43 ml_, 35.8 mmol) was added and the mixture was stirred under nitrogen overnight. Solvent removed in vacuo. Dissolved in dichloromethane (100 ml_) and washed with water. Organic phase dried over Na2SO4 and solvent removed in vacuo. Crude product was purified by recrystallization from heptane/toluene (3:2) to yield the title compound.
1H NMR (CDCI3): δ = 7.46 (m, 6H), 7.28, (m, 9H), 3.75 (t, 2H), 3.68 (t, 2H), 3.62 (t, 2H), 3.28 (t, 2H). LC-MS: m/z = 371 (M+Na); R1 = 2.13 min.
2-[2-(2-Trityloxyethoxy)ethoxymethyl]oxirane:
2-(2-Trityloxyethoxy)ethanol (6.65 g, 19 mmol) was dissolved in dry THF (100 ml_). 60 % NaH (0.764 mg, 19 mmol) was added slowly. The suspension was stirred for 15 min. Epibromohydrin (1 .58 ml_, 19 mmol) was added and the mixture was stirred under nitrogen at room temperature overnight. The reaction was quenched with ice, separated between diethyl ether (300 ml_) and water (300 ml_). The water fase was extracted with dichloromethane. The organic phases were collected, dried (Na2SO4) and solvent removed in vacou.Xo afford an oil which was purified on silical gel column eluted with DCM/MeOH/Et3N (98:1 :1 ) to yield the title compound.
1H NMR (CDCI3): δ = 7.45 (m, 6H), 7.25, (m, 9H), 3.82 (dd, 1 H), 3.68 (m, 6H), 3.45 (dd, 1 H), 3.25 (t, 2H), 3.15 (m, 1 H ), 2.78 (t, 1 H), 2.59 (m, 1 H). LC-MS: m/z = 427 (M+Na); R1 = 2.44 min.
1 ,3-Bis[2-(2-trityloxyethoxy)ethoxy]propan-2-ol:
2-(2-Trityloxyethoxy)ethanol (1 .14 g, 3.28 mmol) was dissolved in dry DMF (5 ml_). 60 % NaH (144 mg, 3.61 mmol) was added slowly and the mixture was stirred under nitrogen at room temperature for 30 min. The mixture is heated to 4O0C. 2-[2-(2- Trityloxyethoxy)ethoxymethyl]oxirane (1 .4 g, 3.28 mmol) was dissolved in dry DMF (5 ml_) and added drop wise to the solution under nitrogen while stirring was maintained. After ended addition the mixture is stirred under nitrogen at 4O0C overnight. The heating is removed and after cooling to room temperature the reaction is quenched with ice and poured into saturated aqueous NaHCO3 (I OO ml_), extracted with diethyl ether (3 x75 ml_). The organic phases are collected, dried (Na2SO4), and solvent removed in vacuo to afford an oil which was purified on silical gel column eluted with EtOAc/Heptane/Et3N (49:50:1 ) to yield the title compound. 1H NMR (CDCI3): δ = 7.45 (m, 12H), 7.25, (m, 18H), 3.95 (m, 1 H), 3.78- 3.45 (m, 16H), 3.22 (t, 4H), LC-MS: m/z = 775 (M+Na); R1 = 2.94 min.
1 ,3-Bis[2-(2-trityloxyethoxy)ethoxy]propan-2-ol (2-cyanoethyl diisopropylphosphoramidite):
1 ,3-Bis[2-(2-trityloxyethoxy)ethoxy]propan-2-ol (0.95 g, 1 .26 mmol) was aveporated twice from dry pyridine and once from dry acetonitrile. Dissolved in dry THF (15 ml_), while stirring under nitrogen, diisopropylethylamin (1.2 ml_, 6.95 mmol) was added. The mixture was coold to O0C with an icebath 2-cyanoethyl diisopropylchlorophosphoramidite (0.39 ml_, 1.77 mmol) was added under nitrogen. The mixture was stirred for 10 minutes at O0C followed by 30 minutes at room temperature. Aqueous NaHCO3 (50 ml_) was added and the mixture extracted with DCMEt3N (98:2) (3x30 ml_). Organic phases were collected, dried (Na2SO4), and solvent removed in vacuo to afford an oil which was purified on silical gel column eluted with EtOAc/Heptane/Et3N (35:60:5) to yield the 703 mg of title compound. 31P-NMR (CDCI3): J149.6 ppm {2-[2-(2-Hydroxyethoxy)ethoxy]-1 -[2-(2-hydroxyethoxy)ethoxymethyl]ethoxy}acetic acid tert- butyl ester:
1 ,3-Bis[2-(2-trityloxyethoxy)ethoxy]propan-2-ol (0.3 g, 0.40 mmol) was evaporated once from dry pyridine and once from dry acetonitrile. Dissolved under nitrogen in dry DMF (2 ml_), 60% NaH (24 mg, 0.6 mmol) was added. The mixture was stirred at room temperature for 15 minutes, terf-butylbromoacetate (0.07 ml_, 0.48 mmol) was added and the mixture was stirred for additional 60 minutes. The reaction was quenched with ice. Separated between diethyl ether (2 x50 ml_) and water (2 x 50 ml_), the organic phases were collected, dried (Na2SO4), and solvent removed in vacuo to afford an oil which was eluted on silical gel column with EtOAc/Heptane/Et3N (49:50:1 ). Fraction containing main product was collected, solvent removed in vacuo and dissolved in 80 % aqueous acetic acid (5 ml_) and stirred at room temperature overnight. Solvent was solvent removed in vacuo. And crude material dissolved in diethyl ether (25 ml_), washed with water (2 x 5ml_). The water phases were collected and the water removed on rotorvap to yield 63 mg of the title compound. 1H NMR (CDCI3): δ = 4.19 (s, 2H), 3.78-3.55 (m, 21 H), 1 .49 (s, 9H).
2-(1 ,3-Bis[2-(2-hydroxyethoxy)ethoxy]propan-2-oxy) acetic acid tert-butyl ester (63 mg, 0.16 mmol) was evaporated twice from dry acetonitrile. 1 ,3-Bis[2-(2-trityloxyethoxy)ethoxy]propan- 2-oxy β-cyanoethyl N,N-diisopropylphosphoramidite (353 mg, 0.37 mmol) was evaporated twice from dry acetonotrile, dissolved on dry acetonitrile (2 ml_) and added. A solution of tetrazole in dry acetonitrile (0.25 M, 2.64 ml_) was added under nitrogen and the mixture was stirred at room temperature for 1 hour. 5.5 ml_ of an I2 -solution (0.1 M in THF/lutidine/H2O 7:2:1 ) was added and the mixture was stirred an additional 1 hour. The reaction mixture was diluted with ethyl acetate (20 ml_) and washed with 2% aqueous sodium sulfite until the iodine colour disappeared. The organic phase was dried (Na2SO4), and solvent removed in vacuo. The residue was dissolved in 80 % aqueous acetic acid (5 ml_) and stirred at room temperature overnight. Solvent was removed in vacuo and the crude material was added diethyl ether (25 ml_) and water (10 ml_). The water phase was collected and water removed in vacuo. Product was purified on reverse phase preparative HPLC C-18 colum, gradient 0- 40 % acetonitrile containing 0.1 % TFA to give the title terf-butyl-protected 2nd generation branched polymer product. LC-MS: m/z = 1 171 (M+Na); 1149 (M+), 1093 (loss of terf-butyl in the MS); Rt = 2.76 min.
Deprotection of β-cyanoethyl groups and removal of tert-butyl ester group, is subsequently done using conventional base and acid treatments as known to the person skilled in the art.
ATTACHMENT OF DENDRIMERS TO ITA:
EXAMPLE 55
N-epsilon26,3-[2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetylamino}ethoxy)ethoxy]- propan-2-yloxy)acetylamino]propanoyl[Arg34]GLP-1 -(7-37)-OH
R34-GLP-1 (7-37) (L.B. Knudsen et al., J. Med. Chem. 2000, 43, 1664-1669. )(1 10 mg; 33 umol) was suspended in water (30 ml). To the unclear suspension was added DIPEA (156 ul; 1 .6 mmol), and the mixture was stirred for 10 min, during which time the solution turned clear. The pH was measured to 10. A solution of N-hydroxysuccinimidyl 3-[2-(1 ,3-bis[2-(2-{2- [2-(2-methoxyethoxy)-ethoxy]acetylamino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]- propanoate (80.4 mg; 99 umol, example 32) in water (6.0 ml) was then added. The reaction mixture became yellow and turned slightly unclear. The mixture was stirred at room temperature for 45 min. Then a solution of glycine (5.3 ml, cone. = 10 mg/ml) was added. The mixture was stirred for 5 min at room temperature. The reaction mixture was then purified over 2 run by preparative HPLC with direct injection (20 ml and 16 ml respectively), using a C18 column (20x2 cm) with a linear gradient of 25-55% water - acetonitril and a flow of 10 ml/min, collecting 10 ml fractions. The individual fractions containing product were analyzed using LC-MS (electorspray) and HPLC (metode A). Samples containing pure compound were pooled to give a total volume of 80 ml of sample with a final concentration of 1 .05 mg/ml as determined from relative absorption measurement at λ = 276 nm. Samples were frozen and stored at -18 °C, until use. Yield: 84.6 mg (67%). MALDI-TOF-MS (α-cyano- 4-hydroxycinnamic acid): m/z = 4080. LC-MS (electrospray): (m/3)+1 = 1261 ; (m/4)+1 = 1021 ; Rt = 3.54 min. HPLC (Method A): Rt = 36.46 min.
EXAMPLE 56
N-epsilon26-3-(1 ,3-Bis{2-[2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetamino}-ethoxy)- ethoxy]propan-2-yloxy)acetylamino]ethoxy])ethoxy}propan-2-yloxy)acetylamino)-propanoyl-
[Arg34]GLP-1 -(7-37)-OH
R34-GLP-1 (7-37) (15.6 mg; 4.5 umol) was suspended in water (10 ml). To the unclear suspension was added DIPEA (86 ul; 0.88 mmol), and the mixture was stirred for 10 min, during which time the solution turned clear. The pH was measured to 10. A solution of N- hydroxysuccinimidyl 3-(1 ,3-bis{2-(2-[2-(1 ,3-bis[2-(2-{2-[2-(2-methoxyethoxy)- ethoxy]acetamino}ethoxy)ethoxy]propan-2-yloxy)acetylamino]ethoxy)ethoxy}propan-2-yloxy)- acetylamino)propanoate (93.0 mg; 53 umol, example 33) in water (2.0 ml) was then added. The reaction mixture remained clear. The mixture was stirred at room temperature for 40 min. Then a solution of glycine (3.0 ml, cone. = 10 mg/ml) was added. The mixture was stirred for 5 min at room temperature. The mixture was purified by preparative RP-HPLC as follows: The total sample volumne (15 ml) was injected on to a C18 column (20x2 cm), and eluted using a linear gradient from 25% -55% water-acetonitril with a flow of 10 ml/min. collecting 10 ml fractions. The individual fractions containing product were analyzed using LC-MS (electorspray) and HPLC (metode A). Samples containing pure compound were pooled to give a total volume of 24 ml of sample with a final concentration of 0.25 mg/ml as determined from relative absorption measurement at λ = 276 nm. Samples were frozen and stored at -18 °C, until use. Yield: 5.8 mg (24%). LC-MS (electrospray): (m/3)+1 = 1672; (m/4)+1 = 1254; (m/5)+1 = 1004; Rt = 3.25 min. HPLC (Method A): Rt = 35.64 min.
Example 57 Preparation of Λ/!37-((S)-2,6-Bis-(2-[2-(2-[2-((S)-2,6-bis-[2-(2-[2-(2-(2-(2-(2-methoxyethoxy)- ethoxy)acetylamino)ethoxy)ethoxy]ethoxy)acetylamino]hexanoylamino)ethoxy]ethoxy)- ethoxy]acetylamino)hexanoyl)[Aib8'22'35,Lys37]GLP-1 (7-37) amide
N >R-N X
1.a Synthesis of the protected peptidyl resin.
Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)- Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Aib-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-lle-Ala- Trp(Boc)-Leu-Val-Lys(Boc)-Aib-Arg(Pmc)-Lys(Dde)-Rink amide resin was prepared according to the Fmoc strategy on an Applied Biosystems 433A peptide synthesizer in 0.25 mmol scale using the manufacturer supplied FastMoc UV protocols which employ HBTU mediated couplings in NMP, and UV monitoring of the deprotection of the Fmoc protection group. To improve the coupling efficiency, Aib residues and residues following Aib, these residues were coupled using HATU instead of HBTU as the coupling reagent. The starting resin (438 mg) used for the synthesis was 4-(2',4'-Dimethoxyphenyl-Fmoc-aminomethyl)- phenoxy resin (Rink amide resin) (Merck Biosciences GmbH, Germany, cat. #: 01 -12-0013) with a substitution capacity of 0.57 mmol / g. The protected amino acid derivatives used were (2S)-6-[1 -(4,4-Dimethyl-2,6-dioxo-cyclohexylidene)-ethylamino]-2-(9 H-fluoren-9-ylmethoxy- carbonylamino)hexanoic acid (Fmoc-Lys(Dde)-OH), Fmoc-Arg(Pmc)-OH, Fmoc-Aib-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH,
Fmoc-lle-OH , Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(tBu)-OH,
Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH and Boc-
His(Boc)-OH
The yield was 1 .37 g of dry peptidyl resin.
1.b Characterisation of the peptidyl resin
The resin was characterized by cleaving off the crude peptide from 50 mg of this resin by treating it for 2 hours with a mixture of 14 μl TIS, 14 μl H2O and 0.5 ml TFA. The resin was removed by filtration and the crude peptide was isolated by precipitation and wash with Et2O. HPLC and LC-MS analysis was performed on the dry precipitate. Analytical results:
1.c Deprotection of Dde The protected peptidyl resin resulting from (1 .a) (1.35 g, 250 μmol) was washed in
NMP:DCM 1 :1 (15 ml) twice. A freshly prepared solution of hydrazine hydrate 2% in NMP (20ml) was added. The reaction mixture was shaken for 12 min at room temperature, and then filtered. The hydrazine treatment was repeated twice. After this the resin was washed extensively with NMP, DCM and NMP.
1.d attachment of branched polymer
The Dde deprotected resin is suspended in NMP (20ml). (S)-2,6-Bis-(2-[2-(2-[2-((S)-2,6-bis-
[2-(2-[2-(2-(2-(2-(2-methoxyethoxy)ethoxy)acetylamino)ethoxy)ethoxy]ethoxy)acetylamino]- hexanoylamino)ethoxy]ethoxy)ethoxy]acetylamino)hexanoic acid preactivated with TSTU as described in example 39 is added together with DIPEA and the suspension is shaken overnight. Then the resin is isolated by filtration and washed extensively with NMP, DCM, 2- propanol, methanol and Et2O and dried in vacuo. 1.e Cleavage of the product
The resin from 1.d is stirred for 3 h at room temperature with a mixture of 350 μl TIS, 350 μl H2O and 14 ml TFA. The resin is removed by filtration and washed with 3 ml TFA. The collected filtrates are concentrated in vacuo, to 5 ml and the crude product is precipitated by addition of 40 ml Et2O followed by centrifugation. The pellet is washed with 40 ml Et2O two times and then air dried.
1.f Purification of product.
The crude peptide is dissolved in H2O/AcOH (40:4) (40ml) and purified by semipreparative HPLC in 2 runs on a 25 mm x 250 mm column packed with 7μ C-18 silica. The column is eluted with a gradient of CH3CN from 40 to 62% against 0.1% TFA / H2O at 10 ml/min at a temperature of 4O 0C for 47min. The peptide containing fractions are collected, diluted with 3 volumes of H2O and lyophilized. The final product obtained is characterized by HPLC.
Compounds of this invention includes:
^NH
Example 58
N-epsilon26,3-[2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetylamino}ethoxy)ethoxy]- propan-2-yloxy)acetylamino]propanoyl [Aib8'22:35]GLP-1 (7-37) amide
2-H-NχlLEG T F I A W L V R-NV>>R-N/V'
Dde-Lys(Fmoc)-Glu(OtBu)-Phe-lle-Ala-Trp(Boc)-Leu-Val- Arg(Pmc)-Aib-Arg(Pmc)-Gly-Rink amide resin was prepared according to the Fmoc strategy on an Applied Biosystems 433A peptide synthesizer in 0.25 mmol scale using the manufacturer supplied FastMoc UV protocols which employ HBTU mediated couplings in NMP, and UV monitoring of the deprotection of the Fmoc protection group. The terminal Fmoc group was removed by treatment with 2% DBU in DMF (3x3 min), and acylated on the lysine side chain, first with Fmoc-AEEAc-OH and after Fmoc deprotection with 3-[2-(1 ,3-bis[2-(2-{2-[2-(2- methoxyethoxy)ethoxy]acetylamino}ethoxy)ethoxy]propan-2-yloxy)acetic acid. The terminal Dde-group was then removed with 10% hydrazin in NMP. The N-terminal of the peptide was then elongated with the Boc-His(Boc)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)- Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Aib-Gln(Trt)-Ala-Ala-sequence using the manufacturer supplied FastMoc UV protocols which employ HBTU mediated couplings in NMP, and UV monitoring of the deprotection of the Fmoc protection group. The peptide was cleaved form the resin using 5% triisopropylsilane and 5% water inTFA. The resin was filtered off, and washed with TFA. The combined filtrates were reduced to a minimal volume, and the peptide was precipitated by addition of cold diethyl ether, and isolated by centrifugation. The precipitate was washed trice with cold diethylether. The crude peptide was purified by RP18-HPLC. The column was eluted with a gradient of CH3CN from 36 to 60% against 0.1 % TFA / H2O at 10 ml/min. The peptide containing fractions were collected, diluted with H2O and lyophilized. LC-MS (electrospray): (m/3)+1 = 1409.8; (m/4)+1 = 1057.0; (m/5)+1 = 846.2; Rt = 3.28 min. HPLC (Method A): Rt = 29.08 min.
Example 59
N-alfa7-formyl,N-epsilon26,3-[2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetylamino}- ethoxy)ethoxy]propan-2-yloxy)acetylamino]propanoyl[Arg34]GLP-1 -(7-37)-OH
EGT FT SDVSSYL EGQ
N-epsilon26,3-[2-(1 ,3-Bis[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]acetylamino}ethoxy)ethoxy]- propan-2-yloxy)acetylamino]propanoyl[Arg34]GLP-1 -(7-37)-OH (Example 55) was dissolved in water (20 ml) and pH was adjusted to 9 with triethylamine. A 1 :1 mixture of acetic anhydride and formic acid was prepared, and 5 ul of this solution (3 eq.) were added while keeping pH at 9 with triethylamine addition. The reaction mixture was stirred for 1 h, then another 5 ul of the 1 :1 mixture of acetic anhydride / formic acid was added. The last step was repeated for one more time, then the mixture was stirred at room temperature for 3h. The product was then purified as described in example 58, to give 9 mg of title material. LC-MS (electrospray): (m/3)+1 = 1370.7; (m/4)+1 = 1028.7; Rt = 3.25 min. HPLC (Method A): Rt = 37.75 min. Method for measuring pulmonary bioavailability.
The present protocol describes the methods and materials used in the development of an anaesthetized rat model for pulmonal delivery of aerosols. The aerosols are generated by use of a nebulizer catheter with a well defined droplet/particle size (mean mass aerodynamic diameter, MMAD). The nebulizer catheter is an extruded multi-lumen catheter that provides fine-particle, baffle-free, aerosols. It incorporates multiple (typically 4-6) gas-lumens around one liquid lumen. Each lumen extends the length of the catheter which tapers to a fine (-0.5 mm diameter) nozzle with tiny orifices at the distal tip. The intimate contact between the gas and liquid at the tip produces a fine aerosol without baffling. The nebulizer catheter is guided through an endotracheal tube and is placed just above the main bronchial branch. The aerosol is deposited in pulses managed from a control unit.
EQUIPMENT
The equipment for pulmonary delivery is obtained from Trudell Medical International (London, Ontario, Canada).
Nebulizer catheters
Nebulizer catheters (Aeroprobe®) are supplied from the manufacturer in a number of different configurations and lengths. These different designs will accommodate a variety of different fluid and flow-rates, as well as provide aerosol particle-sizes that may be as low as 5 μm MMAD (mean mass aerodynamic diameter). In the present experiments a catheter with the following dimensions is used: Outer lumens gas flow of 1 .4 L/min, inner lumen liquid flow of 0.7 ml/min, and MMAD of about 7-8 μm (PN 104504-050) with a length of 50 cm (1 ).
Control unit
LABNeb® Catheter control system (CCS). The Aeroprobe catheter is connected to the control system according to (2).
Air with a pressure of 100 psi is used as supporting gas and maximal fluid pressure, usually 98 psi. A 100 μl syringe is used as reservoir. The LABNeb CCS used a pulse time of 80 msec and a gas delay of 20 msec. Thus, 2.3 ml air and 0.93 μl test solution are delivered in each pulse. ANIMALS
Sprague Dawley S rats weighing between 250 and 350 g. The animals are housed under standardised conditions with free access to food (Altromine 1324) and drinking water. On the experimental day the animals are used in their fed state.
SOLUTION FOR ANAESTHESIA
Hypnorm® (fentanyl 0.2 mg/ml, fluansol 10 mg/ml) is diluted with sterile water 1 +1 . Dormicum® (midazolam 5 mg/ml) is diluted with sterile water 1 + 1 . The two solutions are mixed 1 + 1 .
Surgical procedures and intratracheal administration Anaesthesia is induced by injecting subcutaneously the prepared Hyponorm/Dormicum solution 0.25 ml/100 g BW. An endotracheal tube (PE 240, Becton Dickinson) is inserted and guided to a position about Vz cm above the branch of the two main bronchii. Any heat loss is minimised by wrapping a plastic shield round the rat.
Before applying the test solution into the lungs, it is secured that the syringe and catheter system is free of air bubbles. Before applying the test solution endotracheal^, it is sprayed into a vial to test subsequently the amount of substance administered by the catheter. Then, the catheter is guide through the endotracheal tube leaving 1 -2 mm of the catheter tip free of the tube end and the test solution is aerosolised into the lungs of the anaesthetised rat.
Protraction of GLP-1 derivatives after i.v. or s.c. administration The protraction of a number GLP-1 derivatives of the invention was determined by monitoring the concentration thereof in plasma after sc administration to healthy pigs, using the methods described below. For comparison also the concentration in plasma of GLP-1 (7- 37) after sc. administration was followed. The protraction of other GLP-1 derivatives of the invention can be determined in the same way.
Pharmacokinetic testing of GLP-1 analogues in minipiqs
The test substances were dissolved in a vehicle suitable for subcutaneous or intravenous administration. The concentration was adjusted so the dosing volume was approximately 1 ml.
The study was performed in 12 male Gόttingen minipigs from Ellegaard Gόttingen Minipigs ApS. An acclimatisation period of approximately 10 days was allowed before the animals entered the study. At start of the acclimatisation period the minipigs were about 5 months old and in the weight range of 8-10 kg.
The study was conducted in a suitable animal room with a room temperature set at 21 -230C and the relative humidity to > 50%. The room was illuminated to give a cycle of 12 hours light and 12 hours darkness. Light was from 06.00 to 18.00 h.
The animals were housed in pens with straw as bedding, six together in each pen.
The animals had free access to domestic quality drinking water during the study, but were fasted from approximately 4 pm the day before dosing until approximately 12 hours after dosing. The animals were weighed on arrival and on the days of dosing.
The animals received a single intravenous or subcutaneous injection. The subcutaneous injection was given on the right side of the neck, approximately 5-7 cm from the ear and 7-9 cm from the middle of the neck. The injections were given with a stopper on the needle, allowing 0.5 cm of the needle to be introduced.
Each test substance was given to three animals. Each animal received a dose of 2 nmol/kg body weight.
Six animals were dosed per week while the remaining six were rested.
A full plasma concentration-time profile was obtained from each animal. Blood samples were collected according to the following schedule:
After intravenous administration:
Predose (O), 0.17 (10 minutes), 0.5, 1 , 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 hours after injection.
After subcutaneous administration:
Predose (0), 0.5, 1 , 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 hours after injection. At each sampling time, 2 ml of blood was drawn from each animal. The blood samples were taken from a jugular vein.
The blood samples were collected into test tubes containing a buffer for stabilisation in order to prevent enzymatic degradation of the GLP-1 analogues.
Plasma was immediately transferred to Micronic-tubes. Approximately 200 μl plasma was transferred to each Micronic-tube. The plasma was stored at -2O0C until assayed. The plasma samples were assayed for the content of GLP-1 analogues using a immunoassay.
The plasma concentration-time profiles were analysed by a non-compartmental pharmacokinetic analysis. The following pharmacokinetic parameters were calculated at each occasion: AUC, AUC/Dose, AUCo/oExtrap, Cmax, tmax, λz, ty2, CL, CL/f, Vz, Vz/f and MRT.
Selected compounds of the invention were tested in Danish Landrace pigs.
Pharmacokinetic testing of GLP-1 analogues in pigs Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, app 40 kg) were fasted from the beginning of the experiment. To each pig 0.5 nmol of test compound per kg body weight was administered in a 50 μM isotonic solution (5 mM phosphate, pH 7.4, 0.02% Tween®-20 (Merck), 45 mg/ml mannitol (pyrogen free, Novo Nordisk). Blood samples were drawn from a catheter in vena jugularis. 5 ml of the blood samples were poured into chilled glasses containing 175 μl of the following solution: 0.18 M EDTA, 15000 KIE/ml aprotinin (Novo
Nordisk) and 0.30 mM Valine-Pyrrolidide (Novo Nordisk), pH 7.4. Within 30 min, the samples were centrifuged for 10 min at 5-6000*g. Temperature was kept at 40C. The supernatant was pipetted into different glasses and kept at minus 2O0C until use.
The plasma concentrations of the peptides were determined in a sandwich ELISA or by RIA using different mono- or polyclonal antibodies. Choice of antibodies depends of the GLP-1 derivatives. The time at which the peak concentration in plasma is achieved varies within wide limits, depending on the particular GLP-1 derivative selected.
General assay protocol for sandwich ELISA in 96-wells microtiterplate Coating buffer (PBS): Phosphate buffered saline, pH7.2
Wash-buffer (PBS-wash): Phosphate buffered saline, 0.05 % v/v Tween 20, pH 7.2 Assay-buffer (BSA-buffer): Phosphate buffered saline, 10 g/l Bovin Serum Albumin
(Fluka 05477),0.05 % v/v Tween 20, pH 7.2 Streptavidin-buffer: Phosphate buffered saline, 0.5 M NaCI, 0.05 % v/v Tween 20, pH 7.2
Standard: Individual compounds in a plasma-matrix
A-TNP: Nonsens antibody
AMDEX: Streptavin-horseradish-peroxodase (Amersham RPN4401V)
TMB-substrate: 3,3',5,5'tetramethylbenzidine (<0.02 %), hydrogen peroxide The assay was carried out as follows (volumen/well):
1 .) coat with 100 μl catching antibody 5 μg/ml in PBS-buffer
→ incubate o/n , 4 0C
→ 5x PBS-wash → blocked with last wash in minimum 30 minutes
→then empty the plate
2.) 20 μl sample + 100 μl biotinylated detecting antibody 1 μg/ml in BSA-buffer with 10 μg/ml A- TNP
→ incubate 2 h, room temperature, on a shaker → 5x PBS-wash, then empty the plate 3.) 100 μl AMDEX 1 :8000 in Streptavidin-buffer
→ incubate 45-60 minute, room temperature, on a shaker
→ 5x PBS-wash, then empty the plate 4.) 100 μl TMB-substrate
→ incubate x minute at room temperature on a shaker
→ stop the reaction with 100 μl 4 M H3PO4
Read the absorbance at 450 nm with 620 nm as reference
The concentration in the samples was calculated from standard curves.
General assay protocol for RIA
DB-buffer: 80 mM phosphate buffer, 0.1 % Human serum albumin, 10 mM EDTA,
0.6 mM thiomersal, pH 7.5 FAM-buffer: 40 mM phosphate buffer, 0.1 % Human Serum Albumin,
0.6 mM thiomersal, pH 7.5 Charcoal: 40 mM phosphate buffer, 0.6 mM thiomersal, 16.7 % bovine plasma,
15 g/l activated carbon , pH 7.5
(mix the suspension minimum 1 h before use at 4 0C)
Standard: Individual compounds in a plasma-matrix
The assay was carried out in minisorp tubes 12x75 mm (volumen/tube) as follows:
Mix - incubate 30 min at 4 0C - centrifuge at 3000 rpm, 30 min - immediately after transfer supernatants to new tubes, close with stopper and count on gamma-counter for 1 minute. The concentration in the samples was calculated from individual standard curves.
GLP-1 RADIO RECEPTOR ASSAY (RRA):
The method is a radiometric-ligand binding assay using LEADsee/cer imaging particles. The assay is composed of membrane fragments containing the GLP-1 receptor, unlabeled GLP-1 analogues, human GLP-1 labelled with 125I and PS LEADsee/cer particles coated with wheat germ agglutinin (WGA). Cold and 125l-labelled GLP-1 will compete for the binding to the receptor. When the LEADsee/cer particles are added they will bind to carbohydrates residues on the membrane fragments via the WGA-residues. The proximity between the 125l-molecules and the LEADsee/cer particles causes light emission from the particles. The LEADseeker will image the emitted light and it will be reversibly correlated to the amount of GLP-1 analogue present in the sample.
REAGENTS & MATERIALS:
Pre treatment of animal plasma: Animal plasma was heat treated for 4 hrs at 560C and centrifuged at 10.000 rpm for 10 minutes. Afterwards, Val-Pyr (10 μM) and aprotenin (500
KIE/mL) was added and stored at <-18°C until use.
GLP-1 analogues calibrators: GLP-1 analogues were spiked into heat-treated plasma to produce dilution lines ranging from approximately 1 μM to 1 pM.
GLP-1 RRA assay buffer: 25 mM Na-HEPES (pH=7.5), 2.5 mM CaCI2, 1 mM MgCI2, 50 mM NaCI, 0.1 % ovalbumin, 0.003% tween 20, 0.005% bacitracin, 0.05% NaN3.
GLP-1 receptor suspension: GLP-1 receptor membrane fragments were purified from baby hamster kidney (BHK) cells expressing the human pancreatic GLP-1 receptor. Stored <-80°C until use.
WGA-coupled polystyrene LEADseeker imaging beads (RPNQ0260, Amersham): The beads were reconstituted with GLP-1 RRA assay buffer to a concentration of 13.3 mg/mL The GLP-1 receptor membrane suspension was then added and incubated cold (2-80C) at end- over-end for at least 1 hr prior to use.
[125I]-GLP-I (7-36)amide (Novo Nordisk A/S). Stored <-18°C until use. Ethanol 99.9% vol (De Dansk SpritfabrikkerA/S): Stored <-180C until use. MultiScreen® Solvinert 0.45μm hydrophobic PTFE plates (MSRPN0450, Millipore Corp.) Poly propylene plates (cat. no. 650201, Greiner Bio-One) White polystyrene 384-well plates (cat. no. 781075, Greiner Bio-One)
APPARATUS: Horizontal plate mixer Centrifuge with a standard swinging-bucket microtitre plate rotor assembly UltraVap - Drydown Sample Concentrator (Porvair) LEADseeker™ Multimodality Imaging System (Amersham)
ASSAY PROCEDURE:
Sample preparation: Mount the MultiScreen® Solvinert filter plate on a chemical-comparable receiver plate (i.e. poly propylene plates) to collect the filtrate.
Add 150 μl_ ice-cold ethanol 99.9% into the empty wells of the MultiScreen® Solvinert filter plate followed by 50 μl_ calibrator or plasma sample. Place the storage lid on the filter plate.
Incubate 15 minutes at 18-220C on a horizontal plate mixer. Place the assembled filter and receiver plate, with the lid, into a standard swinging-bucket microtitre plate rotor assembly. The filtrate is then collected in the empty wells of the receiver plate at 1500 rpm for 2 minutes.
Dry down the filtrate by using the UltraVap with heated (4O0C) N> for duration of 15 miuntes.
Reconstitute the dry material by adding 100 μl_ GLP-1 RRA assay buffer into each well. Incubate for 5 minutes on a horizontal mixer.
GLP-1 radio receptor assay:
Use the following pipetting scheme and white polystyrene 384-well plates:
• 35 μl_ GLP-1 RRA assay buffer
• 5 μL reconstituted filtrate. • 10 μL [125I]-GLP-I (7-36)amide. The stock solution was diluted in GLP-1 RRA assay buffer to 20.000 cpm/well prior to use. • 15 μL GLP-1 receptor membrane fragments (-0.5 μg/well) pre-coated to WGA- polystyrene LEADseeker imaging beads (0.2 mg/well)
Seal the plates and incubate over night at 18-220C
The light emission from each wells are detected by using the LEADseeker™ Multimodality Imaging System for duration of 10 minutes.
Stimulation of cAMP formation in a cell line expressing the cloned human GLP-1 receptor.
Purified plasma membranes from a stable transfected cell line, BHK467-12A (tk-ts13), expressing the human GLP-1 receptor was stimulated with GLP-1 and peptide analogues, and the potency of cAMP production was measured using the AlphaScreen™ cAMP Assay Kit from Perkin Elmer Life Sciences.
A stable transfected cell line has been prepared at NN and a high expressing clone was selected for screening. The cells were grown at 5% CO2 in DMEM, 5% FCS, 1 % Pen/Strep and 0.5 mg/ml G418. Cells at approximate 80% confluence were washed 2X with PBS and harvested with
Versene, centrifuged 5 min at 1000 rpm and the supernatant removed. The additional steps were all made on ice. The cell pellet was homogenized by the Ultrathurax for 20-30 sec. in 10 ml of Buffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged 15 min at 20.000 rpm and the pellet resuspended in 10 ml of Buffer 2 (20 mM Na-HEPES, 0.1 mM EDTA, pH=7.4). The suspension was homogenized for 20-30 sec and centrifuged 15 min at 20.000 rpm. Suspension in Buffer 2, homogenization and centrifugation was repeated once and the membranes were resuspended in Buffer 2 and ready for further analysis or stored at -8O0C. The functional receptor assay was carried out by measurering the peptide induced cAMP production by The AlphaScreen Technology. The basic principle of The AlphaScreen Technology is a competition between endogenous cAMP and exogenously added biotin- cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to acceptor beads. Formed cAMP was counted and measured at a AlphaFusion Microplate Analyzer. The EC50 values was calculated using the Graph-Pad Prisme software.

Claims

1 . A conjugate comprising a structurally well-defined branched polymer covalently attached to an insulinotropic agent.
2. A conjugate according to claim 1 represented by the general formula I
ITA-l_4-(L3)m-Y1 (Y2(Y3(Y4(Y5(Y6)r)q)p)s)n (I)
wherein ITA represents an insulinotropic agent from which a hydrogen has been removed from an alpha-amino group present in the insolinotropic agent, or from an epsilon amino group present on a lysine at any position in the insolinotropic agent,
for the 1 st generation of bifurcated compounds,
Y1 is Yb; Y2 is Z; r, q, p, and s are all zero; and n is 2; for the 2nd generation of bifurcated compounds,
Y1 and Y2 are Yb; Y3 is Z; r, q, and p are all zero; s is 4; and n is 2; for the 3rd generation of bifurcated compounds,
Y1 , Y2, and Y3 are all Yb; Y4 is Z; r and q are zero; p is 8; s is 4; and n is 2; for the 4th generation of bifurcated compounds, Y1 , Y2, Y3, and Y4 are all Yb; Y5 is Z; r is zero; q is 16; p is 8; s is 4; and n is 2; and for the 5th generation of bifurcated compounds,
Y1 , Y2, Y3, Y4, and Y5 are all Yb; Y6 is Z; r is 32; q is 16, p is 8; s is 4; and n is 2; wherein
LrB-
Yb is -A-LrX3
L2-B-
for the 1 st generation of trifurcated compounds,
Y1 is Yt; Y2 is Z; r, q, p, and s are all zero; and n is 3; for the 2nd generation of trifurcated compounds,
Y1 and Y2 are Yt; Y3 is Z; r, q, and p are all zero; s is 9; and n is 3; for the 3rd generation of trifurcated compounds,
Y1 , Y2, and Y3 are all Yt; Y4 is Z; r and q are zero; p is 27; s is 9; and n is 3; and for the 4th generation of trifurcated compounds,
Y1 , Y2, Y3, and Y4 are all Yt; Y5 is Z; r is zero; q is 81 ; p is 27; s is 9; and n is 3; wherein
L-B-
Yt is -A-L1-X^ L-B- L-B-
wherein A is -CO-, -C(O)O-, -P(=O)(OR)- or -P(=S)(OR)-, wherein R is hydrogen, alkyl or optionally substituted aryl; and B is -NH- or -O-; with the proviso that when B is -NH-, then A is -CO- or -C(O)O-, and when B is -0-, then A is -P(=O)(OR)- or -P(=S)(OR)-; and wherein the group B of one monomer layer (generation) (exemplified by Y1 , Y2, and Y3) is connected to the group A of the adjacent, following layer where Y has the following number as suffix (exemplified by Y2; Y3, and Y4, respectively) or is connected to Z;
X3 is a nitrogen atom, alkantriyl, arenetriyl, alkantrioxy, an aminocarbonyl moiety of the formula -CO-Nk, an acetamido moiety of the formula -CH2CO-Nk or a moiety of the formula: -CO-NH-Q-NH-CO-
wherein Q is alkantriyl;
X4 is alkantetrayl or arenetetrayl;
L1 is a valence bond, oxy, alkylene, alkyleneoxyalkyl, polyalkoxydiyl, (polyalkoxy)alkyl- carbonyl, oxyalkyl or (polyalkoxy)alkyl;
L2 is a valence bond, oxy, alkylene, alkyleneoxyalkyl, polyalkoxydiyl, (polyalkoxy)alkyl- carbonyl, oxyalkyl or (polyalkoxy);
L3 represents a valence bond, alkylene, oxy, polyalkoxydiyl, oxyalkyl, alkylamino, carbonyl- alkylamino, alkylaminocarbonylalkylamino, carbonylalkylcarbonylamino(polyalkoxy)alkyl- amino, carbonylalkoxyalkylcarbonylamino(polyalkoxy)alkylamino, alkylcarbonylamino(poly- alkoxy)alkylamino, carbonyl(polyalkoxy)alkylamino or carbonylalkoxyalkylamino;
m is zero, 1 , 2 or 3; L4 is selected among a valence bond and a moiety of the formula -CO-L5-CH=N-O-, wherein L5 is a valence bond, alkylene or arylene,
Z is hydrogen, alkyl, alkoxy, hydroxyalkyl, polyalkoxy, oxyalkyl, acyl, polyalkoxyalkyl or polyalkoxyalkylcarbonyl.
3. A conjugate according to claim 2, wherein Y1 is Yb (i.e. bifurcated compounds).
4. A conjugate according to claim 2 or 3, wherein X3 is a branched, trivalent organic radical of one of the following six formulae:
5. A conjugate according to claims 2-4, wherein Y1 is Yt (i.e. trifurcated compounds).
6. A conjugate according to the any of the claims 2-5, wherein X4 is benzen-1 ,3,4,5-tetrayl.
7. A conjugate according to any of the claims 2-6, wherein L1 a valence bond, oxy (-0-), oxymethyl (-OCH2-) or a moiety of the general formula -CH2(OCH2CH2)n-OCH2C(O)-, where n" is an integer from O to 10.
8. A conjugate according to any of the claims 2-7, wherein L2 is a moiety of the formula (-
CH2CH2O-)2, also having the formula: ° , -CH2CH2OCH2CH2O-, -
CH2CH2OCH2CH2OCH2CH2OCH2-, -CH2CH2OCH2CH2- or -CH2CH2-.
9. A conjugate according to any of the claims 2-8, wherein L3 is a valence bond or a divalent linker radical such as those illustrated by the following six formulae: wherein each end of the divalent radicals can be attached to the ITA group.
10. A conjugate according to any of the claims 2-9, wherein L4 and the adjacent L3 is a divalent linker radical such as those illustrated by the following eight formulae:
wherein each ends of the divalent radicals can be connected to the ITA group
1 1 . A conjugate according to any of the claims 2-10, wherein L4 is an oxyiminoalkylcarbonyl moiety in both isomeric (syn and anti) forms, an oxyiminoalkylarylcarbonyl moiety in both isomeric (syn and anti) forms, or a valence bond.
12. A conjugate according to any of the claims 2-11 , wherein Z is a capping agent that can react with a terminal amino group or hydroxy group, preferably a group having one of the following three formulae:
JL H N^NH2
NH
where Me is methyl.
13. A conjugate according to any of the claims 2-12, wherein A is one of the three moieties: - CO-, -P(O)O- and -P(S)O-.
14. A conjugate according to any of the claims 2-13, wherein B is oxy or the moiety -NH-.
15. A conjugate according to any of the preceding claims, wherein the insulinotropic agent is GLP-1 peptide or an exendin-4 peptide.
16. A conjugate according to any of the preceding claims, wherein said insulinotropic agent (ITA) is a DPPIV protected peptide.
17. A conjugate according to any of the preceding claims, wherein said insulinotropic agent (ITA) has an EC50 of less than 1 nM as determined by the functional receptor assay disclosed herein.
18. A conjugate according to any of the preceding claims, wherein said insulinotropic agent (ITA) has an EC50 of less than 300 pM, less than 200 pM or less than 100 pM as determined by the functional receptor assay disclosed herein.
19. A conjugate according to any one of the preceding claims, wherein said insulinotropic agent (ITA) is selected from a peptide comprising the amino acid sequence of the formula (II): Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaai6-Ser-Xaai8-Xaai9-Xaa2o-Glu-Xaa22-Xaa23-Ala-
Xaa25-Xaa26-Xaa27-Phe-lle-Xaa30-Trp-Leu-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-
Xaa4i"Xaa42-Xaa43-Xaa44-Xaa45"Xaa46
Formula (II) (SEQ ID No: 3)
wherein Xaa7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy- histidine, homohistidine, Nα-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
Xaa8 is Ala, D-AIa, GIy, VaI, Leu, He, Lys, Aib, (1 -aminocyclopropyl) carboxylic acid, (1 - aminocyclobutyl) carboxylic acid, (1 -aminocyclopentyl) carboxylic acid, (1 -aminocyclohexyl) carboxylic acid, (1 -aminocycloheptyl) carboxylic acid, or (1 -aminocyclooctyl) carboxylic acid;
Xaaie is VaI or Leu;
Xaais is Ser, Lys or Arg;
Xaaig is Tyr or GIn; Xaa20 is Leu or Met;
Xaa23 is GIn, GIu, Lys or Arg;
Xaa26 is Lys, GIu or Arg;
Xaa30 is Ala, GIu or Arg;
Xaa33 is VaI or Lys;
Xaa34 is Lys, GIu, Asn or Arg;
Xaa35 is GIy or Aib; Xaa36 is Arg, GIy or Lys;
Xaa37 is GIy, Ala, GIu, Pro, Lys, amide or is absent;
Xaa38 is Lys, Ser, amide or is absent.
Xaa39 is Ser, Lys, amide or is absent;
Xaa40 is GIy, amide or is absent; Xaa4i is Ala, amide or is absent;
Xaa42 is Pro, amide or is absent;
Xaa43 is Pro, amide or is absent;
Xaa44 is Pro, amide or is absent;
Xaa45 is Ser, amide or is absent; Xaa46 is amide or is absent ; provided that if Xaa38, Xaa39, Xaa40, Xaa4i, Xaa42, Xaa43, Xaa44, Xaa45 or Xaa46 is absent then each amino acid residue downstream is also absent.
20. A conjugate according to claim 19, wherein said insulinotropic agent (ITA) is a peptide comprising the amino acid sequence of formula (III):
Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaai8-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-
Xaa26-Glu-Phe-lle-Xaa3o-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38
Formula (III) (SEQ ID No: 4) wherein Xaa7 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy- histidine, homohistidine, Nα-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3- pyridylalanine, 2-pyridylalanine or 4-pyridylalanine;
Xaa8 is Ala, D-AIa, GIy, VaI, Leu, He, Lys, Aib, (1 -aminocyclopropyl) carboxylic acid, (1 - aminocyclobutyl) carboxylic acid, (1 -aminocyclopentyl) carboxylic acid, (1 -aminocyclohexyl) carboxylic acid, (1 -aminocycloheptyl) carboxylic acid, or (1 -aminocyclooctyl) carboxylic acid;
Xaais is Ser, Lys or Arg;
Xaa22 is GIy, GIu or Aib;
Xaa23 is GIn, GIu, Lys or Arg;
Xaa26 is Lys, GIu or Arg; Xaa30 is Ala, GIu or Arg;
Xaa34 is Lys, GIu or Arg;
Xaa35 is GIy or Aib;
Xaa36 is Arg or Lys;
Xaa37 is GIy, Ala, GIu or Lys; Xaa38 is Lys, amide or is absent.
21 . A conjugate according to any one of the preceeding claims, wherein said insulinotropic agent (ITA) is selected from GLP-1 (7-35), GLP-1 (7-36), GLP-1 (7-36)-amide, GLP-1 (7-37), GLP-1 (7-38), GLP-1 (7-39), GLP-1 (7-40), GLP-1 (7-41 ) or an analogue thereof.
22. A conjugate according to any one of the preceding claims, wherein said insulinotropic agent (ITA) comprises no more than fifteen amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ), or no more than ten amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ).
23. A conjugate according to the preceeding claim, wherein said insulinotropic agent (ITA) comprises no more than six amino acid residues which have been exchanged, added or deleted as compared to GLP-1 (7-37) (SEQ ID No. 1 ).
24. A conjugate according to any one of the preceding claims, wherein said insulinotropic (ITA) agent comprises no more than 4 amino acid residues which are not encoded by the genetic code.
25. A conjugate according to any one of the preceding claims, wherein said insulinotropic agent (ITA) comprises an Aib residue as the second amino acid residue from the N-terminal.
26. A conjugate according to any one of the preceding claims, wherein the N-terminal amino acid residue (position 7 in formulae Il and III) of said insulinotropic agent (ITA) is selected from the group consisting of D-histidine, desamino-histidine, 2-amino-histidine, β-hydroxy- histidine, homohistidine, Nα-acetyl-histidine , α-fluoromethyl-histidine, α-methyl-histidine, 3- pyridylalanine, 2-pyridylalanine and 4-pyridylalanine.
27. A conjugate according to any one of the preceding claims, wherein said insulinotropic agent (ITA) is selected from the group consisting of [Arg34]GLP-1 (7-37), [Arg26'34]GLP-1 (7-
37)Lys, [Lys36Arg26'34]GLP-1 (7-36), [Aib8'22'35]GLP-1 (7-37), [Aib8'35]GLP-1 (7-37), [Aib8'22]GLP- 1 (7-37), [Aib82235 Arg26'34]GLP-1 (7-37)Lys, [Aib8'35 Arg26'34]GLP-1 (7-37)Lys, [Aib8'22 Arg26'34]GLP-1 (7-37)Lys, [Aib82235 Arg26'34]GLP-1 (7-37)Lys, [Aib8'35 Arg26'34]GLP-1 (7-37)Lys, [Aib8'22'35 Arg26]GLP-1 (7-37)Lys, [Aib8'35 Arg26]GLP-1 (7-37)Lys, [Aib8'22 Arg26]GLP-1 (7-37)Lys, [Aib8'22'35 Arg34]GLP-1 (7-37)Lys, [Aib8'35Arg34]GLP-1 (7-37)Lys, [Aib8'22Arg34]GLP-1 (7-37)Lys,
[Aib8'22'35Ala37]GLP-1 (7-37)Lys, [Aib8'35Ala37]GLP-1 (7-37)Lys, [Aib8'22Ala37]GLP-1 (7-37)Lys, [Aib8'22'35 Lys37]GLP-1 (7-37), [Aib8'35l_ys37]GLP-1 (7-37), [Aib8'22Lys37]GLP-1 (7-37) or derivatives thereof which has been amidated on the C-terminal, exendin-4(1 -39), ZP-10, i.e. [Ser38Lys39]Exendin-4(1 -39)LysLysLysLysLys-amide (SEQ ID No. 5).
28. A conjugate according to any one of the preceding claims, wherein said insulinotropic agent (ITA) is attached to a branched polymer according to any one of the preceding claims via a carboxyl group, an amino group, a keto group, a hydroxyl group, a thiol group or a hydrazide group.
29. A conjugate according to any of the preceding claims, wherein said compound has an EC50 of less than 1000 pM, less than 500 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 50 pM or less than 10 pM as determined by the functional receptor assay disclosed herein.
30. A pharmaceutical composition comprising a compound according to any one of the proceding claims, and a pharmaceutically acceptable excipient.
31. Use of a compound according to any one of the claims 1 -29 for the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers.
32. Use of a compound according to claim 31 for the preparation of a medicament for delaying or preventing disease progression in type 2 diabetes.
33. A method for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive disorders, atheroschlerosis, myocardial infarction, coronary heart disease and other cardiovascular disorders, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcers by administering an effective amount of a compound accordiing to any fo the claims 1 -29.
EP06708301A 2005-02-16 2006-02-16 Insulinotropic agents conjugated with structurally well defined branched polymers Withdrawn EP1853321A2 (en)

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