WO2006048451A1 - Peptides for use in the treatment of obesity - Google Patents

Peptides for use in the treatment of obesity Download PDF

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Publication number
WO2006048451A1
WO2006048451A1 PCT/EP2005/055761 EP2005055761W WO2006048451A1 WO 2006048451 A1 WO2006048451 A1 WO 2006048451A1 EP 2005055761 W EP2005055761 W EP 2005055761W WO 2006048451 A1 WO2006048451 A1 WO 2006048451A1
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Prior art keywords
ethoxy
arg
phe
lys
trp
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PCT/EP2005/055761
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French (fr)
Inventor
Ulrich Sensfuss
Leif Christensen
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Novo Nordisk A/S
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Priority to EP05803336A priority Critical patent/EP1809667A1/en
Priority to JP2007539581A priority patent/JP2008519008A/en
Priority to US11/666,800 priority patent/US20080306008A1/en
Publication of WO2006048451A1 publication Critical patent/WO2006048451A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • 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
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/68—Melanocyte-stimulating hormone [MSH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel peptide compounds which are ligands for one or more melanocortin receptors and which may exert prolonged activity, to the use of the compounds in therapy, to methods of treatment comprising administration of the compounds to patients, and to the use of the compounds in the manufacture of medicaments.
  • Obesity is a well known risk factor for the development of many very common diseases such as atherosclerosis, hypertension, type 2 diabetes (non-insulin dependent diabetes mellitus (NIDDM)), dyslipidaemia, coronary heart disease, and osteoarthritis and various malignan ⁇ cies. It also causes considerable problems through reduced motility and decreased quality of life. The incidence of obesity and thereby also these diseases is increasing throughout the entire industrialised world. Only a few pharmacological treatments are available to date, namely Sibutramine (Abbot; acting via serotonergic and noradrenaline mechanisms) and OrI- istat (Roche Pharm; reducing fat uptake from the gut,). However, due to the important effect of obesity as a risk factor in serious (and even fatal) and common diseases, there is still a need for pharmaceutical compounds useful in the treatment of obesity.
  • NIDDM non-insulin dependent diabetes mellitus
  • obesity implies an excess of adipose tissue.
  • obesity is best viewed as any degree of excess adiposity that imparts a health risk.
  • the distinction between normal and obese individuals can only be approximated, but the health risk imparted by obesity is probably a continuum with increasing adiposity.
  • Proopiomelanocortin is the precursor for ⁇ -endorphin and melanocortin peptides, including melanocyte stimulating hormone ( ⁇ -MSH) and adrenocorticotropin (ACTH). POMC is expressed in several peripheral and central tissues including melanocytes, the pituitary, and neurons of the hypothalamus. The POMC precursor is processed differently in different tissues, resulting in the expression of different melanocortin peptides depending on the site of expression.
  • ⁇ -MSH melanocyte stimulating hormone
  • ACTH adrenocorticotropin
  • a family of five melanocortin receptor subtypes has been identified (melanocortin receptor 1 - 5, also called MC1 , MC2, MC3, MC4 and MC5).
  • the MC1 , MC2 and MC5 are mainly ex ⁇ pressed in peripheral tissues, whereas MC3 and MC4 are mainly centrally expressed; MC3 are, however, also expressed in several peripheral tissues.
  • MC3 receptors In addition to being involved in energy homeostasis, MC3 receptors have also been suggested to be involved in several in- flammatory diseases.
  • An MC3 agonist could have a positive effect on such diseases, e.g. gouty arthritis.
  • MC5 are mainly peripherally expressed, and have been suggested to be in ⁇ volved in exocrine secretion and in inflammation.
  • MC4 have been shown to be involved in the regulation of body weight and feeding behaviour, as MC4 knock-out mice develop obesity [Huzar et al., CeN 88, 131 -141 (1997)].
  • the MC4 receptor has been shown to be involved in the regulation of energy expenditure [Fekete et al., Journal of Neuroscience 20, 1550-1558 (2000)].
  • a MC4 agonist could serve as an anorectic drug or energy expenditure regu ⁇ lating drug and be useful in the treatment of obesity or obesity-related diseases, as well as in the treatment of other diseases, disorders or conditions which may be ameliorated by activa ⁇ tion of MC4 .
  • MC4 antagonists may be useful for treatment of cachexia or anorexia, and for treatment of waisting in frail elderly patients. Furthermore, MC4 antagonists may be used for treatment of chronic pain, neuropathy and neurogenic inflammation.
  • peptides as melanocortin receptor modulators is disclosed in a number of patent documents, e.g. WO 03/006620, US 5731 ,408 and WO 98/27113.
  • Hadley [Pigment Cell Res., 4, 180-185, (1991 )] reports a prolonged effect of specific melanotropic peptides conju ⁇ gated to fatty acids, the prolongation being effected by a transformation of the modulators from being reversibly acting to being irreversibly acting caused by the conjugated fatty acids.
  • the invention relates to compounds of formula Ia, Ib or Ic:
  • Z 1 and Z 2 independently represent GIy, Ser, Thr, GIn, Asn, His, homoArg, Arg, Lys, Dab, Dap or Orn;
  • Z 3 represents Ala, VaI, Leu, He, Met or NIe
  • X 1 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap;
  • X 2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, He, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met, Met(O), Met(O 2 ), GIn, Gln(alkyl), Gln(aryl), Asn, Asn(alkyl), Asn(aryl), Ser, Thr, Cys, Pro, Hyp, Tic, 2-PyAIa, 3-PyAIa, 4-PyAIa, (2-thienyl)alanine, 3-(thienyl)alanine, (4-thiazolyl)Ala, (2-furyl)alanine, (3-furyl)alanine or Phe, wherein the phenyl moiety of said Phe is optionally substituted with a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano;
  • X 3 represents D-Phe, wherein the phenyl moiety in D-Phe may optionally be substituted with one or more substituents selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoro ⁇ methyl and cyano;
  • X 4 represents Trp, 2-NaI, a (3-benzo[b]thienyl)alanine residue or a (S)-2,3,4,9-tetrahydro-1 H- ⁇ -carboline-3-carboxylic acid residue;
  • X 5 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap; wherein X 1 and X 5 are joined, rendering the compound of formula Ia, Ib or Ic cyclic, either via a disulfide bridge deriving from X 1 and X 5 both independently being Cys, homoCys or Pen, or via an amide bond formed between a carboxylic acid in the side-chain of X 1
  • the invention further relates to the use of compounds of the invention in therapy, to pharma ⁇ ceutical compositions comprising compounds of the invention, to methods of treatment com ⁇ prising administration of compounds of the invention to patients in need thereof, and to the use of compounds of the invention in the manufacture of medicaments.
  • C x - y alkyl e.g. Ci 4 - 20 alkyl
  • Ci 4 - 20 alkanoyl, Ci 4 - 20 alkenoyl or Ci 4 - 20 alkynoyl groups as they occur as substituents R 1 in compounds of the present invention embrace straight-chain, branched and/or cyclic alkanoyl, alkenoyl or alkynoyl groups having 14, 15, 16, 17, 18, 19 or 20 carbon atoms (i.e. Ci 4 , C 15 , C 16 , C 17 , C 18 , C 19 or C 20 ).
  • alkyl refers to a straight-chain, branched and/or cyclic, saturated monovalent hydrocarbon radical.
  • Examples hereof include methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • alkenyl refers to a straight-chain, branched and/or cyclic, mono ⁇ valent hydrocarbon radical comprising at least one carbon-carbon double bond. Examples hereof include ethenyl, prop-1 -en-1 -yl, prop-2-en-1 -yl and prop-2-en-2-yl.
  • alkynyl refers to a straight-chain, branched and/or cyclic, monova- lent hydrocarbon radical comprising at least one carbon-carbon triple bond, and it may op ⁇ tionally also comprise one or more carbon-carbon double bonds. Examples hereof include ethynyl, prop-1 -yn-1yl and prop-2-yn-1 -yl.
  • alkanoyl as used herein is intended to indicate a radical of the formula -C(O)-R', wherein R' is alkyl as indicated above.
  • alkenoyl as used herein is intended to indicate a radical of the formula -C(O)-R", wherein R" is alkenyl as indicated above.
  • alkynoyl as used herein is intended to indicate a radical of the formula -C(O)-R'", wherein R'" is alkynyl as indicated above.
  • alkoxy as used herein is intended to indicate a radical of the formula -OR', wherein R' is alkyl as indicated above. Examples hereof include methoxy and ethoxy.
  • aryl is intended to indicate a carbocyclic aromatic ring radical or a fused aromatic ring system radical wherein at least one of the rings is aromatic.
  • Typical aryl groups include phenyl, biphenylyl, naphthyl, and the like.
  • halogen is intended to indicate members of the 7 th main group of the periodic ta ⁇ ble of the elements, which includes fluorine, chlorine, bromine and iodine (corresponding to fluoro, chloro, bromo and iodo substituents, respectively).
  • agonist is intended to indicate a substance (ligand) that ac ⁇ tivates the receptor type in question.
  • the term "antagonist” is intended to indicate a substance (ligand) that blocks, neutralizes or counteracts the effect of an agonist.
  • receptor ligands may be classified as follows:
  • Receptor agonists which activate the receptor; partial agonists also activate the receptor, but with lower efficacy than full agonists.
  • a partial agonist will behave as a receptor partial an ⁇ tagonist, partially inhibiting the effect of a full agonist.
  • Receptor neutral antagonists which block the action of an agonist, but do not affect the re- ceptor-constitutive activity.
  • Receptor inverse agonists which block the action of an agonist and at the same time attenu ⁇ ate the receptor-constitutive activity.
  • a full inverse agonist will attenuate the receptor- constitutive activity completely; a partial inverse agonist will attenuate the receptor- constitutive activity to a lesser extent.
  • antagonist includes neutral antagonists and partial antagonists, as well as inverse agonists.
  • agonist includes full agonists as well as partial agonists.
  • salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts.
  • Acid addition salts include salts of inorganic acids as well as organic acids. Represen ⁇ tative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric and nitric acids, and the like.
  • suitable or ⁇ ganic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cin- namic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene- salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like.
  • inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. (1977) 66, 2, which is incorporated herein by reference.
  • relevant metal salts include lithium, sodium, potassium and magnesium salts, and the like.
  • alkylated ammonium salts include methylammo- nium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium and tetramethylammonium salts, and the like.
  • the term "therapeutically effective amount" of a compound refers to an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and/or its complications. An amount adequate to accomplish this is defined as a “therapeutically effective amount”. Effective amounts for each purpose will depend on the severity of the disease or injury, as well as on the weight and general state of the subject. It will be understood that determination of an appropriate dosage may be achieved using rou- tine experimentation, by constructing a matrix of values and testing different points in the ma ⁇ trix, all of which is within the level of ordinary skill of a trained physician or veterinarian.
  • treatment refers to the man ⁇ agement and care of a patient for the purpose of combating a condition, such as a disease or a disorder.
  • the terms are intended to include the full spectrum of treatments for a given con ⁇ dition from which the patient is suffering, such as administration of the active compound(s) in question to alleviate symptoms or complications thereof, to delay the progression of the dis ⁇ ease, disorder or condition, to cure or eliminate the disease, disorder or condition, and/or to prevent the condition, in that prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder, and includes the administration of the active compound(s) in question to prevent the onset of symptoms or complications.
  • the patient to be treated is preferably a mammal, in particular a human being, but treatment of other animals, such as dogs, cats, cows, horses, sheep, goats or pigs, is within the scope of the invention.
  • solvate refers to a complex of defined stoichiometry formed be ⁇ tween a solute (in casu, a compound according to the present invention) and a solvent.
  • Sol ⁇ vents may include, by way of example, water, ethanol, or acetic acid.
  • amino acid abbreviations used in the present context have the following meanings:
  • D-Ser D-His and so on
  • one aspect of the invention relates to compounds of formula Ia, Ib or Ic:
  • Z 1 and Z 2 independently represent GIy, Ser, Thr, GIn, Asn, His, homoArg, Arg, Lys, Dab, Dap or Orn;
  • Z 3 represents Ala, VaI, Leu, He, Met or NIe;
  • X 1 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap;
  • X 2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, He, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met,
  • Hyp Tic, 2-PyAIa, 3-PyAIa, 4-PyAIa, (2-thienyl)alanine, 3-(thienyl)alanine, (4-thiazolyl)Ala, (2-furyl)alanine, (3-furyl)alanine or Phe, wherein the phenyl moiety of said Phe is optionally substituted with a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano;
  • X 3 represents D-Phe, wherein the phenyl moiety in D-Phe may optionally be substituted with one or more substituents selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoro- methyl and cyano;
  • X 4 represents Trp, 2-NaI, a (3-benzo[b]thienyl)alanine residue or a (S)-2,3,4,9-tetrahydro-1 H- ⁇ -carboline-3-carboxylic acid residue;
  • X 5 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap; wherein X 1 and X 5 are joined, rendering the compound of formula Ia, Ib or Ic cyclic, either via a disulfide bridge deriving from X 1 and X 5 both independently being Cys, homoCys or Pen, or via an amide bond formed between a carboxylic acid in the side-chain of X 1 and an amino group in the side chain of X 5 , or between a carboxylic acid in the side-chain of X 5 and an amino group in the side-chain of X 1 ; each R' independently represents hydrogen or d- 6 alkyl, which may optionally be substituted with one or more amino or hydroxy; with the proviso that the compound of formula Ia, Ib or Ic is not
  • R 1 is Ci 4 -i 8 -alkanoyl.
  • R 1 is 4-(Ci 4 -i 8 alkanoyl- sulfamoyl)butanoyl, such as 4-(hexadecanoylsulfamoyl)butanoyl.
  • S has a structure selected among for ⁇ mulas Na, Nd and lie.
  • Z 3 is NIe.
  • Z 2 is Ser, Thr, Dab or Dap, notably Ser or Dab.
  • Z 1 is His, GIn, Arg, homoArg, Lys, Orn, Dab or Dap, such as His, GIn or Arg.
  • R 1 is Ci 4 -i 8 alkanoyl or 4-(hexadecanoylsulfamoyl)butanoyl; Z ⁇ if present, is His, Arg, homoArg, Lys, GIn, Asn, Orn, Dab or Dap; Z 2 , if present, is Ser, Thr, Dab or Dap; Z 3 is NIe;
  • X 1 is GIu, Asp or Cys
  • X 2 is Ser, Hyp, Cit, Dap, Asn, GIn or (4-thiazolyl)Ala;
  • X 3 is D-Phe;
  • X 4 is Trp;
  • X 5 is Lys when X 1 is GIu or Asp, or X 5 is Cys when X 1 is Cys; and N(R) 2 is NH 2 .
  • particularly interesting compounds include:
  • Z 1 if present, is His, Arg or GIn;
  • the present invention also encompasses combinations of two or more embodiments of com- pounds of the invention as outlined above.
  • the compound of the invention is an agonist of a melanocortin receptor, notably an agonist of MC4.
  • the compound is a selective agonist of MC4.
  • selectivity is to be understood in re- lation to the activity of the compound with respect to MC1 , MC3 and/or MC5. If a compound is a significantly more potent as a MC4 agonist than as a MC1 , MC3 and/or MC5 agonist, it is deemed to be a selective MC4 agonist.
  • the agonistic potency of a compound with respect to MC1 and MC4 may be determined by comparing an MC1 binding assay as described below under "Assay IV" (MC1 ) with a functional MC4 assay as described below under “Assay III” (MC4). If a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times more potent with respect to MC4 than with respect to MC1 , it is deemed to be a selective MC4 agonist with respect to MC1 .
  • the agonistic potency of a compound with re ⁇ spect to MC3, MC4 and MC5 may be determined in functional assays as described in "Assay II" (MC 3 and MC5) and "Assay III” (MC4). If a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times more potent with respect to MC4 than with re ⁇ spect to MC3, it is deemed to be a selective MC4 agonist with respect to MC3. If a com ⁇ pound is more than 10 times, such as more than 50 times, e.g.
  • the compound of the present invention is a selective MC4 agonist with respect to MC1 , with respect to MC3, with respect to MC5, with respect to MC1 and MC3, with respect to MC1 and MC5, with respect to MC3 and MC5 or with respect to MC1 , MC3 and MC5.
  • the compound of the invention is a selective MC4 agonist and a MC3 antagonist.
  • a compound is deemed to be a selective MC4 agonist and a MC3 antagonist if it is a selective MC4 agonist with respect to MC1 and MC5 as discussed above, and it antagonizes MC3 as determined as described in "Assay II".
  • a compound exhibiting an IC 50 value of less than 100 nM, such as less than 10 nM, e.g. less than 5 nM, such as less than 1 nM is deemed to be a MC3 antagonist.
  • the compound of the present invention is both a selective MC3 agonist and a selective MC4 agonist.
  • a compound is deemed to be a selective MC3 and MC4 agonist if it is significantly more potent as an agonist towards MC3 and MC4 than as an agonist toward MC1 and MC5.
  • the selectivity of a compound with respect to MC1 and MC3 may be determined by comparing the potency determined for MC1 as described in "Assay IV" with the potency for MC3 determined as described in "Assay II". If a compound is more than 10 times, such as more than 50 times, e.g.
  • the selectivity of a compound with respect to MC3 and MC5 may be determined by comparing the potency determined as described in "Assay II". If a compound is more than 10 times, such as more the 50 times, e.g. more than 100 times more potent with respect to MC3 than with respect to MC5, it is deemed to be a selective MC3 agonist with respect to MC5.
  • the MC4 selectivity of a compound with respect to MC3 and MC5 is determined as discussed above.
  • Compounds of the present invention may exert a protracted effect, i.e. the period of time in which they exert a biological activity may be prolonged.
  • a protracting effect may be evalu ⁇ ated in a slightly modified "Assay I" in a comparison between a compound of the present in ⁇ vention and the corresponding compound wherein R 1 is hydrogen and S is a bond.
  • the ex- periment is allowed to continue for a period of time, T, until the rats have eaten as much as they did prior to the experiment.
  • T values for compounds of the present invention and the corresponding compounds wherein R 1 is hydrogen and S is a bond are measured, and the difference ⁇ T is calculated.
  • Compounds of the present invention giving rise to ⁇ T above 3 hours, such as above 7 hours, such as above 12 hours, such as above 24 hours, such as above 48 hours, such as above 72 hours, are deemed to exert a protracted effect.
  • compounds of the present invention modulate melanocortin receptors, and they are there ⁇ fore believed to be particularly suited for the treatment of diseases or states which can be treated by a modulation of melanocortin receptor activity.
  • compounds of the present invention are believed to be suited for the treatment of diseases or states via activa ⁇ tion of MC4.
  • the present invention relates to a method of agonizing or activating MC4 in a subject, the method comprising administering to the subject an effective amount of a com- pound of the present invention.
  • the invention provides a method of delaying the progression from impaired glucose tolerance (IGT) to type 2 diabetes, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
  • ITT impaired glucose tolerance
  • the invention provides a method of delaying the progression from type 2 diabetes to insulin-requiring diabetes, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
  • the invention relates to a method of treating obesity or preventing overweight, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
  • the present invention provides a method of regulating appetite, the method comprising administering to a patient in need thereof an effective amount of a com ⁇ pound of the present invention.
  • Another aspect of the invention relates to a method of inducing satiety, the method compris ⁇ ing administering to a patient in need thereof an effective amount of a compound of the pre- sent invention.
  • a further aspect of the invention relates to a method of preventing weight regain after suc ⁇ cessfully having lost weight, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
  • Yet another aspect of the invention relates to a method of increasing energy expenditure, the method comprising administering to a patient in need thereof an effective amount of a com ⁇ pound of the present invention.
  • Still further aspects of the invention include the following:
  • a method of treating a disease or state related to overweight or obesity comprising administering to a patient in need thereof an effective amount of a compound of the pre ⁇ sent invention
  • a method of treating bulimia comprising administering to a patient in need thereof an effective amount of a compound of the present invention
  • a method of treating binge-eating comprising administering to a patient in need thereof an effective amount of a compound of the present invention; a method of treating a disease or state selected from atherosclerosis, hypertension, diabe ⁇ tes, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of prema- ture death, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
  • a disease or state selected from atherosclerosis, hypertension, diabe ⁇ tes, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of prema- ture death
  • compounds of the present invention may be suited for the treatment of diseases in obese or overweight patients.
  • the present invention also provides a method of treating, in an obese patient, a disease or state selected from type 2 diabetes, impaired glu ⁇ cose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of premature death in obese patients, the method comprising administering to an obese patient in need thereof an effective amount of a compound of the present invention.
  • a disease or state selected from type 2 diabetes, impaired glu ⁇ cose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of premature death in obese patients.
  • ITT impaired glu ⁇ cose tolerance
  • administration of compounds of the present invention may be advantageous in the treatment of patients, notably obese or overweight patients, who have undergone, or are to undergo, gastric banding and/or gastric surgery.
  • MC4 agonists could have a positive effect on insulin sensitivity, on drug abuse by modulating the reward system and on hemorrhagic shock.
  • MC3 and MC4 ago ⁇ nists have antipyretic effects, and both have been suggested to be involved in peripheral nerve regeneration.
  • MC4 agonists are also known to reduce stress response.
  • Appropriate routes of administration of compounds of the invention to patients in the context of the invention include parenteral routes such as nasal, pulmonary or sublingual administra ⁇ tion routes, all of which are familiar to persons of skill in the art of drug administration.
  • a compound of the present invention may be administered alone or in combination with one or more (i.e. one or two or three.... etc.) additional compounds of the present invention.
  • a compound of the invention, or a combination of two or more (i.e. two or three or four.... etc.) compounds of the invention may be administered in combination with one or more other therapeutically active agents or com ⁇ pounds (i.e. agents or compounds which are not within the scope of the present invention), either sequentially or concomitantly.
  • a typical dosage of a compound of the invention when employed in a method according to the present invention is in the range of from about 0.001 to about 100 mg/kg body weight per day, e.g.
  • a typical unit dosage form intended for oral ad ⁇ ministration one or more times per day, such as from one to three times per day, may suita ⁇ bly contain from 0.05 to about 1000 mg, e.g. from about 0.1 to about 500 mg, such as from about 0.5 mg to about 200 mg of a compound of the invention.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a com ⁇ pound of the present invention, optionally in combination with one or more additional thera ⁇ Therapeuticically active compounds or substances, together with one or more pharmaceutically ac- ceptable carriers or excipients.
  • the composition may suitably be in unit dosage form com ⁇ prising from about 0.05 mg to about 1000 mg, such as from about 0.1 mg to about 500 mg, e.g. from about 0.5 mg to about 200 mg, of a compound of the present invention.
  • the present invention also relates to the use of a compound of the present invention, option- ally in combination with one or more additional therapeutically active compounds or sub ⁇ stances, in the manufacture of a medicament for the treatment of a disease or condition se ⁇ lected from overweight or obesity, bulimia, binge-eating, atherosclerosis, hypertension, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallblad ⁇ der disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of premature death.
  • ITT impaired glucose tolerance
  • the invention also relates to the use of a compound of the present invention, optionally in combination with one or more additional therapeutically active compounds or substances, in the manufacture of a medicament effective in: delaying the progression from IGT to type 2 diabetes; delaying the progression from type 2 diabetes to insulin-requiring diabetes; regulat- ing appetite; inducing satiety; preventing weight regain after successfully having lost weight; or increasing energy expenditure.
  • compounds of the present invention may be administered or applied in combination with one or more additional therapeutically active compounds or substances.
  • additional compounds or substances may be selected, for example, from antidia ⁇ betic agents, antihyperlipidemic agents, antiobesity agents, antihypertensive agents and agents for the treatment of complications resulting from, or associated with, diabetes.
  • Suitable antidiabetic agents include: insulin; derivatives or analogues of insulin, including de ⁇ rivatives or analogues exhibiting a profile of protracted or prolonged activity, such as those disclosed in WO 95/07931 , WO 97/31022 and WO 2005/012347 (Novo Nordisk A/S), the contents of all of which are incorporated herein by reference; derivatives of GLP-1 (glucagon like peptide-1 ), such as those disclosed in WO 98/08871 (Novo Nordisk A/S), the contents of which are incorporated herein by reference; derivatives of GLP-1 analogues, such as those disclosed in US 6,458,924 (Knudsen et al.), the contents of which are incorporated herein by reference; and orally active hypoglycemic agents.
  • Suitable orally active hypoglycemic agents include: imidazolines; sulfonylureas; biguanides; meglitinides; oxadiazolidinediones; thiazolidinediones; insulin sensitizers; ⁇ -glucosidase in ⁇ hibitors; agents acting on the ATP-dependent potassium channel of the pancreatic ⁇ -cells, e.g.
  • potassium channel openers such as those disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (Novo Nordisk A/S), the contents of which are incorporated herein by ref ⁇ erence; potassium channel openers such as ormitiglinide; potassium channel blockers such as nateglinide or BTS-67582; glucagon antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), the contents of which are incorporated herein by reference; GLP-1 agonists such as those disclosed in WO 00/42026 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), the contents of which are incorporated herein by reference; DPP-IV (dipeptidyl peptidase-IV) inhibitors; PTPase (pro- tein tyrosine phosphatase) inhibitors; glucokinase activators, such as those described in
  • Suitable additional therapeutically active substances include insulin or in- sulin analogues; sulfonylureas, e.g. tolbutamide, chlorpropamide, tolazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide; biguanides, e.g. metformin; and meglitinides, e.g. repaglinide or senaglinide/nateglinide.
  • sulfonylureas e.g. tolbutamide, chlorpropamide, tolazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide
  • biguanides e.g. metformin
  • meglitinides e.g. repaglinide or senaglinide/nateglinide.
  • suitable additional therapeutically active substances include thiazolidin- edione insulin sensitizers, e.g. troglitazone, ciglitazone, pioglitazone, rosiglitazone, isaglita- zone, darglitazone , englitazone, CS-01 1 /CI-1037 or T 174, or the compounds disclosed in WO 97/41097 (DRF-2344), WO 97/41 119, WO 97/41120, WO 00/41121 and WO 98/45292 (Dr. Reddy's Research Foundation), the contents of all of which are incorporated herein by reference.
  • thiazolidin- edione insulin sensitizers e.g. troglitazone, ciglitazone, pioglitazone, rosiglitazone, isaglita- zone, darglitazone , englitazone, CS-01 1 /CI-1037 or T 174
  • Suitable additional therapeutically active substances include insulin sensitizers, e.g. Gl 262570, YM-440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW- 409544, CRE-16336, AR-H049020, LY510929, MBX-102, CLX-0940, GW-501516 and the compounds disclosed in WO 99/19313 (NN622/DRF-2725), WO 00/50414, WO 00/63191 , WO 00/63192 and WO 00/63193 (Dr.
  • insulin sensitizers e.g. Gl 262570, YM-440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW- 409544, CRE-16336, AR-H049020, LY510929, MBX-102, CLX-0940, GW-501516 and the compounds disclosed in WO 99/19313 (NN
  • suitable additional therapeutically active substances include:
  • ⁇ -glucosidase inhibitors e.g. voglibose, emiglitate, miglitol or acarbose;
  • glycogen phosphorylase inhibitors e.g. the compounds described in WO 97/09040 (Novo Nordisk A/S);
  • agents acting on the ATP-dependent potassium channel of the pancreatic ⁇ -cells e.g. tolbu- tamide, glibenclamide, glipizide, glicazide, BTS-67582 or repaglinide;
  • suitable additional therapeutically active substances include antihyperlipidemic agents and antilipidemic agents, e.g. cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
  • agents which are suitable as additional therapeutically active substances include an- tiobesity agents and appetite-regulating agents.
  • Such substances may be selected from the group consisting of CART (cocaine amphetamine regulated transcript) agonists, NPY (neu ⁇ ropeptide Y) antagonists, Y2 and Y4 receptor agonists, MC3 (melanocortin 3) agonists, MC3 (melanocortin 3) antagonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotro ⁇ pin releasing factor binding protein) antagonists, urocortin agonists, ⁇ 3 adrenergic agonists such as CL-316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140, MC1 (melano
  • fluoxetine, seroxat or citalopram se ⁇ rotonin and norepinephrine reuptake inhibitors
  • 5HT serotonin
  • bombesin agonists bombesin agonists, galanin antagonists, growth hormone, growth factors such as prolactin or placental lactogen, growth hormone releasing compounds (growth hormone secretagogues), ghrelin antago ⁇ nists, TRH (thyrotropin releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, chemical uncouplers, leptin agonists, DA (dopamine) agonists (bromocriptin, doprexin), lipase/amylase inhibitors, PPAR modulators, RXR modulators, TR ⁇ agonists, adrenergic CNS stimulating agents, AGRP (agouti-related protein) inhibitors, histamine H3 receptor antagonists such as those disclosed in WO 00/42023,
  • antiobesity agents are bupropion (antidepressant), topiramate (anticonvul ⁇ sant), ecopipam (dopamine D1/D5 antagonist), naltrexone (opioid antagonist), and peptide YY 3 -36 (Batterham et al, Nature 418, 650-654 (2002)).
  • An embodiment of a suitable antiobesity agent for use in a method of the invention as an ad ⁇ ditional therapeutically active substance in combination with a compound of the invention is leptin.
  • a further embodiment of a suitable antiobesity agent is peptide YY 3 - 36 .
  • Additional embodiments of suitable antiobesity agents are serotonin and norepinephrine re ⁇ uptake inhibitors, e.g. sibutramine.
  • Suitable antiobesity agents are lipase inhibitors, e.g. orlistat.
  • Suitable antiobesity agents are adrenergic CNS stimulating agents, e.g. dexamphetamine, amphetamine, phentermine, mazindol, phendimetrazine, di- ethylpropion, fenfluramine or dexfenfluramine.
  • adrenergic CNS stimulating agents e.g. dexamphetamine, amphetamine, phentermine, mazindol, phendimetrazine, di- ethylpropion, fenfluramine or dexfenfluramine.
  • antihyper ⁇ tensive agents examples include antihyper ⁇ tensive agents.
  • antihypertensive agents are ⁇ -blockers such as alprenolol, at ⁇ enolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and ⁇ -blockers such as doxazosin, urapidil, prazosin and terazosin.
  • ⁇ -blockers such as alprenolol, at ⁇ enolol, ti
  • the compound of the present invention may be administered or applied in combination with more than one of the above-mentioned, suitable additional therapeutically active compounds or substances, e.g. in combination with: metformin and a sulfonylurea such as glyburide; a sulfonylurea and acarbose; nateglinide and metformin; acarbose and metformin; a sulfonylurea, metformin and troglitazone; insulin and a sulfonylurea; insulin and metformin; insulin, metformin and a sulfonylurea; insulin and troglitazone; insulin and lovastatin; etc.
  • metformin and a sulfonylurea such as glyburide
  • a sulfonylurea and acarbose nateglinide and metformin
  • one aspect of the present invention provides a pharmaceutical com ⁇ position (formulation) comprising a compound of the present invention.
  • a pharmaceutical com ⁇ position comprising a compound of the present invention.
  • Appropriate embodi ⁇ ments of such formulations will often contain a compound of the invention in a concentration of from 10 '3 mg/ml to 200 mg/ml, such as, e.g., from 10 '1 mg/ml to 100 mg/ml.
  • the pH in such a formulation of the invention will typically be in the range of 2.0 to 10.0.
  • the formulation may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabi ⁇ lizers) and/or surfactant(s).
  • the pharmaceutical formula ⁇ tion is an aqueous formulation, i.e. a formulation comprising water
  • aqueous formulation in the present context may normally be taken to indicate a formulation compris ⁇ ing at least 50 % by weight (w/w) of water.
  • a formulation is typically a solution or a sus ⁇ pension.
  • An aqueous formulation of the invention in the form of an aqueous solution will normally comprise at least 50 % (w/w) of water.
  • an aqueous formulation of the in- vention in the form of an aqueous suspension will normally comprise at least 50 % (w/w) of water.
  • a pharmaceutical composition (formulation) of the invention may be a freeze-dried (i.e. lyophilized) formulation intended for reconstitution by the physician or the patient via addition of solvents and/or diluents prior to use.
  • a pharmaceutical composition (formulation) of the invention may be a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolu ⁇ tion.
  • the invention relates to a pharmaceutical composition (formulation) com ⁇ prising an aqueous solution of a compound of the present invention, and a buffer, wherein the compound of the invention is present in a concentration of 0.1 -100 mg/ml or above, and wherein the formulation has a pH from about 2.0 to about 10.0.
  • the pH of the formulation has a value selected from the list consisting of 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5,
  • the buffer in a buffered pharmaceutical composition of the invention may comprise one or more buffer substances selected from the group consisting of sodium acetate, sodium carbonate, citrates, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, tris(hydroxymethyl)aminomethane (TRIS), bicine, tricine, malic acid, succinates, maleic acid, fumaric acid, tartaric acid and aspartic acid.
  • a pharmaceutical composition of the invention may comprise a pharmaceutically acceptable preservative, e.g.
  • preservatives 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, thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride and chlorphenesine (3p-chlorphenoxypropane-1 ,2-diol).
  • the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml.
  • the preservative is present in a concentration in the range of 0.1 mg/ml to 5 mg/ml, a concentration in the range of 5 mg/ml to 10 mg/ml, or a concentration in the range of 10 mg/ml to 20 mg/ml.
  • the use of a preservative in pharmaceutical compositions is well known to the skilled person. For convenience, reference is made in this respect to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • the formulation further comprises a tonicity-adjusting agent, i.e. a substance added for the purpose of adjusting the tonicity (osmotic pressure) of a liquid formulation (notably an aqueous formulation) or a reconstituted freeze-dried formulation of the invention to a desired level, normally such that the resulting, final liquid formulation is isotonic or substantially isotonic.
  • a tonicity-adjusting agent i.e. a substance added for the purpose of adjusting the tonicity (osmotic pressure) of a liquid formulation (notably an aqueous formulation) or a reconstituted freeze-dried formulation of the invention to a desired level, normally such that the resulting, final liquid formulation is isotonic or substantially isotonic.
  • Suitable tonicity-adjusting agents may be selected from the group consisting of salts (e.g. sodium chloride), sugars and sugar alcohols (e.g. mannitol), amino acids (e.g.
  • glycine histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine
  • alditols e.g. glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3-propanediol or 1 ,3-butanediol
  • polyethyleneglycols e.g. PEG 400
  • Any sugar such as a mono-, di- or polysaccharide, or a water-soluble glucan, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch or carboxymethylcellulose-sodium, may be used; in one embodiment, sucrose may be employed.
  • Sugar alcohols include, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol employed is mannitol.
  • 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 composition (formulation) and does not adversely effect the stabilizing effects achieved using the methods of the invention.
  • the concentration of sugar or sugar alcohol is between about 1 mg/ml and about 150 mg/ml.
  • the tonicity-adjusting agent is present in a concentration of from 1 mg/ml to 50 mg/ml, such as from 1 mg/ml to 7 mg/ml, from 8 mg/ml to 24 mg/ml, or from 25 mg/ml to 50 mg/ml.
  • a pharmaceutical composition of the invention containing any of the tonicity-adjusting agents specifically mentioned above constitutes an embodiment of the invention.
  • the use of a tonicity-adjusting agent in pharmaceutical compositions is well known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 20 th edition, 2000.
  • the formulation further comprises a chelating agent.
  • Suitable chelating agents may be selected, for example, from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof.
  • the concentration of chelating agent will suitably be in the range from 0.1 mg/ml to 5 mg/ml, such as from 0.1 mg/ml to 2 mg/ml or from 2 mg/ml to 5 mg/ml.
  • a pharmaceutical composition of the invention containing any of the chelating agents specifically mentioned above constitutes an 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, 20 th edition, 2000.
  • the formulation further comprises a stabilizer.
  • 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, 20 th edition, 2000.
  • compositions of the invention include stabilized liquid pharmaceutical compositions whose therapeutically active components include an oligo- or polypeptide that possibly exhibits aggregate formation during storage in liquid pharmaceuti ⁇ cal formulations.
  • aggregate formation is meant the formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution, as the result of a physical interaction between the oligo- or polypeptide molecules.
  • the term “during storage” refers to the fact that a liquid pharmaceutical composition or formulation, once prepared, is not normally administered to a subject immediately.
  • dried form is meant the product obtained when a liquid pharmaceutical composition or formulation is dried by freeze-drying (i.e., lyophilization; see, for example, Williams and PoIIi (1984) J ⁇ Parenteral Sci. Technol. 38: 48-59), by spray-drying [see, e.g., Masters (1991 ) in Spray- Drying Handbook (5th edn.; Longman Scientific and Technical, Essex, U.K.), pp. 491 -676; Broadhead et al. (1992) Drug Devel.
  • a pharmaceutical composition of the invention may further comprise an amount of an amino acid base sufficient to decrease aggregate formation by the oligo- or polypeptide during stor ⁇ age of the composition.
  • amino acid base is meant 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 for use in preparing a composition 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 mix ⁇ tures thereof) of a particular amino acid e.g. methionine, histidine, arginine, lysine, isoleu- cine, aspartic acid, tryptophan or threonine, and mixtures thereof
  • a particular amino acid e.g. methionine, histidine, arginine, lysine, isoleu- cine, aspartic acid, tryptophan or threonine, and mixtures thereof
  • the L-stereoisomer of an amino acid is used.
  • Compositions of the invention may also be formulated with analogues of these amino acids.
  • amino acid analogue is meant a derivative of a naturally occurring amino acid that brings about the desired effect of de ⁇ creasing aggregate formation by the oligo- or polypeptide during storage of liquid pharma ⁇ ceutical compositions of the invention.
  • Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl-L-arginine.
  • Suitable methionine analogues in- elude ethionine and buthionine, and suitable cysteine analogues include S-methyl-L-cysteine.
  • amino acid analogues are incorporated into compositions of the invention in either their free base form or their salt form.
  • the amino acids or amino acid analogues are incorporated in a concentration which is sufficient to prevent or delay aggregation of the oligo-or polypeptide.
  • methionine (or another sulfur-containing amino acid or amino acid analogue) may be incorporated in a composition of the invention to inhibit oxidation of methionine residues to methionine sulfoxide when the oligo- or polypeptide act- ing as the therapeutic agent is a peptide comprising at least one methionine residue suscep ⁇ tible to such oxidation.
  • the term "inhibit" in this context refers to minimization of accumulation of methionine-oxidized species over time. Inhibition of methionine oxidation results in in ⁇ creased retention of the oligo- or polypeptide in its proper molecular form.
  • the amount to be added should be an amount sufficient to inhibit oxidation of methionine residues such that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that no more than from about 10% to about 30% of forms of the oligo- or polypeptide wherein methionine is sulfoxidated are present. In general, this can be achieved by incorporating methionine in the composition such that the ratio of added methionine to methionine residues ranges from about 1 :1 to about 1000:1 , such as from about 10:1 to about 100:1 .
  • the formulation further comprises a stabilizer selected from high-molecular-weight polymers and low-molecular-weight compounds.
  • the stabilizer may be selected from substances such as polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose and derivatives thereof (e.g. HPC, HPC-SL, HPC-L or HPMC), cyclodextrins, sulfur- containing substances such as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and various salts (e.g. sodium chloride).
  • PEG 3350 polyethylene glycol
  • PVA polyvinyl alcohol
  • PVpyrrolidone carboxy-/hydroxycellulose and derivatives thereof
  • cyclodextrins e.g. HPC, HPC-SL, HPC-L or HPMC
  • sulfur- containing substances such as monothioglycerol, thioglycolic acid and 2-methylthio
  • compositions of the present invention may also comprise additional stabilizing agents which further enhance stability of a therapeutically active oligo- or polypeptide therein.
  • Stabilizing agents of particular interest in the context of the present invention include, but are not limited to: methionine and EDTA, which protect the peptide against methionine oxidation; and surfactants, notably nonionic surfactants, which protect the polypeptide against aggregation or degradation associated with freeze-thawing or mechanical shearing.
  • the pharmaceutical formulation comprises a surfactant, particularly a nonionic surfactant.
  • a surfactant particularly a nonionic surfactant.
  • examples thereof include ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (e.g. 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, e.g.
  • Tween-20, Tween-40, Tween-80 and Brij-35 monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (e.g. phosphatidyl-serine, phosphatidyl-choline, phosphatidyl- ethanolamine, phosphatidyl-inositol, diphosphatidyl-glycerol and sphingomyelin), derivatives of phospholipids (e.g. dipalmitoyl phosphatidic acid) and lysophospholipids (e.g.
  • cholines ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (eg. cephalins), glyceroglycolipids (e.g. galactopyranoside), sphingoglycolipids (e.g. ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives (e.g.
  • sodium tauro- dihydrofusidate, etc. long-chain fatty acids (e.g. oleic acid or caprylic acid) and salts thereof, acylcarnitines and derivatives, N ⁇ -acylated derivatives of lysine, arginine or histidine, or side- chain acylated derivatives of lysine or arginine, N ⁇ -acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, N ⁇ -acylated derivative of a tripeptide comprising any combination of a neutral amino acid and two charged amino acids, DSS (docusate sodium, CAS registry no.
  • DSS docusate sodium, CAS registry no.
  • the surfactant may also be selected from imidazoline derivatives and mixtures thereof.
  • a pharmaceutical composition of the invention containing any of the surfactants specifically mentioned above constitutes an embodiment of the invention.
  • Additional ingredients may also be present in a pharmaceutical composition (formulation) of the present invention.
  • additional ingredients may include, for example, wetting agents, emulsifiers, antioxidants, bulking agents, metal ions, oleaginous vehicles, proteins (e.g. human serum albumin, gelatine or other proteins) and a zwitterionic species (e.g. an amino acid such as betaine, taurine, arginine, glycine, lysine or histidine).
  • proteins e.g. human serum albumin, gelatine or other proteins
  • a zwitterionic species e.g. an amino acid such as betaine, taurine, arginine, glycine, lysine or histidine.
  • Such additional ingredients should, of course, 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 one or more of several sites, for example at topical sites (e.g. skin and mucosal sites), at sites which bypass absorption (e.g. via administration in an artery, in a vein or in the heart), and at sites which involve absorption (e.g. in the skin, under the skin, in a muscle or in the abdomen).
  • topical sites e.g. skin and mucosal sites
  • sites which bypass absorption e.g. via administration in an artery, in a vein or in the heart
  • sites which involve absorption e.g. in the skin, under the skin, in a muscle or in the abdomen.
  • Administration of pharmaceutical compositions according to the invention to patients in need thereof may be via several routes of administration. These include, for example, lingual, sub ⁇ lingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary (for exam ⁇ ple through the bronchioles and alveoli or a combination thereof), epidermal, dermal, trans ⁇ dermal, vaginal, rectal, ocular (for example through the conjunctiva), uretal and parenteral.
  • routes of administration include, for example, lingual, sub ⁇ lingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary (for exam ⁇ ple through the bronchioles and alveoli or a combination thereof), epidermal, dermal, trans ⁇ dermal, vaginal, rectal, ocular (for example through the conjunctiva), uretal and parenteral.
  • compositions of the present invention may be administered in various dosage forms, for ex ⁇ ample in the form of solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules (e.g.
  • hard gelatine capsules or soft gelatine capsules such as hard gelatine capsules or 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 solutions, in s/fcv-transforming solutions (for example in situ gelling, in situ setting, in situ precipitating or in situ crystallizing), infusion solutions or implants.
  • s/fcv-transforming solutions for example in situ gelling, in situ setting, in situ precipitating or in situ crystallizing
  • compositions of the invention may further be compounded in, or bound to, e,g. via covalent, hydrophobic or electrostatic interactions, a drug carrier, drug delivery system or advanced drug delivery system in order to further enhance the stability of the compound of the present invention, 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 deliv ⁇ ery systems include, but are not limited to: polymers, for example cellulose and derivatives; polysaccharides, for example dextran and derivatives, starch and derivatives; polyvinyl al ⁇ cohol); acrylate and methacrylate polymers; polylactic and polyglycolic acid and block co ⁇ polymers thereof; polyethylene glycols; carrier proteins, for example albumin; gels, for exam- pie thermogelling systems, such as 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 thereof well known to those skilled in the art of phase be ⁇ haviour in lipid-water systems; polymeric micelles; multiple emulsions (self-emulsifying, self- microemulsifying); cyclodextrins and derivatives thereof; and dendrimers.
  • polymers for example cellulose and derivatives
  • compositions of the present invention are useful in the formulation of solids, semisolids, powders and solutions for pulmonary administration of a compound of the present invention, using, for example, a metered dose inhaler, dry powder inhaler or a nebulizer, all of which are devices well known to those skilled in the art.
  • compositions of the present invention are useful in the formulation of controlled-release, sus ⁇ tained-release, protracted, retarded or slow-release drug delivery systems.
  • Compositions of the invention are thus of value in the formulation of parenteral controlled-release and sus ⁇ tained-release systems well known to those skilled in the art (both types of systems leading to a many-fold reduction in the number of administrations required).
  • controlled-release and sustained-release systems for subcutaneous administration.
  • examples of useful controlled re ⁇ lease systems and compositions are those containing hydrogels, oleaginous gels, liquid crys- tals, polymeric micelles, microspheres or nanoparticles,
  • Methods for producing controlled-release systems useful for compositions of the present in ⁇ vention include, but are not limited to, crystallization, condensation, co-crystallization, precipi ⁇ tation, co-precipitation, emulsification, dispersion, high-pressure homogenisation, encapsula- tion, spray-drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes.
  • General reference is made in this context to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000), and Drugs and the Pharmaceutical Sciences, vol. 99: Pro ⁇ tein 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, for example a syringe in the form of a pen device.
  • parenteral administration may be performed by means of an infusion pump.
  • a further option is administration of a composition of the invention which is a liquid (typically aqueous) solution or suspension in the form of a nasal or pulmonary spray.
  • a pharmaceutical composition of the invention can be adapted to trans ⁇ dermal administration (e.g. by needle-free injection or via a patch, such as an iontophoretic patch) or transmucosal (e.g. buccal) administration.
  • stabilized formulation refers to a formulation with increased physical stability, in ⁇ creased chemical stability or increased physical and chemical stability.
  • physical stability in the context of a formulation containing an oligo- or polypeptide refers to the ten ⁇ dency of the peptide to form biologically inactive and/or insoluble aggregates as a result of exposure to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces.
  • Physical stability of aqueous protein formulations is evaluated by means of visual inspection and/or turbidity measure ⁇ ments after exposing the formulation, filled in suitable containers (e.g. cartridges or vials), to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods.
  • the turbidity of a formulation is characterized by a visual score ranking the degree of turbidity, for instance on a scale from 0 to 3 (in that a formulation showing no turbidity corre ⁇ sponds to a visual score 0, whilst a formulation showing visual turbidity in daylight corre ⁇ sponds to visual score 3).
  • a formulation is normally classified physically unstable with re ⁇ spect to aggregation when it shows visual turbidity in daylight.
  • the turbidity of a formulation can be evaluated by simple turbidity measurements well-known to the skilled person.
  • aqueous oligo- or polypeptide formulations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the peptide.
  • the probe is preferably a small molecule that preferentially binds to a non-native conformer of the oligo- or polypeptide.
  • a small-molecular spectroscopic probe of this type 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 possibly also other configurations, 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 form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths in question.
  • small molecules can be used as probes of the changes in peptide structure from native to non-native states.
  • Examples are the "hydrophobic patch" probes that bind preferentially to exposed hydrophobic patches of a polypeptide.
  • the hydrophobic patches are generally bur ⁇ ied within the tertiary structure of a polypeptide in its native state, but become exposed as it begins to unfold or denature.
  • Examples of such small-molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as anthracene, acridine, phenanthroline and the like.
  • Other spectroscopic probes are metal complexes of amino acids, such as cobalt complexes of hy ⁇ drophobic amino acids, e.g. phenylalanine, leucine, isoleucine, methionine, valine, or the like.
  • chemical stability of a pharmaceutical formulation as used herein refers to chemi ⁇ cal covalent changes in oligo- or polypeptide structure leading to formation of chemical deg ⁇ radation products with potentially lower biological potency and/or potentially increased im- munogenicity compared to the original molecule.
  • chemical degradation products can be formed depending on the type and nature of the starting molecule and the environment to which it is exposed. Elimination of chemical degradation can most probably not be com ⁇ pletely avoided and gradually increasing amounts of chemical degradation products may of ⁇ ten be seen during storage and use of oligo- or polypeptide formulations, as is well known to the person skilled in the art.
  • a commonly encountered degradation process is deamidation, a process in which the side-chain amide group in glutaminyl or asparaginyl residues is hydro- lysed to form a free carboxylic acid.
  • Other degradation pathways involve formation of higher molecular weight transformation products wherein two or more molecules of the starting sub ⁇ stance are covalently bound to each other through transamidation and/or disulfide interac ⁇ tions, leading to formation of covalently bound dimer, oligomer or polymer degradation prod ⁇ ucts (see, e.g., Stability of Protein Pharmaceuticals, Ahern. TJ. & Manning M. C, Plenum Press, New York 1992).
  • Oxidation (of for instance methionine residues) may be mentioned as another variant of chemical degradation.
  • the chemical stability of a formulation may be evaluated by measuring the amounts of chemical degradation products at various time-points after exposure to different environmental conditions (in that the formation of degradation products can often be accelerated by, e.g., increasing temperature).
  • the amount of each in- dividual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatographic techniques (e.g. SEC-HPLC and/or RP-HPLC).
  • a “stabilized formulation” refers to a formulation with increased physical stability, increased chemical stability, or increased physical and chemical stability.
  • a pharmaceutical composition (formulation) must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiry date is reached.
  • a pharmaceutical composition (formulation) of the invention should preferably be stable for more than 2 weeks of usage and for more than two years of storage, more preferably for more than 4 weeks of usage and for more than two years of storage, desirably for more than 4 weeks of usage and for more than 3 years of storage, and most preferably for more than 6 weeks of usage and for more than 3 years of storage.
  • Headings and sub-headings are used herein for convenience only, and should not be con ⁇ strued as limiting the invention in any way.
  • Rt values are retention times and the mass values are those detected by the mass spectroscopy (MS) detector and obtained using one of the following HPLC-MS devices (LCMS).
  • Fmoc-Rink resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxypolystyrene resin, Bachem D-2080, Lot 514460; 0.47 mmol/g) was filled into two 60 ml Teflon reactors with frit (per reactor: 3.55 g, 1.67 mmol). The resin in each reactor was washed with DCM (30 ml).
  • DIPEA Ethyldiisopropylamine
  • Remaining solvents were removed by sucking air through the reactors for 4 h.
  • the resins from both reactors were mixed to give 12.785 g of resin Fmoc-Nle-c[Glu-Asn(Trt)-D-Phe- Arg(Pbf)-Trp-Lys]-NH-Rink linker-polystyrene, corresponding to a supposed maximum load ⁇ ing of 0.26 mmol/g, if complete reactions are assumed.
  • Step B for Example 1 2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle- c[Glu-Asn-D-Phe-Arg-Trp-Lys]-NH 2
  • Precipitation with ether The residue was treated with diethyl ether (20 ml) to give a solid pre- cipitate. The ether phase was removed after centrifugation. The solid residue was washed again with diethyl ether (20 ml). After centrifugation and removal of the ether phase, the solid residue was left overnight in order to remove remaining diethyl ether.
  • Examples of further compounds of the invention which may be obtained in a manner analo ⁇ gous to the compound of Example 1 are the compounds of Examples 2-21 , below.
  • the syn ⁇ thesis of the building block employed in the synthesis of the compounds of Examples 13, 19 and 21 is described below:
  • TAC:SPRD rats or Wistar rats from M&B Breeding and Research Centre A/S, Denmark are used for the experiments.
  • the rats have a body weight 200-250 g at the start of experiment.
  • the rats arrive at least 10-14 days before start of experiment with a body weight of 180-200 g.
  • Each dose of compound is tested in a group of 8 rats.
  • a vehicle group of 8 rats is included in each set of testing.
  • the animals When the animals arrive they are housed individually in a reversed light/dark phase (lights off 7:30 am, lights on 7:30 pm), meaning that lights are off during daytime and on during night ⁇ time.
  • rats Since rats normally initiate food intake when light is removed, and eat the major part of their daily food intake during the night, this set-up results in an alteration of the initiation time for food intake to 7:30 am, when lights are switched off.
  • the rats During the acclimatization period of 10-14 days, the rats have free access to food and water. During this period the animals are handled at least 3 times.
  • the rats are returned to their home cages, where they then have access to food and water.
  • the food consumption is recorded individu ⁇ ally every hour for 7 hours, and then after 24 h and sometimes 48 h.
  • the animals are euthanised.
  • the cAMP assays for MC3 and MC5 receptors are performed on cells (either HEK293 or BHK cells) stably expressing the MC3 and MC5 receptors, respectively.
  • the receptors are cloned from cDNA by PCR and inserted into the pcDNA 3 expression vector. Stable clones are selected using 1 mg/ml G418.
  • Cells at approx. 80-90% confluence are washed 3x with PBS, lifted from the plates with Versene and diluted in PBS. They are then centrifuged for 2 min at 1300 rpm, and the super ⁇ natant removed. The cells are washed twice with stimulation buffer, and then resuspended in stimulation buffer to a final concentration of 1 x10 6 or 2x10 6 cells/ml. 25 ⁇ l of cell suspension is added to the microtiter plates containing 25 ⁇ l of test compound or reference compound (all diluted in stimulation buffer). The plates are incubated for 30 minutes at room tempera ⁇ ture (RT) on a plate-shaker set to a low rate of shaking.
  • RT room tempera ⁇ ture
  • the reaction is stopped by adding 25 ⁇ l of acceptor beads with anti-cAMP, and 2 min later 50 ⁇ l of donor beads per well with bioti- nylated cAMP in a lysis buffer.
  • the plates are then sealed with plastic, shaken for 30 minutes and allowed to stand overnight, after which they are counted in an AlphaTM microplate reader.
  • EC 50 values are calculated by non-linear regression analysis of dose/response curves (6 points minimum) using the WindowsTM program GraphPadTM Prism (GraphPadTM Software, USA). All results are expressed in nM.
  • the MC3 receptors are stimulated with 3 nM ⁇ -MSH, and inhibited with increasing amounts of the potential an ⁇ tagonist.
  • the IC 50 value for the antagonist is defined as the concentration that inhibits MC3 stimulation by 50 %.
  • BHK cells expressing the MC4 receptor are stimulated with potential MC4 agonists, and the degree of stimulation of cAMP is measured using the Flash Plate® cAMP assay (NENTM Life Science Products, cat. No. SMP004).
  • the MC4 receptor-expressing BHK cells are produced by transfecting the cDNA encoding MC4 receptor into BHK570/KZ10-20-48, and selecting for stable clones expressing the MC4 receptor.
  • the MC4 receptor cDNA, as well as a CHO cell line expressing the MC4 receptor, may be purchased from EuroscreenTM.
  • the cells are grown in DMEM, 10% FCS, 1 mg/ml G418, 250 nM MTX and 1 % penicillin/streptomycin.
  • Detection Mix 11 ml Detection Buffer + 100 ⁇ l ( ⁇ 2 ⁇ Ci) cAMP [ 125 I] tracer).
  • the plates are then sealed with plastic, shaken for 30 minutes, and al ⁇ lowed to stand overnight (or for 2 hours) and then counted in the Topcounter (2 min/well).
  • the assay procedure is as described in the Flash Plate kit-protocol (Flash Plate® cAMP assay (NENTM Life Science Products, cat. No. SMP004)).
  • Flash Plate® cAMP assay Flash Plate® cAMP assay (NENTM Life Science Products, cat. No. SMP004)
  • the cAMP stan ⁇ dards are diluted in 0.1 % HSA and 0.005% TweenTM 20 and not in
  • EC 50 values are calculated by non-linear regression analysis of dose/response curves (6 points minimum) using the WindowsTM program GraphPadTM Prism (GraphPad Software, USA). All results are expressed in nM.
  • the MC1 receptor binding assay is performed on BHK cell membranes stably expressing the MC1 receptor.
  • the assay is performed in a total volume of 250 ⁇ l: 25 ⁇ l of 125 NDP- ⁇ -MSH (22 pM in final concentration), 25 ⁇ l of test compound/control and 200 ⁇ l of cell membrane (35 ⁇ g/ml).
  • Test compounds are dissolved in DMSO.
  • Radioactively labeled ligand, mem ⁇ branes and test compounds are diluted in buffer: 25 mM HEPES, pH 7.4, 0.1 mM CaCI 2 , 1 mM MgSO 4 , 1 mM EDTA, 0.1 % HSA and 0.005% TweenTM 20.
  • the samples are incubated at 3O 0 C for 90 min in Greiner microtiter plates, separated with GF/B filters that are pre-wetted for 60 min in 0.5% PEI, and washed 2-3 times with NaCI (0.9%) before separation of bound from unbound radiolabeled ligand by filtration. After filtration the filters are washed 10 times with ice-cold 0.9% NaCI. The filters are dried at 5O 0 C for 30 min, sealed, and 30 ⁇ l of Mi- croscint 0 (Packard, cat. No. 6013616) is added to each well. The plates are counted in a Topcounter (1 min/well).
  • the data are analysed by non-linear regression analysis of binding curves, using the Win ⁇ dowsTM program GraphPadTM Prism (GraphPad Software, USA).
  • the assay is performed in a total volume of 200 ⁇ l: 50 ⁇ l of cell suspension, 50 ⁇ l of 125 NDP- ⁇ -MSH ( ⁇ 79 pM in final concentration), 50 ⁇ l of test compound and 50 ⁇ l binding buffer (pH 7) mixed and incubated for 2 h at 25 0 C [binding buffer: 25 mM HEPES (pH 7.0), 1 mM CaCI 2 , 1 mM MgSO 4 , 1 mM EGTA, 0.02% Bacitracin and 0.2% BSA]. Test compounds are dissolved in H 2 O and diluted in binding buffer. Radiolabeled ligand and membranes are diluted in binding buffer.
  • the incubation is stopped by dilution with 5 ml ice-cold 0.9% NaCI, followed by rapid filtration through Whatman GF/C filters pre-treated for 1 hour with 0.5% polyethyleneimine.
  • the filters are washed with 3 x 5 ml ice-cold NaCI.
  • the radioactivity re ⁇ tained on the filters is counted using a Cobra Il auto gamma counter.
  • the data are analysed by non-linear regression analysis of binding curves, using the Win ⁇ dowsTM program GraphPadTM Prism (GraphPad Software, USA).
  • TAC:SPRD rats or Wistar rats from M&B Breeding and Research Centre A/S, Denmark are used. After at least one week of acclimatization, rats are placed individually in metabolic chambers (Oxymax system, Columbus Instruments, Columbus, Ohio, USA; systems cali ⁇ brated daily). During the measurements, animals have free access to water, but no food is provided to the chambers. Lightdark cycle is 12h:12h, with lights being switched on at 6:00. After the animals have spent approx. 2 hours in the chambers (i.e. when the baseline energy expenditure is reached), test compound or vehicle are administered (po, ip or sc), and re- cording is continued in order to establish the action time of the test compound.
  • Oxygen consumption (VO 2 ) is regarded as the major energy expenditure parameter of interest.

Abstract

The present invention relates to novel peptide compounds which are effective in modulating one or more melanocortin receptor types, to the use of the compounds in therapy, to methods of treatment comprising administration of the compounds to patients in need thereof, and to the use of the compounds in the manufacture of medicaments. The compounds of the invention are of particular interest in relation to the treatment of obesity as well as a variety of diseases or conditions associated with obesity.

Description

PEPTIDES FOR USE IN THE TREATMENT OF OBESITY
FIELD OF THE INVENTION
The present invention relates to novel peptide compounds which are ligands for one or more melanocortin receptors and which may exert prolonged activity, to the use of the compounds in therapy, to methods of treatment comprising administration of the compounds to patients, and to the use of the compounds in the manufacture of medicaments.
BACKGROUND OF THE INVENTION
Obesity is a well known risk factor for the development of many very common diseases such as atherosclerosis, hypertension, type 2 diabetes (non-insulin dependent diabetes mellitus (NIDDM)), dyslipidaemia, coronary heart disease, and osteoarthritis and various malignan¬ cies. It also causes considerable problems through reduced motility and decreased quality of life. The incidence of obesity and thereby also these diseases is increasing throughout the entire industrialised world. Only a few pharmacological treatments are available to date, namely Sibutramine (Abbot; acting via serotonergic and noradrenaline mechanisms) and OrI- istat (Roche Pharm; reducing fat uptake from the gut,). However, due to the important effect of obesity as a risk factor in serious (and even fatal) and common diseases, there is still a need for pharmaceutical compounds useful in the treatment of obesity.
The term obesity implies an excess of adipose tissue. In this context, obesity is best viewed as any degree of excess adiposity that imparts a health risk. The distinction between normal and obese individuals can only be approximated, but the health risk imparted by obesity is probably a continuum with increasing adiposity. However, in the context of the present inven¬ tion, individuals with a Body Mass Index (BMI = body weight in kilograms divided by the square of the height in meters) above 25 are to be regarded as obese.
Even mild obesity increases the risk for premature death, diabetes, hypertension, atheroscle- rosis, gallbladder disease and certain types of cancer. In the industrialized western world the prevalence of obesity has increased significantly in the past few decades. Because of the high prevalence of obesity and its health consequences, its treatment should be a high public health priority. When energy intake exceeds energy expenditure, the excess calories are stored in adipose tissue, and if this net positive balance is prolonged, obesity results, i.e. there are two compo¬ nents to weight balance, and an abnormality on either side (intake or expenditure) can lead to obesity.
Proopiomelanocortin (POMC) is the precursor for β-endorphin and melanocortin peptides, including melanocyte stimulating hormone (α-MSH) and adrenocorticotropin (ACTH). POMC is expressed in several peripheral and central tissues including melanocytes, the pituitary, and neurons of the hypothalamus. The POMC precursor is processed differently in different tissues, resulting in the expression of different melanocortin peptides depending on the site of expression. In the anterior lobe of the pituitary, mainly ACTH is produced whereas in the intermediate lobe and the hypothalamic neurons the major peptides are α-MSH, β-MSH, de- sacetyl-α-MSH and β-endorphin. Several of the melanocortin peptides, including ACTH and α-MSH, have been demonstrated to have appetite-suppressing activity when administered to rats by intracerebroventricular injection [Vergoni et al, European Journal of Pharmacology 179, 347-355 (1990)]. An appetite-suppressing effect is also obtained with the artificial cyclic α-MSH analogue, MT-II.
A family of five melanocortin receptor subtypes has been identified (melanocortin receptor 1 - 5, also called MC1 , MC2, MC3, MC4 and MC5). The MC1 , MC2 and MC5 are mainly ex¬ pressed in peripheral tissues, whereas MC3 and MC4 are mainly centrally expressed; MC3 are, however, also expressed in several peripheral tissues. In addition to being involved in energy homeostasis, MC3 receptors have also been suggested to be involved in several in- flammatory diseases. An MC3 agonist could have a positive effect on such diseases, e.g. gouty arthritis. MC5 are mainly peripherally expressed, and have been suggested to be in¬ volved in exocrine secretion and in inflammation. MC4 have been shown to be involved in the regulation of body weight and feeding behaviour, as MC4 knock-out mice develop obesity [Huzar et al., CeN 88, 131 -141 (1997)]. In addition, the MC4 receptor has been shown to be involved in the regulation of energy expenditure [Fekete et al., Journal of Neuroscience 20, 1550-1558 (2000)]. Furthermore, studies of either ectopic central expression of agouti protein (MC1 , MC3 and MC4 antagonist) or over-expression of an endogenously occurring MC3 and MC4 antagonist (agouti gene related protein, AGRP) in mouse brain demonstrated that the over-expression of these two antagonists led to the development of obesity [Kleibig et al., PNAS 92, 4728-4732 (1995)]. Moreover, icv injection of a C-terminal fragment of AGRP in¬ creases feeding and antagonizes the inhibitory effect of α-MSH on food intake.
In humans, several cases of families with obesity which is presumably due to frame shift mu- tations in MC4 have been described [see, e.g., Yeo et al., Nature Genetics 20, 11 1 -112 (1998); Vaisse et al., Nature Genetics 20, 113-114 (1998)].
In conclusion, a MC4 agonist could serve as an anorectic drug or energy expenditure regu¬ lating drug and be useful in the treatment of obesity or obesity-related diseases, as well as in the treatment of other diseases, disorders or conditions which may be ameliorated by activa¬ tion of MC4 .
MC4 antagonists may be useful for treatment of cachexia or anorexia, and for treatment of waisting in frail elderly patients. Furthermore, MC4 antagonists may be used for treatment of chronic pain, neuropathy and neurogenic inflammation.
A large number of patent applications disclose various classes of non-peptidic small mole¬ cules as melanocortin receptor modulators; examples hereof are WO 03/009850, WO 03/007949 and WO 02/081443.
The use of peptides as melanocortin receptor modulators is disclosed in a number of patent documents, e.g. WO 03/006620, US 5731 ,408 and WO 98/27113. Hadley [Pigment Cell Res., 4, 180-185, (1991 )] reports a prolonged effect of specific melanotropic peptides conju¬ gated to fatty acids, the prolongation being effected by a transformation of the modulators from being reversibly acting to being irreversibly acting caused by the conjugated fatty acids.
SUMMARY OF THE INVENTION
The present inventors have surprisingly found that specific peptide conjugates have a high modulating effect on one or more melanocortin receptors, i.e. the MC1 , MC2, MC3, MC4 or MC5. Accordingly, the invention relates to compounds of formula Ia, Ib or Ic:
R1-S-Z1-Z2-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ia] R1-S-Z2-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ib] R1-S-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ic] wherein R1 represents a straight-chain, branched and/or cyclic Ci4-2oalkanoyl, Ci4-2oalkenoyl or Ci4-2oalkynoyl which may optionally be substituted with one or more substituents selected from halogen, hydroxy and aryl, or R1 represents C9-i7-C(O)-NH-S(O)2-(CH2)3-C(O)-; S represents a glycolether-based structure according to one of the formulas Na-IIe
Figure imgf000005_0001
Z1 and Z2 independently represent GIy, Ser, Thr, GIn, Asn, His, homoArg, Arg, Lys, Dab, Dap or Orn;
Z3 represents Ala, VaI, Leu, He, Met or NIe; X1 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap;
X2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, He, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met, Met(O), Met(O2), GIn, Gln(alkyl), Gln(aryl), Asn, Asn(alkyl), Asn(aryl), Ser, Thr, Cys, Pro, Hyp, Tic, 2-PyAIa, 3-PyAIa, 4-PyAIa, (2-thienyl)alanine, 3-(thienyl)alanine, (4-thiazolyl)Ala, (2-furyl)alanine, (3-furyl)alanine or Phe, wherein the phenyl moiety of said Phe is optionally substituted with a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano;
X3 represents D-Phe, wherein the phenyl moiety in D-Phe may optionally be substituted with one or more substituents selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoro¬ methyl and cyano; X4 represents Trp, 2-NaI, a (3-benzo[b]thienyl)alanine residue or a (S)-2,3,4,9-tetrahydro-1 H- β-carboline-3-carboxylic acid residue; X5 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap; wherein X1 and X5 are joined, rendering the compound of formula Ia, Ib or Ic cyclic, either via a disulfide bridge deriving from X1 and X5 both independently being Cys, homoCys or Pen, or via an amide bond formed between a carboxylic acid in the side-chain of X1 and an amino group in the side chain of X5, or between a carboxylic acid in the side-chain of X5 and an amino group in the side-chain of X1 ; each R' independently represents hydrogen or d-6alkyl, which may optionally be substituted with one or more amino or hydroxy; with the proviso that the compound of formula Ia, Ib or Ic is not 2-[2-(octadecanoylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2,
3-(2-{2-[2-(2-(hexadecanoylamino)ethoxy)ethoxy]ethoxy}ethoxy)propionyl-Nle-c[Glu-Hyp-D- Phe-Arg-Trp-Lys]-NH2,
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]- NH2, 2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Arg-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]- NH2 or
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Gln-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]- NH2; and pharmaceutically acceptable salts, prodrugs and solvates thereof.
The invention further relates to the use of compounds of the invention in therapy, to pharma¬ ceutical compositions comprising compounds of the invention, to methods of treatment com¬ prising administration of compounds of the invention to patients in need thereof, and to the use of compounds of the invention in the manufacture of medicaments.
DEFINITIONS
The use of a prefix of the type "Cx-y" preceding the name of a radical, such as in Cx_yalkyl (e.g. Ci4-20alkyl) is intended to indicate a radical of the designated type having from x to y carbon atoms. Thus, straight-chain, branched and/or cyclic Ci4-20alkanoyl, Ci4-20alkenoyl or Ci4-20alkynoyl groups as they occur as substituents R1 in compounds of the present invention embrace straight-chain, branched and/or cyclic alkanoyl, alkenoyl or alkynoyl groups having 14, 15, 16, 17, 18, 19 or 20 carbon atoms (i.e. Ci4, C15, C16, C17, C18, C19 or C20). The term "alkyl" as used herein refers to a straight-chain, branched and/or cyclic, saturated monovalent hydrocarbon radical. Examples hereof include methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The term "alkenyl" as used herein refers to a straight-chain, branched and/or cyclic, mono¬ valent hydrocarbon radical comprising at least one carbon-carbon double bond. Examples hereof include ethenyl, prop-1 -en-1 -yl, prop-2-en-1 -yl and prop-2-en-2-yl.
The term "alkynyl" as used herein refers to a straight-chain, branched and/or cyclic, monova- lent hydrocarbon radical comprising at least one carbon-carbon triple bond, and it may op¬ tionally also comprise one or more carbon-carbon double bonds. Examples hereof include ethynyl, prop-1 -yn-1yl and prop-2-yn-1 -yl.
The term "alkanoyl" as used herein is intended to indicate a radical of the formula -C(O)-R', wherein R' is alkyl as indicated above.
The term "alkenoyl" as used herein is intended to indicate a radical of the formula -C(O)-R", wherein R" is alkenyl as indicated above.
The term "alkynoyl" as used herein is intended to indicate a radical of the formula -C(O)-R'", wherein R'" is alkynyl as indicated above.
The term "alkoxy" as used herein is intended to indicate a radical of the formula -OR', wherein R' is alkyl as indicated above. Examples hereof include methoxy and ethoxy.
In the present context, the term "aryl" is intended to indicate a carbocyclic aromatic ring radical or a fused aromatic ring system radical wherein at least one of the rings is aromatic. Typical aryl groups include phenyl, biphenylyl, naphthyl, and the like.
The term "halogen" is intended to indicate members of the 7th main group of the periodic ta¬ ble of the elements, which includes fluorine, chlorine, bromine and iodine (corresponding to fluoro, chloro, bromo and iodo substituents, respectively).
When two amino acids are said to be bridged, it is intended to indicate that functional groups in the side chains of the two respective amino acids have reacted to form a covalent bond. In the present context, the term "agonist" is intended to indicate a substance (ligand) that ac¬ tivates the receptor type in question.
In the present context, the term "antagonist" is intended to indicate a substance (ligand) that blocks, neutralizes or counteracts the effect of an agonist.
More specifically, receptor ligands may be classified as follows:
Receptor agonists, which activate the receptor; partial agonists also activate the receptor, but with lower efficacy than full agonists. A partial agonist will behave as a receptor partial an¬ tagonist, partially inhibiting the effect of a full agonist.
Receptor neutral antagonists, which block the action of an agonist, but do not affect the re- ceptor-constitutive activity.
Receptor inverse agonists, which block the action of an agonist and at the same time attenu¬ ate the receptor-constitutive activity. A full inverse agonist will attenuate the receptor- constitutive activity completely; a partial inverse agonist will attenuate the receptor- constitutive activity to a lesser extent.
As used herein the term "antagonist" includes neutral antagonists and partial antagonists, as well as inverse agonists. The term "agonist" includes full agonists as well as partial agonists.
In the present context, the term "pharmaceutically acceptable salt" is intended to indicate a salt which is not harmful to the patient. Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Represen¬ tative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric and nitric acids, and the like. Representative examples of suitable or¬ ganic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cin- namic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethylene- salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further ex¬ amples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. (1977) 66, 2, which is incorporated herein by reference. Examples of relevant metal salts include lithium, sodium, potassium and magnesium salts, and the like. Examples of alkylated ammonium salts include methylammo- nium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium and tetramethylammonium salts, and the like.
As used herein, the term "therapeutically effective amount" of a compound refers to an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and/or its complications. An amount adequate to accomplish this is defined as a "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury, as well as on the weight and general state of the subject. It will be understood that determination of an appropriate dosage may be achieved using rou- tine experimentation, by constructing a matrix of values and testing different points in the ma¬ trix, all of which is within the level of ordinary skill of a trained physician or veterinarian.
The terms "treatment", "treating" and other variants thereof as used herein refer to the man¬ agement and care of a patient for the purpose of combating a condition, such as a disease or a disorder. The terms are intended to include the full spectrum of treatments for a given con¬ dition from which the patient is suffering, such as administration of the active compound(s) in question to alleviate symptoms or complications thereof, to delay the progression of the dis¬ ease, disorder or condition, to cure or eliminate the disease, disorder or condition, and/or to prevent the condition, in that prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder, and includes the administration of the active compound(s) in question to prevent the onset of symptoms or complications. The patient to be treated is preferably a mammal, in particular a human being, but treatment of other animals, such as dogs, cats, cows, horses, sheep, goats or pigs, is within the scope of the invention.
As used herein, the term "solvate" refers to a complex of defined stoichiometry formed be¬ tween a solute (in casu, a compound according to the present invention) and a solvent. Sol¬ vents may include, by way of example, water, ethanol, or acetic acid. The amino acid abbreviations used in the present context have the following meanings:
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Amino acid abbreviations beginning with D- followed by a three letter code, such as D-Ser, D-His and so on, refer to the D-enantiomer of the corresponding amino acid, for example D- serine, D-histidine and so on. In the absence of the letter D preceding a three letter code or amino acid name, as in for example Ser (serine), His (histidine) and so on, it is to be under¬ stood that reference is made to the L-enantiomer of the amino acid in question.
DESCRIPTION OF THE INVENTION
As already indicated above, one aspect of the invention relates to compounds of formula Ia, Ib or Ic:
R1-S-Z1-Z2-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ia] R1-S-Z2-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ib] R1-S-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ic] wherein R1 represents a straight-chain, branched and/or cyclic Ci4-2oalkanoyl, Ci4-20alkenoyl or Ci4-20alkynoyl which may optionally be substituted with one or more substituents selected from halogen, hydroxyl and aryl, or R1 represents C9.17-C(O)-NH-S(O)2-(CH2)3-C(O)-; S represents a glycolether-based structure according to one of the formulas Na-IIe
lie
Figure imgf000015_0002
Z1 and Z2 independently represent GIy, Ser, Thr, GIn, Asn, His, homoArg, Arg, Lys, Dab, Dap or Orn; Z3 represents Ala, VaI, Leu, He, Met or NIe;
X1 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap;
X2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, He, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met,
Met(O), Met(O2), GIn, Gln(alkyl), Gln(aryl), Asn, Asn(alkyl), Asn(aryl), Ser, Thr, Cys, Pro,
Hyp, Tic, 2-PyAIa, 3-PyAIa, 4-PyAIa, (2-thienyl)alanine, 3-(thienyl)alanine, (4-thiazolyl)Ala, (2-furyl)alanine, (3-furyl)alanine or Phe, wherein the phenyl moiety of said Phe is optionally substituted with a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano;
X3 represents D-Phe, wherein the phenyl moiety in D-Phe may optionally be substituted with one or more substituents selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoro- methyl and cyano;
X4 represents Trp, 2-NaI, a (3-benzo[b]thienyl)alanine residue or a (S)-2,3,4,9-tetrahydro-1 H- β-carboline-3-carboxylic acid residue;
X5 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap; wherein X1 and X5 are joined, rendering the compound of formula Ia, Ib or Ic cyclic, either via a disulfide bridge deriving from X1 and X5 both independently being Cys, homoCys or Pen, or via an amide bond formed between a carboxylic acid in the side-chain of X1 and an amino group in the side chain of X5, or between a carboxylic acid in the side-chain of X5 and an amino group in the side-chain of X1 ; each R' independently represents hydrogen or d-6alkyl, which may optionally be substituted with one or more amino or hydroxy; with the proviso that the compound of formula Ia, Ib or Ic is not
2-[2-(octadecanoylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2,
3-(2-{2-[2-(2-(hexadecanoylamino)ethoxy)ethoxy]ethoxy}ethoxy)propionyl-Nle-c[Glu-Hyp-D-
Phe-Arg-Trp-Lys]-NH2,
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]- NH2,
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Arg-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-
NH2 or
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Gln-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-
NH2; and pharmaceutically acceptable salts, prodrugs and solvates thereof.
In certain embodiments of compounds of the invention, R1 is Ci4-i8-alkanoyl.
In further embodiments of compounds of the invention, R1 is 4-(Ci4-i8alkanoyl- sulfamoyl)butanoyl, such as 4-(hexadecanoylsulfamoyl)butanoyl.
In other embodiments of compounds of the invention, S has a structure selected among for¬ mulas Na, Nd and lie.
In additional embodiments of compounds of the invention, Z3 is NIe.
In other embodiments of compounds of the invention, Z2 is Ser, Thr, Dab or Dap, notably Ser or Dab.
In further embodiments of compounds of the invention, Z1 is His, GIn, Arg, homoArg, Lys, Orn, Dab or Dap, such as His, GIn or Arg.
Further interesting embodiments of compounds of the invention are those wherein R1 is Ci4-i8alkanoyl or 4-(hexadecanoylsulfamoyl)butanoyl; Z\ if present, is His, Arg, homoArg, Lys, GIn, Asn, Orn, Dab or Dap; Z2, if present, is Ser, Thr, Dab or Dap; Z3 is NIe;
X1 is GIu, Asp or Cys;
X2 is Ser, Hyp, Cit, Dap, Asn, GIn or (4-thiazolyl)Ala; X3 is D-Phe; X4 is Trp;
X5 is Lys when X1 is GIu or Asp, or X5 is Cys when X1 is Cys; and N(R)2 is NH2.
Among such embodiments of the latter type, particularly interesting compounds include:
compounds wherein Z1, if present, is His, Arg or GIn;
compounds wherein Z2, if present, is Ser, Thr or Dab;
compounds wherein X2 is Ser, Hyp, Asn, GIn or Dap, notably compounds wherein X2 is Hyp, compounds wherein X2 is Asn, compounds wherein X2 is GIn, and compounds wherein X2 is Dap; and
compounds wherein S has a structure selected among formulas Na, Nd and lie.
Specific examples of interesting compounds according to the present invention are the fol¬ lowing:
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-Lys]-
NH2 (SEQ ID NO: 1 ),
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Gln-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-Lys]-
NH2 (SEQ ID NO: 2),
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Asn-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 3),
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Hyp-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 4),
3-[2-(2-{2-[2-(hexadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propanoyl-His-Dab-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 5), 2-[2-(2-{2-[2-(hexadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Dap-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 6),
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Arg-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-l_ys]-
NH2 (SEQ ID NO: 7), 2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Asp-Hyp-D-Phe-Arg-Trp-Lys]-
NH2 (SEQ ID NO: 8),
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Dab-Nle- c[Glu-Asn-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 9),
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Ser-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 10),
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Gln-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 11 ),
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Cit-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 12), 2-[2-(4-(hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-Dap-D-Phe-
Arg-Trp-Lys]-NH2 (SEQ ID NO: 13),
{2-[2-(2-(hexadecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}methoxyacetyl-Ser-Nle-c[Asp-
Asn-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 14),
{2-[2-(2-(tetradecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}methoxyacetyl-Ser-Nle-c[Asp- Hyp-D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 15),
4-{2-[2-(2-(octadecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}butanoyl-Dab-Nle-c[Asp-Hyp-
D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 16),
4-{2-[2-(2-(tetradecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}butanoyl-Thr-Nle-c[Glu-Gln-D-
Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 17), 3-(2-{2-[2-(2-(hexadecanoylamino)ethoxy)ethoxy]ethoxy}ethoxy)propionyl-Nle-c[Glu-Dap-D-
Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 18),
2-[2-(4-(hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp-
D-Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 19),
2-[2-(4-(tridecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp-D- Phe-Arg-Trp-Lys]-NH2 (SEQ ID NO: 20), and
2-[2-(4-(hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Cys-Hyp-
D-Phe-Arg-Trp-Cys]-NH2 (SEQ ID NO: 21 ).
The present invention also encompasses combinations of two or more embodiments of com- pounds of the invention as outlined above. In one aspect of the present invention, the compound of the invention is an agonist of a melanocortin receptor, notably an agonist of MC4. In another aspect of the invention, the compound is a selective agonist of MC4. In this context, selectivity is to be understood in re- lation to the activity of the compound with respect to MC1 , MC3 and/or MC5. If a compound is a significantly more potent as a MC4 agonist than as a MC1 , MC3 and/or MC5 agonist, it is deemed to be a selective MC4 agonist. The agonistic potency of a compound with respect to MC1 and MC4 may be determined by comparing an MC1 binding assay as described below under "Assay IV" (MC1 ) with a functional MC4 assay as described below under "Assay III" (MC4). If a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times more potent with respect to MC4 than with respect to MC1 , it is deemed to be a selective MC4 agonist with respect to MC1 . The agonistic potency of a compound with re¬ spect to MC3, MC4 and MC5 may be determined in functional assays as described in "Assay II" (MC 3 and MC5) and "Assay III" (MC4). If a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times more potent with respect to MC4 than with re¬ spect to MC3, it is deemed to be a selective MC4 agonist with respect to MC3. If a com¬ pound is more than 10 times, such as more than 50 times, e.g. more than 100 times more potent with respect to MC4 than with respect to MC5, it is deemed to be a selective MC4 agonist with respect to MC5. In a particular aspect, the compound of the present invention is a selective MC4 agonist with respect to MC1 , with respect to MC3, with respect to MC5, with respect to MC1 and MC3, with respect to MC1 and MC5, with respect to MC3 and MC5 or with respect to MC1 , MC3 and MC5.
In another aspect of the present invention, the compound of the invention is a selective MC4 agonist and a MC3 antagonist. In this context, a compound is deemed to be a selective MC4 agonist and a MC3 antagonist if it is a selective MC4 agonist with respect to MC1 and MC5 as discussed above, and it antagonizes MC3 as determined as described in "Assay II". In the latter assay, a compound exhibiting an IC50 value of less than 100 nM, such as less than 10 nM, e.g. less than 5 nM, such as less than 1 nM, is deemed to be a MC3 antagonist.
In a further aspect of the present invention, the compound of the present invention is both a selective MC3 agonist and a selective MC4 agonist. In this context, a compound is deemed to be a selective MC3 and MC4 agonist if it is significantly more potent as an agonist towards MC3 and MC4 than as an agonist toward MC1 and MC5. The selectivity of a compound with respect to MC1 and MC3 may be determined by comparing the potency determined for MC1 as described in "Assay IV" with the potency for MC3 determined as described in "Assay II". If a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times more potent with respect to MC3 than with respect to MC1 , it is deemed to be a selective MC3 agonist with respect to MC1 . The selectivity of a compound with respect to MC3 and MC5 may be determined by comparing the potency determined as described in "Assay II". If a compound is more than 10 times, such as more the 50 times, e.g. more than 100 times more potent with respect to MC3 than with respect to MC5, it is deemed to be a selective MC3 agonist with respect to MC5. The MC4 selectivity of a compound with respect to MC3 and MC5 is determined as discussed above.
Compounds of the present invention may exert a protracted effect, i.e. the period of time in which they exert a biological activity may be prolonged. A protracting effect may be evalu¬ ated in a slightly modified "Assay I" in a comparison between a compound of the present in¬ vention and the corresponding compound wherein R1 is hydrogen and S is a bond. The ex- periment is allowed to continue for a period of time, T, until the rats have eaten as much as they did prior to the experiment. T values for compounds of the present invention and the corresponding compounds wherein R1 is hydrogen and S is a bond are measured, and the difference ΔT is calculated. Compounds of the present invention giving rise to ΔT above 3 hours, such as above 7 hours, such as above 12 hours, such as above 24 hours, such as above 48 hours, such as above 72 hours, are deemed to exert a protracted effect.
Compounds of the present invention modulate melanocortin receptors, and they are there¬ fore believed to be particularly suited for the treatment of diseases or states which can be treated by a modulation of melanocortin receptor activity. In particular, compounds of the present invention are believed to be suited for the treatment of diseases or states via activa¬ tion of MC4.
In one aspect, the present invention relates to a method of agonizing or activating MC4 in a subject, the method comprising administering to the subject an effective amount of a com- pound of the present invention.
In another aspect, the invention provides a method of delaying the progression from impaired glucose tolerance (IGT) to type 2 diabetes, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention. In a further aspect, the invention provides a method of delaying the progression from type 2 diabetes to insulin-requiring diabetes, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
In an additional aspect, the invention relates to a method of treating obesity or preventing overweight, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
In a still further aspect, the present invention provides a method of regulating appetite, the method comprising administering to a patient in need thereof an effective amount of a com¬ pound of the present invention.
Another aspect of the invention relates to a method of inducing satiety, the method compris¬ ing administering to a patient in need thereof an effective amount of a compound of the pre- sent invention.
A further aspect of the invention relates to a method of preventing weight regain after suc¬ cessfully having lost weight, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
Yet another aspect of the invention relates to a method of increasing energy expenditure, the method comprising administering to a patient in need thereof an effective amount of a com¬ pound of the present invention.
Still further aspects of the invention include the following:
a method of treating a disease or state related to overweight or obesity, the method compris¬ ing administering to a patient in need thereof an effective amount of a compound of the pre¬ sent invention;
a method of treating bulimia, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention;
a method of treating binge-eating, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention; a method of treating a disease or state selected from atherosclerosis, hypertension, diabe¬ tes, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of prema- ture death, the method comprising administering to a patient in need thereof an effective amount of a compound of the present invention.
In particular, compounds of the present invention may be suited for the treatment of diseases in obese or overweight patients. Accordingly, the present invention also provides a method of treating, in an obese patient, a disease or state selected from type 2 diabetes, impaired glu¬ cose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of premature death in obese patients, the method comprising administering to an obese patient in need thereof an effective amount of a compound of the present invention.
Moreover, administration of compounds of the present invention may be advantageous in the treatment of patients, notably obese or overweight patients, who have undergone, or are to undergo, gastric banding and/or gastric surgery.
In addition, MC4 agonists could have a positive effect on insulin sensitivity, on drug abuse by modulating the reward system and on hemorrhagic shock. Furthermore, MC3 and MC4 ago¬ nists have antipyretic effects, and both have been suggested to be involved in peripheral nerve regeneration. MC4 agonists are also known to reduce stress response.
Appropriate routes of administration of compounds of the invention to patients in the context of the invention include parenteral routes such as nasal, pulmonary or sublingual administra¬ tion routes, all of which are familiar to persons of skill in the art of drug administration.
In all of the therapeutic methods disclosed above, a compound of the present invention may be administered alone or in combination with one or more (i.e. one or two or three.... etc.) additional compounds of the present invention. Moreover, a compound of the invention, or a combination of two or more (i.e. two or three or four.... etc.) compounds of the invention, may be administered in combination with one or more other therapeutically active agents or com¬ pounds (i.e. agents or compounds which are not within the scope of the present invention), either sequentially or concomitantly. A typical dosage of a compound of the invention when employed in a method according to the present invention is in the range of from about 0.001 to about 100 mg/kg body weight per day, e.g. from about 0.01 to about 50 mg/kg body weight per day, such as from about 0.05 to about 10 mg/kg body weight per day, administered in one or more doses, such as from 1 to 3 doses. The exact dosage will depend upon the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated, any concomitant diseases to be treated and other factors evident to those skilled in the art.
Compounds of the invention may conveniently be formulated in unit dosage form using tech¬ niques well known to those skilled in the art. A typical unit dosage form intended for oral ad¬ ministration one or more times per day, such as from one to three times per day, may suita¬ bly contain from 0.05 to about 1000 mg, e.g. from about 0.1 to about 500 mg, such as from about 0.5 mg to about 200 mg of a compound of the invention.
In a further aspect, the invention relates to a pharmaceutical composition comprising a com¬ pound of the present invention, optionally in combination with one or more additional thera¬ peutically active compounds or substances, together with one or more pharmaceutically ac- ceptable carriers or excipients. The composition may suitably be in unit dosage form com¬ prising from about 0.05 mg to about 1000 mg, such as from about 0.1 mg to about 500 mg, e.g. from about 0.5 mg to about 200 mg, of a compound of the present invention.
The present invention also relates to the use of a compound of the present invention, option- ally in combination with one or more additional therapeutically active compounds or sub¬ stances, in the manufacture of a medicament for the treatment of a disease or condition se¬ lected from overweight or obesity, bulimia, binge-eating, atherosclerosis, hypertension, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallblad¬ der disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of premature death.
The invention also relates to the use of a compound of the present invention, optionally in combination with one or more additional therapeutically active compounds or substances, in the manufacture of a medicament effective in: delaying the progression from IGT to type 2 diabetes; delaying the progression from type 2 diabetes to insulin-requiring diabetes; regulat- ing appetite; inducing satiety; preventing weight regain after successfully having lost weight; or increasing energy expenditure.
As described above, compounds of the present invention may be administered or applied in combination with one or more additional therapeutically active compounds or substances. Suitable additional compounds or substances may be selected, for example, from antidia¬ betic agents, antihyperlipidemic agents, antiobesity agents, antihypertensive agents and agents for the treatment of complications resulting from, or associated with, diabetes.
Suitable antidiabetic agents include: insulin; derivatives or analogues of insulin, including de¬ rivatives or analogues exhibiting a profile of protracted or prolonged activity, such as those disclosed in WO 95/07931 , WO 97/31022 and WO 2005/012347 (Novo Nordisk A/S), the contents of all of which are incorporated herein by reference; derivatives of GLP-1 (glucagon like peptide-1 ), such as those disclosed in WO 98/08871 (Novo Nordisk A/S), the contents of which are incorporated herein by reference; derivatives of GLP-1 analogues, such as those disclosed in US 6,458,924 (Knudsen et al.), the contents of which are incorporated herein by reference; and orally active hypoglycemic agents.
Suitable orally active hypoglycemic agents include: imidazolines; sulfonylureas; biguanides; meglitinides; oxadiazolidinediones; thiazolidinediones; insulin sensitizers; α-glucosidase in¬ hibitors; agents acting on the ATP-dependent potassium channel of the pancreatic β-cells, e.g. potassium channel openers such as those disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (Novo Nordisk A/S), the contents of which are incorporated herein by ref¬ erence; potassium channel openers such as ormitiglinide; potassium channel blockers such as nateglinide or BTS-67582; glucagon antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), the contents of which are incorporated herein by reference; GLP-1 agonists such as those disclosed in WO 00/42026 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), the contents of which are incorporated herein by reference; DPP-IV (dipeptidyl peptidase-IV) inhibitors; PTPase (pro- tein tyrosine phosphatase) inhibitors; glucokinase activators, such as those described in WO 2004/002481 (Novo Nordisk), the contents of which are incorporated herein by reference, and in WO 02/08209 (Hoffmann La Roche); inhibitors of hepatic enzymes involved in stimu¬ lation of gluconeogenesis and/or glycogenosis; glucose uptake modulators; GSK-3 (glyco¬ gen synthase kinase-3) inhibitors; compounds modifying lipid metabolism, such as antihyper- lipidemic agents and antilipidemic agents; compounds lowering food intake; as well as PPAR (peroxisome proliferator-activated receptor) agonists and RXR (retinoid X receptor) agonists such as ALRT-268, LG-1268 or LG-1069.
Other examples of suitable additional therapeutically active substances include insulin or in- sulin analogues; sulfonylureas, e.g. tolbutamide, chlorpropamide, tolazamide, glibenclamide, glipizide, glimepiride, glicazide or glyburide; biguanides, e.g. metformin; and meglitinides, e.g. repaglinide or senaglinide/nateglinide.
Further examples of suitable additional therapeutically active substances include thiazolidin- edione insulin sensitizers, e.g. troglitazone, ciglitazone, pioglitazone, rosiglitazone, isaglita- zone, darglitazone , englitazone, CS-01 1 /CI-1037 or T 174, or the compounds disclosed in WO 97/41097 (DRF-2344), WO 97/41 119, WO 97/41120, WO 00/41121 and WO 98/45292 (Dr. Reddy's Research Foundation), the contents of all of which are incorporated herein by reference.
Additional examples of suitable additional therapeutically active substances include insulin sensitizers, e.g. Gl 262570, YM-440, MCC-555, JTT-501 , AR-H039242, KRP-297, GW- 409544, CRE-16336, AR-H049020, LY510929, MBX-102, CLX-0940, GW-501516 and the compounds disclosed in WO 99/19313 (NN622/DRF-2725), WO 00/50414, WO 00/63191 , WO 00/63192 and WO 00/63193 (Dr. Reddy's Research Foundation), and in WO 00/23425, WO 00/23415, WO 00/23451 , WO 00/23445, WO 00/23417, WO 00/23416, WO 00/63153, WO 00/63196, WO 00/63209, WO 00/63190 and WO 00/63189 (Novo Nordisk A/S), the con¬ tents of all of which are incorporated herein by reference.
Still further examples of suitable additional therapeutically active substances include:
α-glucosidase inhibitors, e.g. voglibose, emiglitate, miglitol or acarbose;
glycogen phosphorylase inhibitors, e.g. the compounds described in WO 97/09040 (Novo Nordisk A/S);
glucokinase activators;
agents acting on the ATP-dependent potassium channel of the pancreatic β-cells, e.g. tolbu- tamide, glibenclamide, glipizide, glicazide, BTS-67582 or repaglinide; Other suitable additional therapeutically active substances include antihyperlipidemic agents and antilipidemic agents, e.g. cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
Further agents which are suitable as additional therapeutically active substances include an- tiobesity agents and appetite-regulating agents. Such substances may be selected from the group consisting of CART (cocaine amphetamine regulated transcript) agonists, NPY (neu¬ ropeptide Y) antagonists, Y2 and Y4 receptor agonists, MC3 (melanocortin 3) agonists, MC3 (melanocortin 3) antagonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotro¬ pin releasing factor binding protein) antagonists, urocortin agonists, β3 adrenergic agonists such as CL-316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140, MC1 (melano¬ cortin 1 ) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (chole- cystokinin) agonists, serotonin reuptake inhibitors (e.g. fluoxetine, seroxat or citalopram), se¬ rotonin and norepinephrine reuptake inhibitors, 5HT (serotonin) agonists, bombesin agonists, galanin antagonists, growth hormone, growth factors such as prolactin or placental lactogen, growth hormone releasing compounds (growth hormone secretagogues), ghrelin antago¬ nists, TRH (thyrotropin releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, chemical uncouplers, leptin agonists, DA (dopamine) agonists (bromocriptin, doprexin), lipase/amylase inhibitors, PPAR modulators, RXR modulators, TR β agonists, adrenergic CNS stimulating agents, AGRP (agouti-related protein) inhibitors, histamine H3 receptor antagonists such as those disclosed in WO 00/42023, WO 00/63208 and WO 00/64884, the contents of all of which are incorporated herein by reference, exendin-4, GLP- 1 agonists and ciliary neurotrophic factor.
Further suitable antiobesity agents are bupropion (antidepressant), topiramate (anticonvul¬ sant), ecopipam (dopamine D1/D5 antagonist), naltrexone (opioid antagonist), and peptide YY3-36 (Batterham et al, Nature 418, 650-654 (2002)).
An embodiment of a suitable antiobesity agent for use in a method of the invention as an ad¬ ditional therapeutically active substance in combination with a compound of the invention is leptin.
A further embodiment of a suitable antiobesity agent is peptide YY3-36. Additional embodiments of suitable antiobesity agents are serotonin and norepinephrine re¬ uptake inhibitors, e.g. sibutramine.
Other embodiments of suitable antiobesity agents are lipase inhibitors, e.g. orlistat.
Still further embodiments of suitable antiobesity agents are adrenergic CNS stimulating agents, e.g. dexamphetamine, amphetamine, phentermine, mazindol, phendimetrazine, di- ethylpropion, fenfluramine or dexfenfluramine.
Other examples of suitable additional therapeutically active compounds include antihyper¬ tensive agents. Examples of antihypertensive agents are β-blockers such as alprenolol, at¬ enolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, 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.
In certain embodiments of the uses and methods of the present invention, the compound of the present invention may be administered or applied in combination with more than one of the above-mentioned, suitable additional therapeutically active compounds or substances, e.g. in combination with: metformin and a sulfonylurea such as glyburide; a sulfonylurea and acarbose; nateglinide and metformin; acarbose and metformin; a sulfonylurea, metformin and troglitazone; insulin and a sulfonylurea; insulin and metformin; insulin, metformin and a sulfonylurea; insulin and troglitazone; insulin and lovastatin; etc.
PHARMACEUTICAL COMPOSITIONS
As already mentioned, one aspect of the present invention provides a pharmaceutical com¬ position (formulation) comprising a compound of the present invention. Appropriate embodi¬ ments of such formulations will often contain a compound of the invention in a concentration of from 10'3 mg/ml to 200 mg/ml, such as, e.g., from 10'1 mg/ml to 100 mg/ml. The pH in such a formulation of the invention will typically be in the range of 2.0 to 10.0. The formulation may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabi¬ lizers) and/or surfactant(s). In one embodiment of the invention the pharmaceutical formula¬ tion is an aqueous formulation, i.e. a formulation comprising water, and the term "aqueous formulation" in the present context may normally be taken to indicate a formulation compris¬ ing at least 50 % by weight (w/w) of water. Such a formulation is typically a solution or a sus¬ pension. An aqueous formulation of the invention in the form of an aqueous solution will normally comprise at least 50 % (w/w) of water. Likewise, an aqueous formulation of the in- vention in the form of an aqueous suspension will normally comprise at least 50 % (w/w) of water.
In another embodiment, a pharmaceutical composition (formulation) of the invention may be a freeze-dried (i.e. lyophilized) formulation intended for reconstitution by the physician or the patient via addition of solvents and/or diluents prior to use.
In a further embodiment, a pharmaceutical composition (formulation) of the invention may be a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolu¬ tion.
In a further aspect, the invention relates to a pharmaceutical composition (formulation) com¬ prising an aqueous solution of a compound of the present invention, and a buffer, wherein the compound of the invention is present in a concentration of 0.1 -100 mg/ml or above, and wherein the formulation has a pH from about 2.0 to about 10.0.
In another embodiment of the invention, the pH of the formulation has a value selected from the list consisting of 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6,
5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,
9.9 and 10.0.
In a further embodiment, the buffer in a buffered pharmaceutical composition of the invention may comprise one or more buffer substances selected from the group consisting of sodium acetate, sodium carbonate, citrates, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, tris(hydroxymethyl)aminomethane (TRIS), bicine, tricine, malic acid, succinates, maleic acid, fumaric acid, tartaric acid and aspartic acid. Each one of these specific buffers constitutes an alternative embodiment of the invention. In another embodiment, a pharmaceutical composition of the invention may comprise a pharmaceutically acceptable preservative, e.g. one or more preservatives 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, thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride and chlorphenesine (3p-chlorphenoxypropane-1 ,2-diol). Each one of these specific preservatives constitutes an alternative embodiment of the invention. In a further embodiment of the invention the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In still further embodiments of such a pharmaceutical composition of the invention, the preservative is present in a concentration in the range of 0.1 mg/ml to 5 mg/ml, a concentration in the range of 5 mg/ml to 10 mg/ml, or a concentration in the range of 10 mg/ml to 20 mg/ml. The use of a preservative in pharmaceutical compositions is well known to the skilled person. For convenience, reference is made in this respect to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.
In a further embodiment of the invention the formulation further comprises a tonicity-adjusting agent, i.e. a substance added for the purpose of adjusting the tonicity (osmotic pressure) of a liquid formulation (notably an aqueous formulation) or a reconstituted freeze-dried formulation of the invention to a desired level, normally such that the resulting, final liquid formulation is isotonic or substantially isotonic. Suitable tonicity-adjusting agents may be selected from the group consisting of salts (e.g. sodium chloride), sugars and sugar alcohols (e.g. mannitol), amino acids (e.g. glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine), alditols [e.g. glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1 ,3-propanediol or 1 ,3-butanediol], polyethyleneglycols (e.g. PEG 400) and mixtures thereof.
Any sugar, such as a mono-, di- or polysaccharide, or a water-soluble glucan, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch or carboxymethylcellulose-sodium, may be used; in one embodiment, sucrose may be employed. Sugar alcohols (polyols derived from mono-, di-, oligo- or polysaccharides) include, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one embodiment, the sugar alcohol employed is mannitol. 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 composition (formulation) and does not adversely effect the stabilizing effects achieved using the methods of the invention. In one embodiment, the concentration of sugar or sugar alcohol is between about 1 mg/ml and about 150 mg/ml.
In further embodiments, the tonicity-adjusting agent is present in a concentration of from 1 mg/ml to 50 mg/ml, such as from 1 mg/ml to 7 mg/ml, from 8 mg/ml to 24 mg/ml, or from 25 mg/ml to 50 mg/ml. A pharmaceutical composition of the invention containing any of the tonicity-adjusting agents specifically mentioned above constitutes an embodiment of the invention. The use of a tonicity-adjusting agent in pharmaceutical compositions is well known to the skilled person. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 20th edition, 2000.
In a still further embodiment of a pharmaceutical composition (formulation) of the invention, the formulation further comprises a chelating agent. Suitable chelating agents may be selected, for example, from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof. The concentration of chelating agent will suitably be in the range from 0.1 mg/ml to 5 mg/ml, such as from 0.1 mg/ml to 2 mg/ml or from 2 mg/ml to 5 mg/ml. A pharmaceutical composition of the invention containing any of the chelating agents specifically mentioned above constitutes an 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, 20th edition, 2000.
In another embodiment of a pharmaceutical composition (formulation) of the invention, the formulation further comprises a stabilizer. 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, 20th edition, 2000.
More particularly, particularly useful compositions of the invention include stabilized liquid pharmaceutical compositions whose therapeutically active components include an oligo- or polypeptide that possibly exhibits aggregate formation during storage in liquid pharmaceuti¬ cal formulations. By "aggregate formation" is meant the formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution, as the result of a physical interaction between the oligo- or polypeptide molecules. The term "during storage" refers to the fact that a liquid pharmaceutical composition or formulation, once prepared, is not normally administered to a subject immediately. Rather, following preparation, it is pack¬ aged for storage, whether in a liquid form, in a frozen state, or in a dried form for later recon- stitution into a liquid form or other form suitable for administration to a subject. By "dried form" is meant the product obtained when a liquid pharmaceutical composition or formulation is dried by freeze-drying (i.e., lyophilization; see, for example, Williams and PoIIi (1984) J^ Parenteral Sci. Technol. 38: 48-59), by spray-drying [see, e.g., Masters (1991 ) in Spray- Drying Handbook (5th edn.; Longman Scientific and Technical, Essex, U.K.), pp. 491 -676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18: 1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11 : 12-20], or by air-drying [see, e.g., Carpenter and Crowe (1988) Crvobioloαv 25: 459-470; and Roser (1991 ) Biopharm. 4: 47-53]. Aggregate formation by an oligo- or polypeptide during storage of a liquid pharmaceutical composition can adversely affect biological activity of that peptide, resulting in loss of therapeutic efficacy of the phar¬ maceutical composition. Furthermore, aggregate formation may cause other problems, such as blockage of tubing, membranes or pumps when the oligo- or polypeptide-containing pharmaceutical composition is administered using an infusion system.
A pharmaceutical composition of the invention may further comprise an amount of an amino acid base sufficient to decrease aggregate formation by the oligo- or polypeptide during stor¬ age of the composition. By "amino acid base" is meant 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 for use in preparing a composition 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 mix¬ tures thereof) of a particular amino acid (e.g. methionine, histidine, arginine, lysine, isoleu- cine, aspartic acid, tryptophan or 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 em- bodiment, the L-stereoisomer of an amino acid is used. Compositions of the invention may also be formulated with analogues of these amino acids. By "amino acid analogue" is meant a derivative of a naturally occurring amino acid that brings about the desired effect of de¬ creasing aggregate formation by the oligo- or polypeptide during storage of liquid pharma¬ ceutical compositions of the invention. Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl-L-arginine. Suitable methionine analogues in- elude ethionine and buthionine, and suitable cysteine analogues include S-methyl-L-cysteine. As with the amino acids per se, amino acid analogues are incorporated into compositions of the invention 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 incorporated in a concentration which is sufficient to prevent or delay aggregation of the oligo-or polypeptide.
In a particular embodiment of the invention, methionine (or another sulfur-containing amino acid or amino acid analogue) may be incorporated in a composition of the invention to inhibit oxidation of methionine residues to methionine sulfoxide when the oligo- or polypeptide act- ing as the therapeutic agent is a peptide comprising at least one methionine residue suscep¬ tible to such oxidation. The term "inhibit" in this context refers to minimization of accumulation of methionine-oxidized species over time. Inhibition of methionine oxidation results in in¬ creased retention of the oligo- or polypeptide in its proper molecular form. Any stereoisomer of methionine (L or D) or combinations thereof can be used. The amount to be added should be an amount sufficient to inhibit oxidation of methionine residues such that the amount of methionine sulfoxide is acceptable to regulatory agencies. Typically, this means that no more than from about 10% to about 30% of forms of the oligo- or polypeptide wherein methionine is sulfoxidated are present. In general, this can be achieved by incorporating methionine in the composition such that the ratio of added methionine to methionine residues ranges from about 1 :1 to about 1000:1 , such as from about 10:1 to about 100:1 .
In a further embodiment of the invention the formulation further comprises a stabilizer selected from high-molecular-weight polymers and low-molecular-weight compounds. Thus, for example, the stabilizer may be selected from substances such as polyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycellulose and derivatives thereof (e.g. HPC, HPC-SL, HPC-L or HPMC), cyclodextrins, sulfur- containing substances such as monothioglycerol, thioglycolic acid and 2-methylthioethanol, and various salts (e.g. sodium chloride). A pharmaceutical composition of the invention containing any of the stabilizers specifically mentioned above constitutes an embodiment of the invention.
Pharmaceutical compositions of the present invention may also comprise additional stabilizing agents which further enhance stability of a therapeutically active oligo- or polypeptide therein. Stabilizing agents of particular interest in the context of the present invention include, but are not limited to: methionine and EDTA, which protect the peptide against methionine oxidation; and surfactants, notably nonionic surfactants, which protect the polypeptide against aggregation or degradation associated with freeze-thawing or mechanical shearing.
Thus, in a further embodiment of the invention, the pharmaceutical formulation comprises a surfactant, particularly a nonionic surfactant. Examples thereof include ethoxylated castor oil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitan fatty acid esters, polyoxypropylene-polyoxyethylene block polymers (e.g. 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, e.g. Tween-20, Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylated derivatives thereof, diglycerides or polyoxyethylene derivatives thereof, alcohols, glycerol, lectins and phospholipids (e.g. phosphatidyl-serine, phosphatidyl-choline, phosphatidyl- ethanolamine, phosphatidyl-inositol, diphosphatidyl-glycerol and sphingomyelin), derivatives of phospholipids (e.g. dipalmitoyl phosphatidic acid) and lysophospholipids (e.g. palmitoyl lysophosphatidyl-L-serine and 1 -acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine or threonine) and alkyl, alkyl ester and alkyl ether derivatives of lysophosphatidyl and phosphatidylcholines, e.g. lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar head group, i.e. cholines, ethanolamines, phosphatidic acid, serines, threonines, glycerol, inositol, and the positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (eg. cephalins), glyceroglycolipids (e.g. galactopyranoside), sphingoglycolipids (e.g. ceramides, gangliosides), dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives (e.g. sodium tauro- dihydrofusidate, etc.), long-chain fatty acids (e.g. oleic acid or caprylic acid) and salts thereof, 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. [577-1 1 -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- sulfonates) monovalent surfactants, zwitterionic surfactants (e.g. N-alkyl-N,N- dimethylammonio-1 -propanesulfonates, 3-cholamido-1 -propyldimethylammonio-1 - propanesulfonate, cationic surfactants (quaternary ammonium bases) (e.g. cetyl- trimethylammonium bromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecyl β- D-glucopyranoside), poloxamines (e.g. Tetronic's), which are tetrafunctional block copolymers derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine. The surfactant may also be selected from imidazoline derivatives and mixtures thereof. A pharmaceutical composition of the invention containing any of the surfactants specifically mentioned above constitutes an 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, 20th edition, 2000.
Additional ingredients may also be present in a pharmaceutical composition (formulation) of the present invention. Such additional ingredients may include, for example, wetting agents, emulsifiers, antioxidants, bulking agents, metal ions, oleaginous vehicles, proteins (e.g. human serum albumin, gelatine or other proteins) and a zwitterionic species (e.g. an amino acid such as betaine, taurine, arginine, glycine, lysine or histidine). Such additional ingredients should, of course, 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 one or more of several sites, for example at topical sites (e.g. skin and mucosal sites), at sites which bypass absorption (e.g. via administration in an artery, in a vein or in the heart), and at sites which involve absorption (e.g. in the skin, under the skin, in a muscle or in the abdomen).
Administration of pharmaceutical compositions according to the invention to patients in need thereof may be via several routes of administration. These include, for example, lingual, sub¬ lingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary (for exam¬ ple through the bronchioles and alveoli or a combination thereof), epidermal, dermal, trans¬ dermal, vaginal, rectal, ocular (for example through the conjunctiva), uretal and parenteral. Compositions of the present invention may be administered in various dosage forms, for ex¬ ample in the form of solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules (e.g. hard gelatine capsules or 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 solutions, in s/fcv-transforming solutions (for example in situ gelling, in situ setting, in situ precipitating or in situ crystallizing), infusion solutions or implants.
Compositions of the invention may further be compounded in, or bound to, e,g. via covalent, hydrophobic or electrostatic interactions, a drug carrier, drug delivery system or advanced drug delivery system in order to further enhance the stability of the compound of the present invention, 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 deliv¬ ery systems include, but are not limited to: polymers, for example cellulose and derivatives; polysaccharides, for example dextran and derivatives, starch and derivatives; polyvinyl al¬ cohol); acrylate and methacrylate polymers; polylactic and polyglycolic acid and block co¬ polymers thereof; polyethylene glycols; carrier proteins, for example albumin; gels, for exam- pie thermogelling systems, such as 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 thereof well known to those skilled in the art of phase be¬ haviour in lipid-water systems; polymeric micelles; multiple emulsions (self-emulsifying, self- microemulsifying); cyclodextrins and derivatives thereof; and dendrimers.
Compositions of the present invention are useful in the formulation of solids, semisolids, powders and solutions for pulmonary administration of a compound of the present invention, using, for example, a metered dose inhaler, dry powder inhaler or a nebulizer, all of which are devices well known to those skilled in the art.
Compositions of the present invention are useful in the formulation of controlled-release, sus¬ tained-release, protracted, retarded or slow-release drug delivery systems. Compositions of the invention are thus of value in the formulation of parenteral controlled-release and sus¬ tained-release systems well known to those skilled in the art (both types of systems leading to a many-fold reduction in the number of administrations required). Of particular value are controlled-release and sustained-release systems for subcutaneous administration. Without limiting the scope of the invention, examples of useful controlled re¬ lease systems and compositions are those containing hydrogels, oleaginous gels, liquid crys- tals, polymeric micelles, microspheres or nanoparticles,
Methods for producing controlled-release systems useful for compositions of the present in¬ vention include, but are not limited to, crystallization, condensation, co-crystallization, precipi¬ tation, co-precipitation, emulsification, dispersion, high-pressure homogenisation, encapsula- tion, spray-drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made in this context to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000), and Drugs and the Pharmaceutical Sciences, vol. 99: Pro¬ tein 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, for example a syringe in the form of a pen device. Alternatively, parenteral administration may be performed by means of an infusion pump. A further option is administration of a composition of the invention which is a liquid (typically aqueous) solution or suspension in the form of a nasal or pulmonary spray. As a still further option, a pharmaceutical composition of the invention can be adapted to trans¬ dermal administration (e.g. by needle-free injection or via a patch, such as an iontophoretic patch) or transmucosal (e.g. buccal) administration.
The term "stabilized formulation" refers to a formulation with increased physical stability, in¬ creased chemical stability or increased physical and chemical stability. The term "physical stability" in the context of a formulation containing an oligo- or polypeptide refers to the ten¬ dency of the peptide to form biologically inactive and/or insoluble aggregates as a result of exposure to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. Physical stability of aqueous protein formulations is evaluated by means of visual inspection and/or turbidity measure¬ ments after exposing the formulation, filled in suitable containers (e.g. cartridges or vials), to mechanical/physical stress (e.g. agitation) at different temperatures for various time periods. Visual inspection of formulations is performed in a sharp focused light with a dark back- ground. The turbidity of a formulation is characterized by a visual score ranking the degree of turbidity, for instance on a scale from 0 to 3 (in that a formulation showing no turbidity corre¬ sponds to a visual score 0, whilst a formulation showing visual turbidity in daylight corre¬ sponds to visual score 3). A formulation is normally classified physically unstable with re¬ spect to aggregation when it shows visual turbidity in daylight. Alternatively, the turbidity of a formulation can be evaluated by simple turbidity measurements well-known to the skilled person. Physical stability of aqueous oligo- or polypeptide formulations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the peptide. The probe is preferably a small molecule that preferentially binds to a non-native conformer of the oligo- or polypeptide. One example of a small-molecular spectroscopic probe of this type 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 possibly also other configurations, 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 form. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths in question.
Other small molecules can be used as probes of the changes in peptide structure from native to non-native states. Examples are the "hydrophobic patch" probes that bind preferentially to exposed hydrophobic patches of a polypeptide. The hydrophobic patches are generally bur¬ ied within the tertiary structure of a polypeptide in its native state, but become exposed as it begins to unfold or denature. Examples of such small-molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as anthracene, acridine, phenanthroline and the like. Other spectroscopic probes are metal complexes of amino acids, such as cobalt complexes of hy¬ drophobic amino acids, e.g. phenylalanine, leucine, isoleucine, methionine, valine, or the like.
The term "chemical stability" of a pharmaceutical formulation as used herein refers to chemi¬ cal covalent changes in oligo- or polypeptide structure leading to formation of chemical deg¬ radation products with potentially lower biological potency and/or potentially increased im- munogenicity compared to the original molecule. Various chemical degradation products can be formed depending on the type and nature of the starting molecule and the environment to which it is exposed. Elimination of chemical degradation can most probably not be com¬ pletely avoided and gradually increasing amounts of chemical degradation products may of¬ ten be seen during storage and use of oligo- or polypeptide formulations, as is well known to the person skilled in the art. A commonly encountered degradation process is deamidation, a process in which the side-chain amide group in glutaminyl or asparaginyl residues is hydro- lysed to form a free carboxylic acid. Other degradation pathways involve formation of higher molecular weight transformation products wherein two or more molecules of the starting sub¬ stance are covalently bound to each other through transamidation and/or disulfide interac¬ tions, leading to formation of covalently bound dimer, oligomer or polymer degradation prod¬ ucts (see, e.g., Stability of Protein Pharmaceuticals, Ahern. TJ. & Manning M. C, Plenum Press, New York 1992). Oxidation (of for instance methionine residues) may be mentioned as another variant of chemical degradation. The chemical stability of a formulation may be evaluated by measuring the amounts of chemical degradation products at various time-points after exposure to different environmental conditions (in that the formation of degradation products can often be accelerated by, e.g., increasing temperature). The amount of each in- dividual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatographic techniques (e.g. 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 pharmaceutical composition (formulation) must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiry date is reached.
A pharmaceutical composition (formulation) of the invention should preferably be stable for more than 2 weeks of usage and for more than two years of storage, more preferably for more than 4 weeks of usage and for more than two years of storage, desirably for more than 4 weeks of usage and for more than 3 years of storage, and most preferably for more than 6 weeks of usage and for more than 3 years of storage.
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).
Headings and sub-headings are used herein for convenience only, and should not be con¬ strued as limiting the invention in any way.
The use of any and all examples, or exemplary language (including "for instance", "for exam- pie", "e.g." and "such as") in the present specification is intended merely to better illuminate the invention, and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any non- claimed element as being 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 present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto, as permitted by applicable law.
EXAMPLES
List of abbreviations employed
AcOH acetic acid
BSA bovine serum albumin
DCM dichloromethane
DIC diisopropylcarbodiimide DIPEA ethyldiisopropylamine
DMAP 4-N,N-dimethylaminopyridine
DMEM Dulbecco's Modified Eagle's Medium
DMF N,N-dimethylformamide
DMSO dimethyl sulfoxide EGTA 1 ,2-di(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic acid
FCS fetal calf serum
Fmoc 9-fluorenylmethyloxycarbonyl
HEPES 2-[4-(2-hydroxyethyl)-piperazin-1 -yl]-ethanesulfonic acid
HOAt 1 -hydroxy-7-azabenzotriazole HOBt 1 -hydroxybenzotriazole
HSA human serum albumin
MeCN acetonitrile
MeOH methanol α-MSH α-form of melanocyte-stimulating hormone
MTX methotrexate
NEt3 triethylamine
NMP N-methylpyrrolidone
PBS phosphate-buffered saline
PEI polyethyleneimine
PhMe toluene
PyBop (benzotriazol-i -yloxy)tripyrrolidino-phosphonium hexafluorophosphate
All compounds of the present invention can be synthesized by those skilled in the art using standard coupling and deprotection steps. A description of all necessary tools and synthetic methods including standard abbreviations for peptide synthesis can be found in "The Fine Art Of Solid Phase Synthesis", 2002/3 Catalogue, Novabiochem.
In the examples listed below, Rt values are retention times and the mass values are those detected by the mass spectroscopy (MS) detector and obtained using one of the following HPLC-MS devices (LCMS).
LCMS (system 1) Agilent 1 100 Series, electrospray; column: Waters XTerra® Ci8 5 μm 3.0x50mm; wa- ter/acetonitrile containing 0.05 % TFA; gradient: 5 % → 100 % acetonitrile from 0 to 6.75 min, elution until t = 9.0 min; flow 1 .5 ml/min.
LCMS (system 2)
Sciex API-150 Ex Quadrupole MS, electrospray, m/z = 200 to m/z = 1500; column: Waters XTerra® MS Ci8 5μm 3.0x50mm; elution with a mixture of solution A (water containing 0.1 % TFA) and solution B (acetonitrile containing 0.08 % TFA); gradient: 5 % → 20 % solution B from 1 .0 to 3.0 min, 20 % → 50 % solution B from 3.0 to 16.0 min, 50 % → 90 % solution B from 16.0 to 18.0 min, elution until t = 18.0 min; flow 1 .5 ml/min.
A typical example of a synthesis procedure which includes a cyclization step is as follows: Example 1
2-[2-(Hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-Lys]- NH2
Figure imgf000041_0001
Step A for Example 1 : protected peptide resin Fmoc-Nle-c[Glu-Asn(Trt)-D-Phe- Arg(Pbf)-Trp-Lys]-NH-Rink linker-polystyrene
Fmoc-Rink resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxypolystyrene resin, Bachem D-2080, Lot 514460; 0.47 mmol/g) was filled into two 60 ml Teflon reactors with frit (per reactor: 3.55 g, 1.67 mmol). The resin in each reactor was washed with DCM (30 ml).
Removal of Fmoc: The resin was shaken with a solution of 20 % piperidine in NMP (25 ml) for 20 min and then washed with NMP/DCM 1 :1 (5x 30 ml).
Acylation with Fmoc-Lvs(Mtt)-OH: In a separate glass vial, the Fmoc-amino acid (10.0 mmol) was mixed with NMP (12.5 ml), DCM (22.5 ml) and a 1 M solution (10.0 ml, 10.0 mmol) of 1 - hydroxybenzotriazol (HOBt) in NMP. To the resulting clear solution, DIC (1 .56 ml, 10.0 mmol) was quickly added and the solution was shaken immediately thereafter. The solution was left to stand in a closed vial for 15 min. 25 ml (5.0 mmol HOBt ester) of this solution was added to each reactor and the resin was shaken for 21/2 hours. Ethyldiisopropylamine (DIPEA) (per reactor 0.856 ml, 5.0 mmol) was added and the mixture was shaken for 18 h. The resin was washed with NMP/DCM 1 :1 (4x 30 ml).
Removal of Fmoc: As described above
Acylation with Fmoc-Trp(Boc)-OH: In a separate glass vial, the Fmoc-amino acid (10.0 mmol) was mixed with NMP (12.5 ml), DCM (22.5 ml) and 1 M HOBt-NMP solution (10.0 ml, 10.0 mmol). To the resulting clear solution, DIC (1 .56 ml, 10.0 mmol) was quickly added and the solution was shaken immediately thereafter. The solution was left to stand in a closed vial for 30 min. 25 ml (5.0 mmol HOBt ester) of this solution was added to each reactor and the resin was shaken for 21/2 h. The liquids were filtered off and the resin was washed with NMP/DCM 1 :1 (4x 30 ml).
In a similar manner, the following amino acids were successively attached to the resin: Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(2-phenylisopropyloxy)- OH and Fmoc-Nle-OH. Coupling with Fmoc-Glu(2-phenylisopropyloxy)-OH was performed by using HOAt instead of HOBt, and DIPEA (5.0 mmol per reactor added after HOAt ester for- mation). The resulting Fmoc-protected resin was extensively washed with DCM .
Selective side-chain deprotection of Lvs and GIu: The resin was shaken with a solution of 2 vol-% TFA and 3 vol-% triisopropylsilane in DCM (25 ml) for 10 min and the liquid was fil¬ tered off. This procedure was repeated another seven times. The resin was washed with DCM (4x 30 ml), 10 % DIPEA in DCM (2x 30 ml) and DCM (2x 30 ml).
Side-chain cvclisation of Lvs with GIu: In a separate glass vial, PyBOP (5.205 g = 10.0 mmol) was mixed with 1 M HOBt-NMP solution (10.0 ml = 10.0 mmol), DCM (25 ml) and NMP (15 ml). 25 ml (containing 5.0 mmol PyBOP/HOBt) of this solution was added to each reactor, followed by DIPEA (per reactor 1 .71 ml = 10.0 mmol). The resin was shaken for 15 h. The liquids were filtered off and the resin was washed with NMP/DCM 1 :1 (4x 30 ml).
Capping of non-acylated amino groups: The resin was shaken with a solution of Boc anhy¬ dride (10 mmol per reactor) in DCM (25 ml per reactor) for 1 h. The liquids were filtered off and the resin was washed with DCM (3x 30 ml), DCM/MeOH 2:1 (2x 30 ml), THF (4x 30 ml) and DCM (3x 30 ml).
Remaining solvents were removed by sucking air through the reactors for 4 h. The resins from both reactors were mixed to give 12.785 g of resin Fmoc-Nle-c[Glu-Asn(Trt)-D-Phe- Arg(Pbf)-Trp-Lys]-NH-Rink linker-polystyrene, corresponding to a supposed maximum load¬ ing of 0.26 mmol/g, if complete reactions are assumed. Step B for Example 1 : 2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle- c[Glu-Asn-D-Phe-Arg-Trp-Lys]-NH2
A 10 ml Teflon reactor with frit was charged with resin Fmoc-Nle-c[Glu-Asn(Trt)-D-Phe- Arg(Pbf)-Trp-Lys]-NH-Rink linker-polystyrene (0.385 g, theoretically 0.10 mmol, obtained by step A described above). The resin was washed with DCM (2x 3 ml).
Removal of Fmoc: The resin was shaken with a solution of 20 % piperidine in NMP (2.5 ml) for 20 min and then washed with NMP/DCM 1 :1 (5x 3 ml).
Acylation with Fmoc-Ser(tBu)-OH: In a separate glass vial, the Fmoc-amino acid (0.5 mmol) was mixed with NMP (0.65 ml), DCM (1 .15 ml) and 1 M HOBt-NMP solution (0.5 ml, 0.5 mmol). To the resulting clear solution, DIC (0.078 ml, 0.5 mmol) was quickly added and the solution was shaken immediately thereafter. The solution was left to stand in a closed vial for 30 min and then added to the resin. The mixture was shaken for 3V∑ h. The liquids were fil¬ tered off and the resin was washed with NMP/DCM 1 :1 (4x 3 ml).
In a similar manner, the following carboxylic acids were successively attached to the resin: Fmoc-His(Trt)-OH, Fmoc-NH-(CH2)2-O-(CH2)2-O-CH2-CO2H, and hexadecanoic acid. Finally, the resin was washed with NMP/DCM 1 :1 (2x 3 ml), DCM/MeOH 2:1 (4x 3 ml), THF (4x 3 ml) and DCM (3x 3ml).
Cleavage from the resin: The resin was shaken for 2 h with a premixed solution (3.5 ml) con¬ taining TFA (95 vol-%), triisopropylsilane (2.5 vol-%l) and water (2.5 vol-%). The mixture was filtered and the filtrate was collected in a glass vial. The resin was washed with 2x 3 ml DCM/TFA 2:1 and the filtrates were collected. The combined filtrate solution was concen¬ trated to give a reddish-brown oily suspension.
Precipitation with ether: The residue was treated with diethyl ether (20 ml) to give a solid pre- cipitate. The ether phase was removed after centrifugation. The solid residue was washed again with diethyl ether (20 ml). After centrifugation and removal of the ether phase, the solid residue was left overnight in order to remove remaining diethyl ether.
Purification: The crude product precipitated from ether was dissolved in a mixture of acetoni- trile (8 ml), acetic acid (0.4 ml) and water to give a total volume of about 21 ml. The resulting dim liquid was filtered and the clear filtrate was injected into a Gilson preparative HPLC de¬ vice. Elution was performed with water/acetonitrile containing 0.1 % TFA with a gradient from 44 % to 56 % acetonitrile. The eluate was collected as fractions of 5 ml (peak fractions) or 12 ml (non-peak fractions), respectively. Relevant fractions were checked by analytical HPLC. Fractions containing the pure target peptide were mixed, concentrated under reduced pres¬ sure and diluted with de-ionised water to give a clear solution (29 ml). After addition of 1 M aqueous HCI (0.6 ml), the solution was dispensed into glass vials. The vials were capped with Millipore glass fibre prefilters. Freeze-drying for three days afforded the peptide hydrochloride (21 .6 mg, 13 % yield) as a white solid.
Analytical HPLC (Waters Symmetry300 C18, 5 μm, 3.9 x 150 mm; 42 5C; water/acetonitrile containing 0.05 % TFA; gradient: 5 % → 95 % acetonitrile from 0 to 15 min; flow 1 ml/min): tR = 11 .36 min (99 % purity by UV 214 nm and 94 % purity by UV 254 nm) LCMS (system 1 ): Rt = 4.18 min; ((m+2)/2) = 791
Examples of further compounds of the invention which may be obtained in a manner analo¬ gous to the compound of Example 1 are the compounds of Examples 2-21 , below. The syn¬ thesis of the building block employed in the synthesis of the compounds of Examples 13, 19 and 21 is described below:
Example 2
2-[2-(Hexadecanoylamino)ethoxy]ethoxyacetyl-Gln-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-Lys]-
NH2
Figure imgf000044_0001
LCMS (system 1 ): Rt = 4.49 min; ((m+2)/2) = 787. Example 3
2-[2-(2-{2-[2-(Tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Asn-D-Phe-Arg-Trp-Lys]-NH2
Q
Figure imgf000045_0001
LCMS (system 1 ): Rt = 4.30 min; ((m+2)/2) = 738.
Example 4
2-[2-(2-{2-[2-(Tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Hyp-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000045_0002
LCMS (system 1 ): Rt = 4.34 min; ((m+2)/2) = 737.
Example 5
3-[2-(2-{2-[2-(Hexadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propanoyl-His-Dab-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000045_0003
LCMS (system 2): Rt = 14.22 min; ((m+2)/2) = 848. Example 6
2-[2-(2-{2-[2-(Hexadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Dap-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000046_0001
LCMS (system 2): Rt = 15.61 min; ((m+2)/2) = 738.
Example 7
2-[2-(Hexadecanoylamino)ethoxy]ethoxyacetyl-Arg-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-Lys]- NH2
Figure imgf000046_0002
Example 8
2-[2-(Hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Asp-Hyp-D-Phe-Arg-Trp-Lys]- NHP
Figure imgf000046_0003
Example 9
2-[2-(2-{2-[2-(Tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Dab-Nle- c[Glu-Asn-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000047_0001
Example 10
2-[2-(2-{2-[2-(Tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Ser-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000047_0002
LCMS (system 2): Rt = 15.51 min; ((m+2)/2) = 724.
Example 11
2-[2-(2-{2-[2-(Tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Gln-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000047_0003
LCMS (system 2): Rt = 15.07 min; ((m+2)/2) = 745. Example 12
2-[2-(2-{2-[2-(Tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Cit-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000048_0001
5 LCMS (system 2): Rt = 14.94 min; ((m+2)/2) = 760.
Example 13
2-[2-(4-(Hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-Dap-D-Phe- Arg-Trp-Lys]-NH2
Figure imgf000048_0002
LCMS (system 2): Rt = 17.01 min; ((m+2)/2) = 740.
Example 14
{2-[2-(2-(Hexadecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}methoxyacetyl-Ser-Nle-c[Asp- 15 Asn-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000048_0003
O Example 15
{2-[2-(2-(Tetradecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}methoxyacetyl-Ser-Nle-c[Asp- Hyp-D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000049_0001
Example 16
4-{2-[2-(2-(Octadecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}butanoyl-Dab-Nle-c[Asp-Hyp- D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000049_0002
Example 17
4-{2-[2-(2-(Tθtradθcanoylamino)ethoxy)ethoxy]ethylcarbamoyl}butanoyl-Thr-Nle-c[Glu-Gln-D- Phe-Arg-Trp-Lys]-NH2
Figure imgf000049_0003
H O Example 18
3-(2-{2-[2-(2-(Hexadecanoylamino)ethoxy)ethoxy]ethoxy}ethoxy)propionyl-Nle-c[Glu-Dap-D- Phe-Arg-Trp-Lys]-NH
Figure imgf000050_0001
Example 19
2-[2-(4-(Hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp- D-Phe-Arg-Trp-Lys]-NH2
Figure imgf000050_0002
LCMS (system 2): Rt = 16.09 min; ((m+2)/2) = 865.
Example 20
2-[2-(4-(Tridecanoylsulfamoyl)butanoylamino)θthoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp-D- Phe-Arg-Trp-Lys]-NH2
Figure imgf000050_0003
LCMS (system 2): Rt = 13.30 min; ((m+2)/2) = 844. Example 21
2-[2-(4-(Hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Cys-Hyp- D-Phe-Arg-Trp-Cys]-NH2
Figure imgf000051_0001
LCMS (system 1 ): Rt = 4.57 min; ((m+2)/2) = 848.
Preparation of 4-(hexadecanoylsulfamoyl)butyric acid for use in the syntheses of the title compounds of Examples 13, 19 and 21
Figure imgf000051_0002
The synthesis of this building block has been described previously in WO 2004/099246 (Novo Nordisk A/S). Briefly, 4-sulfamoylbutyric acid methyl ester is prepared from commer¬ cially available 4-sulfamoylbutyric acid and acylated with palmitic chloride in the presence of 4-dimethylaminopyridine. The resulting acylsulfonamide is isolated. Saponification with so- dium hydroxide and recrystallisation affords 4-(hexadecanoylsulfamoyl)butyric acid.
PHARMACOLOGICAL METHODS
Assay (I) - Experimental protocol for efficacy testing on appetite with MC4 analogues, using an ad libitum fed rat model.
TAC:SPRD rats or Wistar rats from M&B Breeding and Research Centre A/S, Denmark are used for the experiments. The rats have a body weight 200-250 g at the start of experiment. The rats arrive at least 10-14 days before start of experiment with a body weight of 180-200 g. Each dose of compound is tested in a group of 8 rats. A vehicle group of 8 rats is included in each set of testing. When the animals arrive they are housed individually in a reversed light/dark phase (lights off 7:30 am, lights on 7:30 pm), meaning that lights are off during daytime and on during night¬ time. Since rats normally initiate food intake when light is removed, and eat the major part of their daily food intake during the night, this set-up results in an alteration of the initiation time for food intake to 7:30 am, when lights are switched off. During the acclimatization period of 10-14 days, the rats have free access to food and water. During this period the animals are handled at least 3 times. The experiment is conducted in the rats' home cages. Immediately before dosing the rats are randomised to the various treatment groups (n=8) by body weight. They are dosed according to body weight at between 7:00 am and 7:45 am, with a 1 -3 mg/kg solution administered intraperitoneal^ (ip), orally (po) or subcutaneously (sc). The time of dosing is recorded for each group. After dosing, the rats are returned to their home cages, where they then have access to food and water. The food consumption is recorded individu¬ ally every hour for 7 hours, and then after 24 h and sometimes 48 h. At the end of the ex¬ perimental session, the animals are euthanised.
The individual data are recorded in Microsoft excel sheets. Outliers are excluded after apply¬ ing the Grubbs statistical evaluation test for outliers, and the result is presented graphically using the GraphPad Prism program.
Assay (II) - Melanocortin receptor 3 and 5 (MC3 and MC5) cAMP functional assay using the AlphaScreen™ cAMP detection kit
The cAMP assays for MC3 and MC5 receptors are performed on cells (either HEK293 or BHK cells) stably expressing the MC3 and MC5 receptors, respectively. The receptors are cloned from cDNA by PCR and inserted into the pcDNA 3 expression vector. Stable clones are selected using 1 mg/ml G418.
Cells at approx. 80-90% confluence are washed 3x with PBS, lifted from the plates with Versene and diluted in PBS. They are then centrifuged for 2 min at 1300 rpm, and the super¬ natant removed. The cells are washed twice with stimulation buffer, and then resuspended in stimulation buffer to a final concentration of 1 x106 or 2x106 cells/ml. 25 μl of cell suspension is added to the microtiter plates containing 25 μl of test compound or reference compound (all diluted in stimulation buffer). The plates are incubated for 30 minutes at room tempera¬ ture (RT) on a plate-shaker set to a low rate of shaking. The reaction is stopped by adding 25 μl of acceptor beads with anti-cAMP, and 2 min later 50 μl of donor beads per well with bioti- nylated cAMP in a lysis buffer. The plates are then sealed with plastic, shaken for 30 minutes and allowed to stand overnight, after which they are counted in an Alpha™ microplate reader.
EC50 values are calculated by non-linear regression analysis of dose/response curves (6 points minimum) using the Windows™ program GraphPad™ Prism (GraphPad™ Software, USA). All results are expressed in nM.
For measuring antagonistic activity in the MC3 functional cAMP assay, the MC3 receptors are stimulated with 3 nM α-MSH, and inhibited with increasing amounts of the potential an¬ tagonist. The IC50 value for the antagonist is defined as the concentration that inhibits MC3 stimulation by 50 %.
Assay (III) - Melanocortin receptor 4 (MC4) cAMP assay
BHK cells expressing the MC4 receptor are stimulated with potential MC4 agonists, and the degree of stimulation of cAMP is measured using the Flash Plate® cAMP assay (NEN™ Life Science Products, cat. No. SMP004).
The MC4 receptor-expressing BHK cells are produced by transfecting the cDNA encoding MC4 receptor into BHK570/KZ10-20-48, and selecting for stable clones expressing the MC4 receptor. The MC4 receptor cDNA, as well as a CHO cell line expressing the MC4 receptor, may be purchased from Euroscreen™. The cells are grown in DMEM, 10% FCS, 1 mg/ml G418, 250 nM MTX and 1 % penicillin/streptomycin.
Cells at approx. 80-90% confluence are washed 3x with PBS, lifted from the plates with
Versene and diluted in PBS. They are then centrifuged for 2 min at 1300 rpm, and the super¬ natant removed. The cells are washed twice with stimulation buffer, and resuspended in stimulation buffer to a final concentration of 0.75x106 cells/ml (consumption thereof: 7 ml per 96-well microtiter plate). 50 μl of cell suspension is added to the Flash Plate containing 50 μl of test compound or reference compound (all diluted in H2O). The mixture is shaken for 5 minutes and then allowed to stand for 25 minutes at RT. The reaction is stopped by addition of 100 μl Detection Mix per well (Detection Mix = 11 ml Detection Buffer + 100 μl (~2μCi) cAMP [125I] tracer). The plates are then sealed with plastic, shaken for 30 minutes, and al¬ lowed to stand overnight (or for 2 hours) and then counted in the Topcounter (2 min/well). In general, the assay procedure is as described in the Flash Plate kit-protocol (Flash Plate® cAMP assay (NEN™ Life Science Products, cat. No. SMP004)). However, the cAMP stan¬ dards are diluted in 0.1 % HSA and 0.005% Tween™ 20 and not in stimulation buffer.
EC50 values are calculated by non-linear regression analysis of dose/response curves (6 points minimum) using the Windows™ program GraphPad™ Prism (GraphPad Software, USA). All results are expressed in nM.
Assay (IV) - Melanocortin receptor 1 (MC1) binding assay
The MC1 receptor binding assay is performed on BHK cell membranes stably expressing the MC1 receptor. The assay is performed in a total volume of 250 μl: 25 μl of 125NDP-α-MSH (22 pM in final concentration), 25 μl of test compound/control and 200 μl of cell membrane (35 μg/ml). Test compounds are dissolved in DMSO. Radioactively labeled ligand, mem¬ branes and test compounds are diluted in buffer: 25 mM HEPES, pH 7.4, 0.1 mM CaCI2, 1 mM MgSO4, 1 mM EDTA, 0.1 % HSA and 0.005% Tween™ 20. The samples are incubated at 3O0C for 90 min in Greiner microtiter plates, separated with GF/B filters that are pre-wetted for 60 min in 0.5% PEI, and washed 2-3 times with NaCI (0.9%) before separation of bound from unbound radiolabeled ligand by filtration. After filtration the filters are washed 10 times with ice-cold 0.9% NaCI. The filters are dried at 5O0C for 30 min, sealed, and 30 μl of Mi- croscint 0 (Packard, cat. No. 6013616) is added to each well. The plates are counted in a Topcounter (1 min/well).
The data are analysed by non-linear regression analysis of binding curves, using the Win¬ dows™ program GraphPad™ Prism (GraphPad Software, USA).
Assay (V) - Melanocortin receptor 4 (MC4) binding assay
In vitro 125NDP-Q-MSH binding to recombinant BHK cells expressing human MC4 receptor (filtration assay). The assay is performed in 5 ml minisorb vials (Sarstedt No. 55.526) or in 96-well filterplates (Millipore MADVN 6550), and using BHK cells expressing the human MC4 receptor (obtained from Professer Wikberg, Uppsala, Sweden). The BHK cells are kept at -8O0C until assay, and the assay is run directly on a dilution of this cell suspension, without further preparation. The suspension is diluted to give maximally 10% specific binding, i.e. to approx. 50-100 fold dilu¬ tion. The assay is performed in a total volume of 200 μl: 50 μl of cell suspension, 50 μl of 125NDP-α-MSH (~ 79 pM in final concentration), 50 μl of test compound and 50 μl binding buffer (pH 7) mixed and incubated for 2 h at 250C [binding buffer: 25 mM HEPES (pH 7.0), 1 mM CaCI2, 1 mM MgSO4, 1 mM EGTA, 0.02% Bacitracin and 0.2% BSA]. Test compounds are dissolved in H2O and diluted in binding buffer. Radiolabeled ligand and membranes are diluted in binding buffer. The incubation is stopped by dilution with 5 ml ice-cold 0.9% NaCI, followed by rapid filtration through Whatman GF/C filters pre-treated for 1 hour with 0.5% polyethyleneimine. The filters are washed with 3 x 5 ml ice-cold NaCI. The radioactivity re¬ tained on the filters is counted using a Cobra Il auto gamma counter.
The data are analysed by non-linear regression analysis of binding curves, using the Win¬ dows™ program GraphPad™ Prism (GraphPad Software, USA).
Assay (Vl) - Evaluation of energy expenditure
TAC:SPRD rats or Wistar rats from M&B Breeding and Research Centre A/S, Denmark are used. After at least one week of acclimatization, rats are placed individually in metabolic chambers (Oxymax system, Columbus Instruments, Columbus, Ohio, USA; systems cali¬ brated daily). During the measurements, animals have free access to water, but no food is provided to the chambers. Lightdark cycle is 12h:12h, with lights being switched on at 6:00. After the animals have spent approx. 2 hours in the chambers (i.e. when the baseline energy expenditure is reached), test compound or vehicle are administered (po, ip or sc), and re- cording is continued in order to establish the action time of the test compound. Data for each animal (oxygen consumption, carbon dioxide production and flow rate) are collected every 10-18 min for a total of 22 hours (2 hours of adaptation (baseline) and 20 hours of measure¬ ment). Correction for changes in O2 and CO2 content in the inflowing air is made in each 10- 18 min cycle.
Data are calculated per metabolic weight [(kg body weight) 075] for oxygen consumption and carbon dioxide production, and per animal for heat. Oxygen consumption (VO2) is regarded as the major energy expenditure parameter of interest.

Claims

1 . A compound of formula Ia, Ib or Ic:
R1-S-Z1-Z2-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ia] R1-S-Z2-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ib] R1-S-Z3-c[X1-X2-X3-Arg-X4-X5]N(R')2 [Ic] wherein R1 represents a straight-chain, branched and/or cyclic Ci4-2oalkanoyl, Ci4-20alkenoyl or Ci4-20alkynoyl which may optionally be substituted with one or more substituents selected from halogen, hydroxyl and aryl, or R1 represents C9-i7-C(O)-NH-S(O)2-(CH2)3-C(O)-; S represents a glycolether-based structure according to one of the formulas Na-IIe
Figure imgf000056_0001
lie
Figure imgf000056_0002
Z1 and Z2 independently represent GIy, Ser, Thr, GIn, Asn, His, homoArg, Arg, Lys, Dab, Dap or Orn;
Z3 represents Ala, VaI, Leu, He, Met or NIe;
X1 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap;
X2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, He, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met,
Met(O), Met(O2), GIn, Gln(alkyl), Gln(aryl), Asn, Asn(alkyl), Asn(aryl), Ser, Thr, Cys, Pro,
Hyp, Tic, 2-PyAIa, 3-PyAIa, 4-PyAIa, (2-thienyl)alanine, 3-(thienyl)alanine, (4-thiazolyl)Ala,
(2-furyl)alanine, (3-furyl)alanine or Phe, wherein the phenyl moiety of said Phe is optionally substituted with a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano;
X3 represents D-Phe, wherein the phenyl moiety in D-Phe may optionally be substituted with one or more substituents selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoro- methyl and cyano;
X4 represents Trp, 2-NaI, a (3-benzo[b]thienyl)alanine residue or a (S)-2,3,4,9-tetrahydro-1 H- β-carboline-3-carboxylic acid residue;
X5 represents GIu, Asp, Cys, homoCys, Pen, Lys, Orn, Dab or Dap; wherein X1 and X5 are joined, rendering the compound of formula Ia, Ib or Ic cyclic, either via a disulfide bridge deriving from X1 and X5 both independently being Cys, homoCys or Pen, or via an amide bond formed between a carboxylic acid in the side-chain of X1 and an amino group in the side chain of X5, or between a carboxylic acid in the side-chain of X5 and an amino group in the side-chain of X1 ; each R' independently represents hydrogen or d-6alkyl, which may optionally be substituted with one or more amino or hydroxy; with the proviso that the compound of formula Ia, Ib or Ic is not
2-[2-(octadecanoylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2,
3-(2-{2-[2-(2-(hexadecanoylamino)ethoxy)ethoxy]ethoxy}ethoxy)propionyl-Nle-c[Glu-Hyp-D-
Phe-Arg-Trp-Lys]-NH2, 2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-
NH2,
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Arg-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-
NH2 or
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Gln-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]- NH2; and pharmaceutically acceptable salts, prodrugs and solvates thereof.
2. A compound according to claim 1 , wherein R1 is Ci4-i8alkanoyl.
3. A compound according to claim 1 , wherein R1 is 4-(Ci4-i8alkanoylsulfamoyl)butanoyl.
4. A compound according to claim 3, wherein R1 is 4-(hexadecanoylsulfamoyl)butanoyl.
5. A compound according to any one of claims 1 -4, wherein S has a structure selected among formulas Na, Nd and lie.
6. A compound according to any one of claims 1 -5, wherein Z3 is NIe.
7. A compound according to any one of claims 1 -6, wherein Z2 is Ser, Thr, Dab or Dap.
8. A compound according to any one of claims 1 -7, wherein Z2 is Ser or Dab.
9. A compound according to any one of claims 1 -8, wherein Z1 is His, GIn, Arg, homoArg, Lys, Orn, Dab or Dap.
10. A compound according to any one of claims 1 -9, wherein Z1 is His, GIn or Arg.
1 1 . A compound according to claim 1 wherein
R1 is Ci4-i8alkanoyl or 4-(hexadecanoylsulfamoyl)butanoyl; Z\ if present, is His, Arg, homoArg, Lys, GIn, Asn, Orn, Dab or Dap;
Z2, if present, is Ser, Thr, Dab or Dap;
Z3 is NIe;
X1 is GIu, Asp or Cys;
X2 is Ser, Hyp, Cit, Dap, Asn, GIn or (4-thiazolyl)Ala; X3 is D-Phe;
X4 is Trp;
X5 is Lys when X1 is GIu or Asp, or X5 is Cys when X1 is Cys; and
N(FT)2 is NH2.
12. A compound according to claim 11 , wherein Z1, if present, is His, Arg or GIn.
13. A compound according to claim 11 or 12, wherein Z2, if present, is Ser, Thr or Dab.
14. A compound according to claim 12 or 13, wherein X2 is Ser, Hyp, Asn, GIn or Dap.
15. A compound according to claim 14, wherein X2 is Hyp.
16. A compound according to claim 14, wherein X2 is Asn.
17. A compound according to claim 14, wherein X2 is GIn.
18. A compound according to claim 14, wherein X2 is Dap.
19. A compound according to any one of claims 11 -18, wherein S has a structure selected among formulas Na, Nd and lie.
20. A compound according to claim 1 , selected from the group consisting of: 2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-Lys]- NH2, 2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Gln-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-Lys]-
NH2,
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Asn-D-Phe-Arg-Trp-Lys]-NH2,
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu- Hyp-D-Phe-Arg-Trp-Lys]-NH2,
3-[2-(2-{2-[2-(hexadecanoylamino)ethoxy]ethoxy}ethoxy)ethoxy]propanoyl-His-Dab-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2,
2-[2-(2-{2-[2-(hexadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Dap-D-Phe-Arg-Trp-Lys]-NH2, 2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-Arg-Ser-Nle-c[Glu-Asn-D-Phe-Arg-Trp-Lys]-
NH2,
2-[2-(hexadecanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Asp-Hyp-D-Phe-Arg-Trp-Lys]-
NH2,
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Dab-Nle- c[Glu-Asn-D-Phe-Arg-Trp-Lys]-NH2,
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Ser-D-Phe-Arg-Trp-Lys]-NH2,
2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Gln-D-Phe-Arg-Trp-Lys]-NH2, 2-[2-(2-{2-[2-(tetradecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-
Cit-D-Phe-Arg-Trp-Lys]-NH2,
2-[2-(4-(hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-Dap-D-Phe-
Arg-Trp-Lys]-NH2,
{2-[2-(2-(hexadecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}methoxyacetyl-Ser-Nle-c[Asp- Asn-D-Phe-Arg-Trp-Lys]-NH2, {2-[2-(2-(tetradecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}methoxyacetyl-Ser-Nle-c[Asp-
Hyp-D-Phe-Arg-Trp-Lys]-NH2,
4-{2-[2-(2-(octadecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}butanoyl-Dab-Nle-c[Asp-Hyp-
D-Phe-Arg-Trp-Lys]-NH2, 4-{2-[2-(2-(tetradecanoylamino)ethoxy)ethoxy]ethylcarbamoyl}butanoyl-Thr-Nle-c[Glu-Gln-D-
Phe-Arg-Trp-Lys]-NH2,
3-(2-{2-[2-(2-(hexadecanoylamino)ethoxy)ethoxy]ethoxy}ethoxy)propionyl-Nle-c[Glu-Dap-D-
Phe-Arg-Trp-Lys]-NH2,
2-[2-(4-(hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp- D-Phe-Arg-Trp-Lys]-NH2,
2-[2-(4-(tridecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Glu-Hyp-D-
Phe-Arg-Trp-Lys]-NH2, and
2-[2-(4-(hexadecanoylsulfamoyl)butanoylamino)ethoxy]ethoxyacetyl-His-Ser-Nle-c[Cys-Hyp-
D-Phe-Arg-Trp-Cys]-NH2.
21 . A method of delaying the progression from IGT to type 2 diabetes, comprising adminis¬ tering to a patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combination with one or more additional therapeutically active compounds.
22. A method of delaying the progression from type 2 diabetes to insulin-requiring diabetes, comprising administering to a patient in need thereof an effective amount of a compound ac¬ cording to any one of claims 1 -20, optionally in combination with one or more additional therapeutically active compounds.
23. A method of treating obesity or preventing overweight, comprising administering to a pa¬ tient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combination with one or more additional therapeutically active compounds.
24. A method of regulating appetite, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combina¬ tion with one or more additional therapeutically active compounds.
25. A method of inducing satiety, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combina¬ tion with one or more additional therapeutically active compounds.
26. A method of preventing weight gain after successfully having lost weight, comprising ad¬ ministering to a patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combination with one or more additional therapeutically ac¬ tive compounds.
27. A method of increasing energy expenditure, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combination with one or more additional therapeutically active compounds.
28. A method of treating a disease or state related to overweight or obesity, comprising ad- ministering to a patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combination with one or more additional therapeutically ac¬ tive compounds.
29. A method of treating bulimia, comprising administering to a patient in need thereof an ef- fective amount of a compound according to any one of claims 1 -20, optionally in combination with one or more additional therapeutically active compounds.
30. A method of treating binge-eating, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combi- nation with one or more additional therapeutically active compounds.
31 . A method of treating a disease or state selected from atherosclerosis, hypertension, dia¬ betes, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart dis¬ ease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of premature death, comprising administering to a patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combination with one or more additional therapeutically active compounds.
32. A method of treating, in an obese patient, a disease or state selected from type 2 diabe- tes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder dis- ease, gall stone, osteoarthritis, cancer, sexual dysfunction and risk of premature death, com¬ prising administering to an obese patient in need thereof an effective amount of a compound according to any one of claims 1 -20, optionally in combination with one or more additional therapeutically active compounds.
33. A method according to any one of claims 21 -32, wherein said additional therapeutically active compound is selected from antidiabetic agents, antihyperlipidemic agents, antiobesity agents, antihypertensive agents and agents for the treatment of complications resulting from, or associated with, diabetes.
34. A method according to any one of claims 21 -33, wherein said compound according to any one of claims 1 -20 is administered to said patient in a unit dosage form comprising from about 0.05 mg to about 1000 mg of said compound.
35. A method of activating MC4 in a subject, the method comprising administering to said subject an effective amount of a compound according to any one of claims 1 -20.
36. A method according to any one of claims 21 -35, wherein said compound according to any one of claims 1 -20 is administered parenterally by nasal, pulmonary or sublingual ad- ministration.
37. A compound according to any one of claims 1 -20 for use in therapy.
38. A pharmaceutical composition comprising a compound according to any one of claims 1 -20.
39. The use of a compound according to any one of claims 1 -20 in the manufacture of a me¬ dicament for: delaying the progression from IGT to type 2 diabetes; delaying the progression from type 2 diabetes to insulin-requiring diabetes; treating obesity or preventing overweight; regulating appetite; inducing satiety; preventing weight regain after successful weight loss; increasing energy expenditure; treating a disease or state related to overweight or obesity; treating bulimia; treating binge-eating; treating atherosclerosis, hypertension, type 2 diabe¬ tes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder dis¬ ease, gall stone, os-teoarthritis, cancer, sexual dysfunction or risk of premature death; or treating, in an obese patient, a disease or state selected from type 2 diabetes, impaired glu- cose tolerance (IGT), dyspilidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction or risk of premature death.
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