WO2011104379A1 - Peptides for treatment of obesity - Google Patents

Peptides for treatment of obesity Download PDF

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Publication number
WO2011104379A1
WO2011104379A1 PCT/EP2011/052910 EP2011052910W WO2011104379A1 WO 2011104379 A1 WO2011104379 A1 WO 2011104379A1 EP 2011052910 W EP2011052910 W EP 2011052910W WO 2011104379 A1 WO2011104379 A1 WO 2011104379A1
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Prior art keywords
pro
lys
glu
arg
compound according
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PCT/EP2011/052910
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French (fr)
Inventor
Kilian Waldemar Conde Frieboes
Birgit Schjellerup Wulff
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Novo Nordisk A/S
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Publication of WO2011104379A1 publication Critical patent/WO2011104379A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—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]
    • 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 peptides with improved solubility which are specific to one or more melanocortin receptors, to the use of said peptides in therapy, to methods of treatment comprising administration of said peptides to patients, and to the use of said peptides in the manufacture of medicaments.
  • Obesity is a well known risk factor for the development of common diseases such as atherosclerosis, hypertension, type 2 diabetes, dyslipidaemia, coronary heart disease, gallbladder disease, osteoarthritis, premature death, certain types of cancer and various other malignancies. It also causes considerable problems through reduced motility and decreased quality of life.
  • the prevalence of obesity has increased significantly in the past few decades. Only a few pharmacological treatments are available to date, namely Sibutramine (Abbot, acting via serotonergic and noradrenaline mechanisms), Orlistat (Roche and GlaxoSmithKline, works by reducing fat uptake from the gut). Because obesity represents a very high risk factor in serious and even fatal common diseases, its treatment should be a high public health priority and there is a need for pharmaceutical compounds useful in the treatment of obesity.
  • Pro-opiomelanocortin is the precursor of the melanocortin family of pep- tides, which include o, ⁇ - and ⁇ -melanocyte stimulating hormone (MSH) peptides and adrenocorticotropic hormone (ACTH), as well as other peptides such as ⁇ -endorphin.
  • MSH melanocortin family of pep- tides
  • ACTH adrenocorticotropic hormone
  • POMC is expressed in neurons of the central and peripheral nervous system and in the pituitary.
  • MC1-5 receptors Five melanocortin receptor subtypes, MC1-5 receptors have been identified. MC1 , MC2 and MC5 receptors are mainly expressed in peripheral tissues, whereas MC3 and MC4 receptors are mainly centrally expressed. MC3 receptors are also expressed in several pe- ripheral tissues. In addition to being involved in energy homeostasis, MC3 receptors have also been suggested to be involved in several inflammatory diseases. It has been suggested that MC5 receptors are involved in exocrine secretion and in inflammation.
  • MC4 receptors have been shown to be involved in the regulation of body weight and feeding behavior, as MC4 knock-out mice develop obesity [Huzar et al., Cell 88, 131-141 (1997)] and common variants near MC4 receptor have been found to be associated with fat mass, weight and risk of obesity [Loos et al. Nat Genet., 40(6):768-75 (2008)]. Furthermore, studies with mice showed that overexpression in the mouse brain of the melanocortin receptor antagonists agouti protein and agouti-related protein (AGRP), led to the development of obesity [Kleibig et al., PNAS 92, 4728-4732 (1995)]. Moreover, icv injection of a C-terminal fragment of AGRP increases feeding and antagonizes the inhibitory effect of ⁇ -MSH on food intake.
  • AGRP agouti protein and agouti-related protein
  • MC4 receptor agonists could serve as anorectic drugs and/or energy expenditure increasing drugs 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 activation of MC4 receptor.
  • MC4 receptor antagonists may be useful in the treatment of cachexia or anorexia, of waisting in frail elderly patients, chronic pain, neuropathy and neurogenic inflammation.
  • melanocortin receptor modulators of which 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 98/271 13. Hadley [Pigment Cell Res.
  • the present invention relates to novel peptides which are specific to one or more melanocortin receptors with improved water solubility at neutral pH, to the use of said peptides in therapy, to methods of treatment comprising administration of said peptides to patients, and to the use of said peptides in the manufacture of medicaments.
  • the invention relates pep- tide agonists highly specific for melanocortin receptor wherein the compound is a peptide according to formula I:
  • R 1 represents Ci -3 alkanoyl
  • X1 represents Asp, Glu, Lys, Orn, Dab, Dap, Cys, homo-Cys or Penicillamine.
  • X2 represents Asp, Glu, Lys, Orn, Dab, Dap, Cys, homo-Cys or Penicillamine.
  • X 1 and X 2 are joined, rendering the compound of formula I cyclic, either via a disulfide bridge, X 1 and X 2 being independently Cys, homo-Cys or Penicillamine, or via a lactam bridge, either from X 1 being Asp or Glu and X 2 being Lys, Orn, Dab or Dap, or from X 1 being Lys, Orn, Dab or Dap and X 2 being Asp or Glu.
  • Z 1 represents Pro, D-Pro, Hyp or D-Hyp
  • Z 2 represents Pro, D-Pro, Hyp or D-Hyp
  • Z 3 represents Lys, Arg , His, D-Lys, D-Arg or D-His;
  • Z 4 represents Glu, Asp, D-Glu, D-Asp, Ser or D-Ser;
  • Z 5 represents Lys(bis carboxymethyl
  • Z 6 is absent or represents a peptide fragment comprising one to four amino acid residues derived from Met, D-Met, Leu, D-Leu, Nle, D-Nle, lie, D-lle, Val or D-Val;
  • R 2 represents -OR or -NR 2 , R being hydrogen or Ci -3 alkyl.
  • the invention further relates to the use of compounds of the invention in therapy, to pharmaceutical compositions comprising compounds of the invention, and to the use of compounds of the invention in the manufacture of medicaments. DESCRIPTION OF THE INVENTION
  • a compound according to embodiment 1 wherein X1 represents Cys, homo-Cys or Penicillamine.
  • a compound according to embodiment 1 selected from the group consisting of:
  • the present invention also encompasses combinations of two or more embodiments of compounds 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 relation 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 binding affinity of a compound with respect to MC1 , MC3, MC5 and MC4 may be determined by comparing the Ki from an MC1 , MC3 or MC5 binding assay as described below under "Assay IV” (MC1 ), “Assay VII” (MC3) and “Assay VIII” (MC5), respectively, with Ki from an MC4 binding assay as described below under “Assay V” (MC4). If the binding affinity of a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times greater () with respect to MC4 than with respect to MC1 , it is deemed to be a selective MC4 agonist with respect to MC1 .
  • binding affinity of a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times greater (higher) with respect to MC4 than with respect to MC3, it is deemed to be a selective MC4 agonist with respect to MC3. If the binding affinity of a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times greater with respect to MC4 than with respect to MC5, it is deemed to be a selective MC4 agonist with respect to MC5.
  • the agonistic potency of a compound with respect to MC3, MC4 and MC5 may be determined in functional assays as described in "Assay II” (MC3 and MC5), "Assay IX” (MC3) 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 respect to MC3, it is deemed to be a selective MC4 agonist with respect to MC3. If a compound 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 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 binding affinity determined for MC1 as described in "Assay IV" with the binding affinity for MC3 determined as described in "Assay VII". If the binding affinity of 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 affinity determined as described in "Assay VII and VIII". If the binding affinity of a compound is more than 10 times, such as more the 50 times, e.g. more than 100 times greater with re- spect to MC3 than with respect to MC5, it is deemed to be a selective MC3 agonist with re- spect to MC5.
  • the MC4 selectivity of a compound with respect to MC3 and MC5 is determined as discussed above.
  • compounds of the present invention modulate melanocortin receptors, and they are therefore 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 activation of MC4.
  • ITT impaired glucose tolerance
  • a method of delaying the progression from IGT to type 2 diabetes comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of delaying the progression from non-insulin-requiring type 2 diabetes to insulin-requiring type 2 diabetes comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of treating obesity or preventing overweight comprising administering to a patient in need thereof an effective amount of a compound according to any of embodi- ments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of regulating appetite comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of inducing satiety comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of preventing weight gain after successfully having lost weight comprising administering to a patient in need thereof an effective amount of a compound accord- ing to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of increasing energy expenditure comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1- 28, optionally in combination with one or more additional therapeutically active compounds.
  • a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart dis-ease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea and risk of premature death.
  • ITT impaired glucose tolerance
  • dyslipidemia CAD
  • coronary heart dis-ease CAD
  • gallbladder disease gall stone
  • osteoarthritis cancer
  • cancer sexual dysfunction
  • hypthalamic amenorrhea hypthalamic amenorrhea and risk of premature death.
  • 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 according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of treating bulimia comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of treating binge-eating comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
  • a method of treating a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea and risk of premature death comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
  • a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea and risk of premature death
  • a method for providing neuronal protection for having an effect on ischemic heart disease, cerebral ischemia or anti-inflammatory effects and for the treatment of autoimmune diseases, e.g. multiple sclerosis., comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
  • a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea, risk of premature death, neuronal protection, effect in ischemic heart disease or anti-inflammatory effects.
  • a method of treating, in an obese patient, a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea, risk of premature death, neuronal protection, effect in ischemic heart disease or anti-inflammatory effects, comprising administering to an obese patient in need thereof an effective amount of a compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
  • said additional therapeutically active compound is selected from antidiabetic agents, antihyperlipi- demic agents, antiobesity agents, antihypertensive agents and agents for the treatment of complications resulting from, or associated with, diabetes.
  • a method of activating MC4 in a subject comprising administering to said subject an effective amount of a compound according to any of embodiments 1-28.
  • 62. A method according to any of embodiments 36-42, 48-52, 54, 56 and 58-61 , wherein said compound according to any of embodiments 1 -28 is administered parenterally, orally, nasally, buccally or sublingually.
  • a pharmaceutical composition comprising a compound according to any of em- bodiments 1-35, 43-47, 53 and 55.
  • a pharmaceutical composition according to embodiment 64 further comprising one or more additional therapeutically active compounds or substances.
  • a compound according to any of embodiments 1 -28 for use in therapy is 69.
  • a compound according to any of embodiments 1-28 in the manufacture of a medicament for delaying the progression from impaired glucose tolerance (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 diabetes, IGT, dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea or risk of premature death; or treating, in an obese patient, a disease or state selected from type 2 diabetes, IGT, dyspilidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, risk of premature death; for providing neuronal pro- tection, for having an effect on ischemic heart disease,
  • IGT impaired
  • MC4 agonists Compounds of the invention that act as 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 agonists have antipyretic effects, and both have been suggested to be involved in peripheral nerve regeneration. MC4 agonists are also known to reduce stress response. In addition to treating drug abuse, treating or preventing hemorrhagic shock, and reducing stress response, compounds of the invention may also be of value in treating alcohol abuse, treating stroke, treating ischemia and protecting against neuronal damage.
  • the compound of the present invention may be administered alone. However, it may also be administered in combination with one or more additional therapeutically active agents, substances or compounds, 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, preferably from about 0.01 to about 10mg/kg body weight, more preferably from about 0.01 to about 5 mg/kg body weight per day, e.g. from about 0.05 to about 10 mg/kg body weight per day or from about 0.03 to about 5mg/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.
  • a typical unit dosage form intended for oral administration one or more times per day, such as from one to three times per day, may suitably contain from about 0.05 to about 1000mg, preferably from about 0.1 to about 500mg, such as from about 0.5 to about 200mg of a compound of the invention.
  • Compounds of the invention comprise compounds that are believed to be well- suited to administration with longer intervals than, for example, once daily, thus, appropriately formulated compounds of the invention may be suitable for, e.g., twice-weekly or once- weekly administration by a suitable route of administration, such as one of the routes disclosed herein.
  • compounds of the present invention may be administered or applied in combination with one or more additional therapeutically active compounds or substances, and suitable additional compounds or substances may be selected, for example, from antidiabetic 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, insulin derivatives or analogues, GLP-1 (glucagon like peptide-1 ) derivatives or analogues [such as those disclosed in WO 98/08871 (Novo Nordisk A/S), which is incorporated herein by reference, or other GLP-1 analogues such as exenatide (Byetta, Eli Lilly/Amylin; AVE0010, Sanofi-Aventis), taspoglutide (Roche), albiglutide (Syncria, GlaxoSmithKline), amylin, amylin analogues (e.g. SymlinTM/Pramlintide) as well as orally active hypoglycemic agents.
  • GLP-1 glucagon like peptide-1
  • analogues such as those disclosed in WO 98/08871 (Novo Nordisk A/S), which is incorporated herein by reference, or other GLP-1 analogues such as exenatide (B
  • Suitable orally active hypoglycemic agents include: metformin, imidazolines; sulfonylureas; biguanides; meglitinides; oxadiazolidinediones; thiazolidinediones; insulin sensitizers; oglucosidase inhibitors; 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) which are incorporated herein by reference; potassium channel openers such as ormitiglinide; potassium channel blockers such as nateglinide or BTS-67582; glucagon receptor antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), all of which are incorporated herein by reference; GLP-1 receptor agonists such as those disclosed in WO 00/42026 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference; amylin analogues (agonists on the amylin receptor); DPP- IV (dipeptidyl peptidase-IV) inhibitors; PTPase (protein tyrosine phosphatase) inhibitors; glu- cokinase activ
  • Suitable additional therapeutically active substances include insulin or insulin analogues; sulfonylureas, e.g. tolbutamide, chlorpropamide, tolazamide, gliben- clamide, glipizide, glimepiride, glicazide or glyburide; biguanides, e.g. metformin; and meglit- inides, e.g. repaglinide or senaglinide/nateglinide.
  • suitable additional therapeutically active substances include thiazolidinedione insulin sensitizers, e.g.
  • troglitazone ciglitazone, pioglitazone, rosiglitazone, isaglitazone, darglitazone, englitazone, CS-01 1/CI-287 or T 174, or the compounds disclosed in WO 97/41097 (DRF-2344), WO 97/41 1 19, WO 97/41 120, WO 00/41 121 and WO 98/45292 (Dr. Reddy's Research Foundation), the contents of all of which are incorporated herein by reference.
  • 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 (NN622/
  • suitable additional therapeutically active substances include: oglucosidase 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 ⁇ -cells, e.g. tolbutamide, glibenclamide, glipizide, glicazide, BTS-67582 or repag- linide;
  • oglucosidase 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
  • antihyperlipi- demic agents and antilipidemic agents, e.g. cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
  • antilipidemic agents e.g. cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol or dextrothyroxine.
  • Such substances may be selected from the group consisting of CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y receptor 1 and/or 5) antagonists, MC3 (melanocortin receptor 3) agonists, MC3 antagonists, MC4 (melanocortin receptor 4) agonists, orexin receptor antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin agonists, neuromedin U analogues (agonists on the neuromedin U receptor subtypes 1 and 2), ⁇ 3 adrenergic agonists such as CL-316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140
  • CART cocaine amphetamine regulated transcript
  • NPY neuropeptide Y receptor 1 and
  • antiobesity agents are bupropion (antidepressant), topiramate (anti- convulsant), ecopipam (dopamine D1/D5 antagonist) and naltrexone (opioid antagonist), and combinations thereof. Combinations of these antiobesity agents would be e.g.: phenter- mine+topiramate, bupropion sustained release (SR)+naltrexone SR, zonisamide SR and bupropion SR.
  • suitable antiobesity agents for use in a method of the invention as additional therapeutically active substances in combination with a compound of the invention are leptin and analogues or derivatives of leptin.
  • Suitable antiobesity agents are serotonin and norepinephrine reuptake inhibitors, e.g. sibutramine.
  • Suitable antiobesity agents are lipase inhibitors, e.g. orlistat. Still further embodiments of suitable antiobesity agents are adrenergic CNS stimu- lating agents, e.g. dexamphetamine, amphetamine, phentermine, mazindol, phendi- metrazine, diethylpropion, fenfluramine or dexfenfluramine.
  • antihypertensive agents examples include antihypertensive agents.
  • antihypertensive agents are ⁇ -blockers such as alpre- nolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting en- zyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, ni- modipine, diltiazem and verapamil, and oblockers such as doxazosin, urapidil, prazosin and terazosin.
  • ⁇ -blockers such as alpre- nolol, atenolol, timolo
  • 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
  • a compound of the invention for a purpose related to treatment or prevention of obesity or overweight, i.e. related to reduction or prevention of excess adiposity, it may be of relevance to employ such administration in combination with surgical intervention for the purpose of achieving weight loss or preventing weight gain, e.g. in combination with bariatric surgical intervention.
  • Examples of frequently used bariatric surgical techniques include, but are not limited to, the following: vertical banded gastroplasty (also known as "stomach stapling"), wherein a part of the stomach is stapled to create a smaller pre-stomach pouch which serves as a new stom- ach; gastric banding, e.g. using an adjustable gastric band system (such as the Swedish Adjustable Gastric Band (SAGB), the LAP-BANDTM or the MIDbandTM), wherein a small pre- stomach pouch which is to serve as a new stomach is created using an elastomeric (e.g. silicone) band which can be adjusted in size by the patient ; and gastric bypass surgery, e.g. "Roux-en-Y” bypass wherein a small stomach pouch is created using a stapler device and is connected to the distal small intestine, the upper part of the small intestine being reattached in a Y-shaped configuration.
  • SAGB Swedish Adjustable Gastric Band
  • Another technique which is within the scope of the term "bariatric surgery” and variants thereof (e.g. "weight-loss surgery”, “weight-loss surgical intervention” “weight-loss surgical procedure”, “bariatric surgical intervention”, “bariatric surgical procedure” and the like) as employed in the context of the present invention is gastric balloon surgery, wherein an inflatable device resembling a balloon is introduced into the stomach and then inflated, the purpose being to reduce the accessible volume within the stomach to create a sensation of satiety in the patient at an earlier stage than normal during food intake, and thereby cause a reduction in food intake by the patient.
  • the EndoBarrier Gastrointestinal Liner works by creating a physical barrier between ingested food and the intestinal wall, which may change how hormonal signals that originate in the intestine are activated, mimicking the effects of gastric bypass pro- cedure. Weight loss is enhanced by combining the EndoBarrier Gastrointestinal Liner with the customizable EndoBarrier Flow Restrictor. Combination of the EndoBarrier Gastrointestinal Liner with the EndoBarrier Flow Restrictor was shown to affect multiple mechanisms of action.
  • Non-limiting ex- amples of additional, irreversible and consequently generally less frequently employed techniques of relevance in the present context include biliopancreatic diversion and sleeve gastrectomy (the latter of which may also be employed in conjunction with duodenal switch), both of which entail surgical resection of a substantial portion of the stomach.
  • a compound of the invention may take place for a period prior to carrying out the bariatric surgical intervention in question and/or for a period of time subsequent thereto. In many cases it may be preferable to begin administration of a compound of the invention after bariatric surgical intervention has taken place.
  • MC4 agonists long-acting melanocortin 4 receptor agonists
  • MC4 agonists comprising a peptide part and an albumin binding fatty acid or alkyltetrazole chain as described in e.g. WO2007/009894, WO2008/087186 and WO2008/087187.
  • These compounds have more basic than acidic residues, resulting in good solubility at acidic pH, but poor solubility at neutral or weakly basic pH. Solubility at pH from 6 to 9 is considered to be an advantage, since this could improve local tolerance and make it possible to combine the MC4 agonist with other drugs, soluble only at neutral to weakly basic pH.
  • the compounds of the present invention have a novel chemical structure.
  • the compounds of the present invention comprise a macrocyclic peptide part, a linear peptide part linked to the C-terminus of the macrocycle.
  • the peptide backbone has some similarity with the hormone ⁇ -MSH.
  • the primary function of the macrocyclic peptide part is to bind to the MC4 receptor, resulting in activation of the MC4 receptor, with selectivity for MC4 over MC1 , MC3 and MC5 receptors.
  • the linear peptide part is also important for receptor binding and selectivity and is therefore limited to certain amino acid sequences.
  • the desired balance between MC4 potency and receptor selectivity is achieved by the covalent combination of macrocyclic peptide part, linear peptide part linked to the C-terminus of the macrocycle, according to the above- described embodiments of compounds of the invention.
  • the compounds of the present invention have high MC4 receptor potency and higher MC4 receptor selectivity in relation to previously disclosed peptides in the art.
  • the compounds of the present invention can be a soluble MC4 receptor agonist, for example with solubility of at least 0.2 mmol/l, at least 0.5 mmol/l, at least 2 mmol/l, at least 4 mmol/l, at least 8 mmol/l, at least 10 mmol/l, or at least 15 mmol/l, at pH 7.5.
  • soluble refers to the solubility of a compound in water or in an aqueous salt or aqueous buffer solution, for example a 10 mM phosphate solution, or in an aqueous solution containing other compounds, but no organic solvents.
  • obesity implies an excess of adipose tissue.
  • BMI body weight in kilograms divided by the square of the height in meters
  • C x-y alkyl e.g. C 6- 2oalkyl
  • alkyl refers to a straight-chain, branched and/or cyclic, saturated monovalent hydrocarbon radical.
  • alkenyl refers to a straight-chain, branched and/or cyclic, monovalent hydrocarbon radical comprising at least one carbon-carbon double bond.
  • alkynyl refers to a straight-chain, branched and/or cyclic, monovalent hydrocarbon radical comprising at least one carbon-carbon triple bond, and it may optionally also comprise one or more carbon-carbon double bonds.
  • alkylene refers to a straight-chain, branched and/or cyclic, saturated bivalent hydrocarbon radical.
  • alkenylene refers to a straight-chain, branched and/or cyclic, bivalent hydrocarbon radical comprising at least one carbon-carbon double bond.
  • alkynylene refers to a straight-chain, branched and/or cyclic, bivalent hydrocarbon radical comprising at least one carbon-carbon triple bond, and it may optionally also comprise one or more carbon-carbon double bonds.
  • alkoxy as used herein is intended to indicate a radical of the formula - OR', wherein R' is alkyl as indicated above.
  • 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 aro- matic.
  • 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 table of the elements, which includes fluorine, chlorine, bromine and iodine (corresponding to fluoro, chloro, bromo and iodo substituents, respectively).
  • tetrazol-5-yl is intended to indicate 1 H-tetrazol-5-yl or 2H-tetrazol-5-yl.
  • common rules for peptide nomenclature based on the three letter amino acid code apply, unless exceptions are specifically indicated. Briefly, the central portion of the amino acid structure is represented by the three letter code (e.g. Ala, Lys) and L-configuration is assumed, unless D-configuration is specifically indicated by "D-" followed by the three letter code (e.g. D-Ala, D-Lys).
  • a substituent at the amino group replaces one hydrogen atom and its name is placed before the three letter code, whereas a C-terminal substituent replaces the carboxylic hydroxy group and its name appears after the three letter code.
  • agonist is intended to indicate a substance (ligand) that activates 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 antagonist, partially inhibiting the effect of a full agonist.
  • Receptor neutral antagonists which block the action of an agonist, but do not affect the receptor-constitutive activity.
  • Receptor inverse agonists which block the action of an agonist and at the same time attenuate 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 accept- able acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts.
  • Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hy- droiodic, phosphoric, sulfuric and nitric acids, and the like.
  • suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethyl- ene-salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like.
  • compositions include the pharmaceutically acceptable salts listed in J. Pharm. Sci. (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 routine experimentation, by constructing a matrix of values and testing different points in the matrix, all of which is within the level of ordinary skill of a trained physician or veterinarian.
  • treatment refers to the management 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 condition from which the patient is suffering, such as administration of the active compound ⁇ ) in question to alleviate symptoms or complications thereof, to delay the progression of the disease, 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 between a solute (in casu, a compound according to the present invention) and a solvent.
  • Solvents may include, by way of example, water, ethanol, or acetic acid.
  • amino acid abbreviations used in the present context have the following meanings:
  • BCMA [bis(carboxymethyl)amino]acetyl
  • one aspect of the present invention provides a pharmaceutical composition (formulation) comprising a compound of the present invention.
  • a pharmaceutical composition comprising a compound of the present invention.
  • Appropriate embodiments 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), stabilizers) and/or surfactant(s).
  • the pharmaceutical formulation is an aqueous formulation, i.e.
  • aqueous formulation in the present context may normally be taken to indicate a formulation comprising at least 50 % by weight (w/w) of water.
  • a formulation is typically a solution or a suspension.
  • 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 invention 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 dissolution.
  • the invention relates to a pharmaceutical composition (formula- tion) comprising 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.
  • a pharmaceutical composition comprising 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, 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,
  • 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.
  • 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, tarta
  • 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).
  • 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-hydroxy
  • 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 pharmaceutical 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” I 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 packaged for storage, whether in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject.
  • dried form is meant the product obtained when a liquid pharmaceutical composition or formulation is dried by freeze-drying (i.e., lyophilization; see, for example, Williams and Polli (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: 1 169-1206; and Mumenthaler et al. (1994) Pharm. Res.
  • freeze-drying i.e., lyophilization
  • 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
  • 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 storage 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 pre- sent 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 mixtures thereof) of a particular amino acid e.g. methionine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan or threonine, and mixtures thereof
  • a particular amino acid e.g. methionine, histidine, arginine, lysine, isoleucine, 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 decreasing aggregate formation by the oligo- or polypeptide during storage of liquid pharmaceutical compositions of the invention.
  • Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl-L-arginine.
  • Suitable methionine ana- logues include 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 acting as the therapeutic agent is a peptide comprising at least one methionine residue susceptible to such oxidation.
  • the term "inhibit" in this context refers to minimization of ac- cumulation of methionine-oxidized species over time. Inhibition of methionine oxidation results in increased 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.
  • 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 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.
  • lingual, sublingual, buccal in the mouth, oral, in the stomach and intestine, nasal, pulmonary (for example through the bronchioles and alveoli or a combination thereof), epidermal, der- mal, transdermal, vaginal, rectal, ocular (for example through the conjunctiva), uretal and parenteral.
  • compositions of the present invention may be administered in various dosage forms, for example in the form of solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, cap- sules (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 situ- transforming solutions (for example in situ gelling, in situ setting, in situ precipitating or in situ crystallizing), infusion solutions or implants.
  • in situ- 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 compli- ance, or any combination thereof.
  • carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to: polymers, for example cellulose and derivatives; polysaccharides, for example dextran and derivatives, starch and derivatives; polyvinyl alcohol); acrylate and methacrylate polymers; polylactic and polyglycolic acid and block co-polymers thereof; polyethylene glycols; carrier proteins, for example albumin; gels, for example thermogelling systems, 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 behavior in lipid-water systems; polymeric micelles; multiple emulsions (self- emulsifying, self-microemulsifying); cyclodextrins and derivatives thereof; and dendrimers.
  • polymers for example cellulose and derivatives
  • polysaccharides for example dextran and derivatives, star
  • 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, sustained-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 sustained-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 subcu- taneous administration.
  • examples of useful controlled release systems and compositions are those containing hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles,
  • Methods for producing controlled-release systems useful for compositions of the present invention include, but are not limited to, crystallization, condensation, co- crystallization, precipitation, co-precipitation, emulsification, dispersion, high-pressure ho- mogenisation, encapsulation, spray-drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes.
  • Crystallization condensation, co- crystallization, precipitation, co-precipitation, emulsification, dispersion, high-pressure ho- mogenisation, encapsulation, spray-drying, microencapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes.
  • 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 can 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 transdermal administration (e.g. by needle-free injection or via a patch, such as an iontopho- retic patch) or transmucosal (e.g. buccal) administration.
  • stabilized formulation refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.
  • physical stability in the context of a formulation containing an oligo- or polypeptide refers to the tendency 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 sur- faces 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 measurements 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.
  • suitable containers e.g. cartridges or vials
  • the turbidity of a formulation is characterized by a visual score ranking the de- gree of turbidity, for instance on a scale from 0 to 3 (in that a formulation showing no turbidity corresponds to a visual score 0, whilst a formulation showing visual turbidity in daylight corresponds to visual score 3).
  • a formulation is normally classified physically unstable with respect 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.
  • hydrophobic patch probes that bind preferentially to exposed hydrophobic patches of a polypeptide.
  • the hydrophobic patches are generally buried 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, spec- troscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenan- throline and the like.
  • Other spectroscopic probes are metal complexes of amino acids, such as cobalt complexes of hydrophobic amino acids, e.g. phenylalanine, leucine, isoleucine, methionine, valine, or the like.
  • chemical stability of a pharmaceutical formulation as used herein refers to chemical covalent changes in oligo- or polypeptide structure leading to formation of chemical degradation products with potentially lower biological potency and/or potentially increased immunogenicity 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 completely avoided and gradually increasing amounts of chemical degradation products may often 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 hydrolysed 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 substance are covalently bound to each other through transamidation and/or disulfide interactions, leading to formation of covalently bound dimer, oligomer or polymer degradation products (see, e.g., Stability of Protein Pharmaceuticals, Ahern. T.J. & 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 individual 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.
  • MC1 melanocortin receptor subtype 1 also denoted melanocortin receptor 1
  • MC2 melanocortin receptor subtype 2 also denoted melanocortin receptor 2
  • MC3 melanocortin receptor subtype 3 also denoted melanocortin receptor 3
  • MC4 melanocortin receptor subtype 4 also denoted melanocortin receptor 4
  • MC5 melanocortin receptor subtype 5 also denoted melanocortin receptor 5
  • PhiPr 2-phenylisopropyl 1 -methyl-1 -phenyl-ethyl
  • the peptide is synthesized according to the Fmoc strategy on an Applied Biosystems 433 peptide synthesizer on a 0.25 mmol or 1.0 mmol scale using the manufacturer supplied FastMoc UV protocols which employ the Fmoc protected amino acid (4 equivalents), HOBt (4 equivalents), HBTU (4 equivalents) and DIPEA (8 equivalents) in NMP, and UV monitoring of the deprotection of the Fmoc protection group. Piperidine in NMP is used for deprotection of the Fmoc protected amino acids.
  • the resin is extensively washed with DCM.
  • the resin is then washed with a premixed solution of DCM-triisopropylsilane-water- mercaptoethanol (92.5:2.5:2.5:2.5).
  • a mixture of TFA-triisopropylsilane-water- mercaptoethanol (92.5:2.5:2.5:2.5; at least 40 ml per mmol of resin) is added, and the mixture agitated for 3 hours before the resin is drained and the filtrate is collected.
  • the resin is washed with TFA-triisopropylsilane-water-mercaptoethanol (92.5:2.5:2.5:2.5) and the filtrate is collected.
  • ice-cold diethyl ether (10 x the volume of the cleavage mixture) is added and the resulting precipitate is filtered off, was
  • the crude peptide is dissolved in a suitable mixture of water and MeCN or N- methylformamide and purified by reversed-phase preparative HPLC (Waters Deltaprep 4000 or Gilson) on a column containing C18-silica gel. Elution is performed with an increasing gradient of MeCN in water containing 0.1 % TFA. Relevant fractions are checked by analytical HPLC or UPLC. Fractions containing the pure target peptide are mixed and concentrated under reduced pressure. The resulting solution is analyzed (HPLC, LCMS) and the product is quantified using a chemiluminescent nitrogen specific HPLC detector (Antek 8060 HPLC- CLND) or by measuring UV-absorption at 280 nm.
  • Fmoc-Lys(bis(tert-butoxycarbonylmet yl))-OH A solution of benzyl chloroformate (8.8 ml, 61.3 mmol) in DCM (50 mL) was added dropwise to a stirred solution of Fmoc-Lys(Boc)-OH (50 g, 53,6 mmol), DIPEA (27 ml, 78 mmol) and DMAP (650 mg, 5.3 mmol) in DCM (250 mL) at 0 °C. The mixture was stirred at 0 °C for 24 hrs; then it was washed with 5% aqueous citric acid and water (200 mL).
  • Fmoc-Lys(bis(ferf-butoxycarbonylmethyl))-OBn 54.24 g, 79 mmol was dissolved in methanol (500 ml_). Palladium on carbon (5 wt%, 3.35 g) was added to the solution. The suspension was stirred under hydrogen atmosphere at room temperature. After 3 hrs, the mixture was filtered through Celite and the filtrate was concentrated. The crude product was purified by flash column chromatography (silica gel, DCM/methanol 95:5) to afford the title compound Fmoc-Lys(bis(ferf-butoxycarbonylmethyl))-OH as white solid.
  • mice Male Sprague Dawley rats from Taconic Europe are used for the experiments.
  • the rats have a body weight of 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 180-200g.
  • the rats are housed in reversed light cycle (dark from 10 am to 10 pm two rats in each cage).
  • rats are moved to the FeedWin system, where the rats are placed in individual cages for acclimatisation. 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 have free access to food and water. During that period the animals are handled at least 3 times. 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.
  • Rats (app. 250g) are dosed at day 1 of the experiment, according to the table below, in the morning before onset of light. After dosing water-, food-intake and activity are registered by the FeedWin system. Data is collected each 15 minutes for 48 hours.
  • FeedWin The effects on food intake is measured using the FeedWin system (Ellegards Systems, Faaborg, Denmark) which contains 32 stations for individual and continuous registration of food and water intakes.
  • One station is defined by 1 cage with a metal lid plus 2 scales, one for food-intake and one for water-intake.
  • Food and water intake is estimated by measurements of the disappearance of preloaded amounts of food and water that are placed on the 2 scales on each side of the cage. Food intake data are recorded electronically. Movements of the animal during recordings of food intake are registered by passive infrared sensors (PIR) placed on top of the cage. These sensors detect body heat within a protected area. In addition two PIR cameras (one for food and one for water) register the time and number of episodes spent eating and drinking.
  • PIR passive infrared sensors
  • 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 supernatant removed. The cells are washed twice with stimulation buffer (5mM HEPES, 0.1 % ovalbumin, 0.005% TweenTM 20 and 0.5mM IBMX, pH 7.4), and then resuspended in stimulation buffer to a final concentration of 1x10 6 or 2x10 s cells/ml. 25 ⁇ of cell suspension is added to the microtiter plates containing 25 ⁇ of test compound or reference compound (all diluted in stimulation buffer).
  • stimulation buffer 5mM HEPES, 0.1 % ovalbumin, 0.005% TweenTM 20 and 0.5mM IBMX, pH 7.4
  • the plates are incubated for 30 minutes at room temperature (RT) on a plate-shaker set to a low rate of shaking.
  • the reaction is stopped by adding 25 ⁇ of acceptor beads with anti-cAMP, and 2 min later 50 ⁇ 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 5 o 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 a-MSH, and inhibited by increasing the amount of potential antagonist.
  • 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.
  • 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 supernatant removed. The cells are washed twice with stimulation buffer, and resuspended in stimulation buffer to a final concentration of 2x10 6 cells/ml (consumption thereof: 7 ml per 96-well microtiter plate). 50 ⁇ of cell suspension is added to the Flash Plate containing 50 ⁇ of test compound or reference compound (all diluted in PBS, 0.1 % HSA and 0.005% Tween). The mixture is shaken for 5 minutes and then allowed to stand for 25 minutes at RT.
  • the re- action is stopped by addition of 100 ⁇ Detection Mix per well (Detection Mix 1 1 ml Detection Buffer + 100 ⁇ ( ⁇ 2 ⁇ ) cAMP [ 125 l] tracer).
  • the plates are then sealed with plastic, shaken for 30 minutes, and allowed to stand overnight (or for 2 hours) and then counted in the Topcounter (2 min/well).
  • the assay procedure and the buffers are generally as described in the Flash Plate kit-protocol (Flash Plate® cAMP assay (NENTM Life Science Products, cat. No. SMP004)). However the cAMP standards are diluted in PBS with 0.1 % HSA and 0.005% TweenTM 20 and not in stimulation buffer.
  • the MC1 receptor binding assay was performed on BHK cell membranes stably expressing the MC1 receptor.
  • the assay was performed in a total volume of 250 ⁇ : 25 ⁇ of 125 NDP-a-MSH (22 pM in final concentration), 25 ⁇ of test compound/control and 200 ⁇ of cell membrane (25 ⁇ g/ml).
  • Test compounds were dissolved in DMSO.
  • Radioactively labeled ligand, membranes and test compounds were diluted in buffer: 25 mM HEPES, pH 7.4, 0.1 mM CaCI 2 , 1 mM MgS0 4 , 1 mM EDTA, 0.1 % HSA and 0.005% TweenTM 20.
  • HSA may be substituted with ovalbumin.
  • the samples were incubated at 30°C for 90min. in Costar round-botton microtiter plates. Incubation was terminated by filtration on a Packard harvester filtermate. Rapid filtration through Packard Unifilter-96 GF/B filters pre-treated with polyetylenimine (PerkinElmer 6005277). The filters were washed with ice-cold 0.9%NaCI 8- 10 times. The plates were air-dried at app. 55°c for 30min, and 50 ⁇ Microscint 0 (Packard, cat. No. 6013616) was added to each well. The plates were counted in a Topcounter (1 min/well). The data were analysed by non-linear regression analysis of binding curves, using the WindowsTM program GraphPadTM Prism (GraphPad Software, USA).
  • the assay was performed in 5 ml minisorb vials (Sarstedt No. 55.526) or in 96-well filter- plates (Millipore MADVN 6550), and using BHK cells expressing the human MC4 receptor using BHK cells stably expressing the human MC4 receptor.
  • the membranes were prepared from frozen or fresh cells that were homogenized in 20 mM HEPES pH 7.1 , 5 mM MgCI 2 and 1 mg/ml bacitracin and centrifuged at 15000 rpm at 4°C, 10 min in a Sorvall RC 5B plus, SS- 34 rotor.
  • the supernatant was discarded, and the pellets were re-suspended in buffer, homogenized and centrifuged two more times.
  • the final pellets were resuspended in the buffer mentioned above, and the protein concentration was measured and adjusted with buffer to 14 to 17 mg/ml and the membrane preparation were kept at -80°C until assay.
  • the assay was run directly on a dilution of this cell membrane suspension, without any further preparation.
  • the BHK cell membranes are kept at -80°C until assay, and the assay is run directly on a dilution of this cell membrane suspension, without further preparation.
  • the suspension is diluted to give maximally 10% specific binding, i.e. to approx. 50-100 fold dilution.
  • the assay was performed in a total volume of 200 ⁇ : 50 ⁇ of cell suspension, 50 ⁇ of 125 NDP-a-MSH ( « 79 pM in final concentration), 50 ⁇ of test compound and 50 ⁇ binding buffer (pH 7) mixed and incubated for 2 h at 25°C [binding buffer: 25 mM HEPES, pH 7.0, 1 mM CaCI 2 , 1 mM MgSC-4, 1 mM EGTA, 0.02% Bacitracin, 0.005% TweenTM 20 and 0.1 % HSA or, alternatively, 0.1 % ovalbumin (Sigma; catalogue No. A-5503)]. Test compounds were dissolved in DMSO and diluted in binding buffer.
  • Radiolabeled ligand and membranes were diluted in binding buffer. The incubation was stopped by dilution with 2 X 100 ⁇ ice-cold 0.9% NaCI. The radioactivity retained on the filters was counted using a Cobra II auto gamma counter.
  • 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. Light:dark 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 recording is continued in order to establish the action time of the test compound.
  • the MC3 receptor binding assay is performed on BHK cell membranes stably expressing the human MC3 receptor.
  • the human MC3 receptor is cloned by PCR and subcloned into pcDNA3 expression vector.
  • Cells stably expressing the human MC3 receptor are generated by transfecting the expression vector into BHK cells and using G418 to select for MC3 clones.
  • the BHK MC3 clones are cultured in DMEM with glutamax, 10% FCS, 1 % pen/strep and 1 mg/ml G418 at 37°C and 5% C0 2 .
  • the binding is performed on a membrane preparation prepared in the following way: The cells are rinsed with PBS and incubated with Versene for approximately 5 min before harvesting. The cells are flushed with PBS and the cell-suspension is centrifuged for 10 min at 2800xG. The pellet is resuspended in 20ml buffer (20mM Tris pH 7.2 + 5mM EDTA + 1 mg/ml Bacitracin (Sigma B-0125)) and homogenized with a glass-teflon homogenizer, 10 times and low speed. The cell suspension is centrifuged at 4°C, 4100xG for 20min. Pellet is resuspended in buffer and the membranes are diluted to a protein concentration of 1 mg/ml in buffer, aliquoted and kept at -80°C until use.
  • the assay is performed in a volume of 100 ⁇ . Mix in the following order 25 ⁇ test compound, 25 ⁇ 125 l-NDP-a-MSH (app. 60 000 cpm/well ⁇ 0.25nM in final concentration) and 50 ⁇ membranes (30 ⁇ g/well) and incubate in Costar round-bottom wells microtiter plate, (catalogue number 3365). Test-compounds are dissolved in DMSO or H 2 0.
  • Radioligand, membranes and test compounds are diluted in buffer; (25 mM HEPES pH 7.4, 1 mM CaCI2, 5 mM MgS04, 0.1 % Ovalbumin (Sigma A-5503), 0.005% Tween-20 and 5% Hydroxypropyl- ⁇ - cyclodextrin 97%, (Acros organics, code 297561000).
  • the assay mixture is incubated for 1 h at 20-25°C. Incubation is terminated by filtration on a Packard harvester filtermate 196. Rapid filtration through Packard Unifilter-96 GF/B filters pre-treated for 1 h with 0.5% poly- ethylenimine is carried out.
  • the filters are washed with ice-cold 0.9% NaCI 8-10 times.
  • the plate is air dried at 55°C for 30 min, and 50 ⁇ Microscint 0 (Packard) is added.
  • the radioactivity retained on the filter is counted using a Packard TopCount.NXT.
  • IC 50 values are calculated by non-linear regression analysis of binding curves (6 points minimum) using the windows program GraphPad Prism, GraphPad software, USA. Ki- values were calculated according to the Cheng-Prusoff equation [Y-C. Cheng and W.H. Pru- soff, Biochem. Pharmacol. 22 (1973) pp. 3099-3108].
  • the MC5 receptor binding assay is performed on BHK cell membranes stably expressing the human MC3 receptor.
  • the human MC5 receptor is cloned by PCR and subcloned into pcDNA3 expression vector.
  • Cells stably expressing the human MC5 receptor are generated by transfecting the expression vector into BHK cells and using G418 to select for MC5 clones.
  • the BHK MC5 clones are cultured in DMEM with glutamax, 10% FCS, 1 % pen/strep and 1 mg/ml G418 at 37°C and 5% C0 2 .
  • the binding is performed on a membrane preparation prepared in the following way:
  • the cells are rinsed with PBS and incubated with Versene for approximately 5 min before harvesting.
  • the cells are flushed with PBS and the cell suspension is centrifuged for 10 min at 2800xG.
  • the pellet is resuspended in 20ml buffer (20mM Tris pH 7.2 + 5mM EDTA + 1 mg/ml Bacitracin (Sigma B-0125)) and homogenized with a glass-teflon homogenizer, 10 times and low speed.
  • the cell-suspension is centrifuged at 4°C, 4100xG for 20min.
  • Pellet is resuspended in buffer and the membranes are diluted to a protein concentration of 1 mg/ml in buffer, aliquoted and kept at -80°C until use.
  • the assay is performed in a volume of 100 ⁇ .
  • Mix in the following order 25 ⁇ test- compound, 25 ⁇ 125 l-NDP-a-MSH (app. 60 000 cpm/well ⁇ 0.25nM in final concentration) and 50 ⁇ membranes (10 ⁇ g/well) and incubate incubation in Costar round-bottom wells microtiter plate, catalogue number 3365: Test-compounds are dissolved in DMSO or H 2 0.
  • Radioligand, membranes and test-compounds are diluted in buffer; (25 mM HEPES pH 7.4, 1 mM CaCI2, 5 mM MgS04, 0.1 % Ovalbumin (Sigma A-5503) , 0.005% Tween-20 and 5% Hydroxypro- pyl-3-cyclodextrin, (97%, Acros organics, code 297561000).
  • the assay mixture is incubated for 1 h at 20-25°C. Incubation is terminated by filtration on a Packard harvester filtermate 196. Rapid filtration through Packard Unifilter-96 GF/B filters pre-treated for 1 h with 0.5% poly- ethylenimine is carried out.
  • the filters are washed with ice-cold 0.9% NaCI 8-10 times.
  • the plate is air dried at 55°C for 30 min, and 50 ⁇ Microscint 0 (Packard) is added.
  • the radioactivity retained on the filter is counted using a Packard TopCount.NXT.
  • IC 50 values are calculated by non-linear regression analysis of binding curves (6 points minimum) using the windows program GraphPad Prism, GraphPad software, USA. Ki- values were calculated according to the Cheng-Prusoff equation [Y-C. Cheng and W.H. Pru- soff, Biochem. Pharmacol. 22 (1973) pp. 3099-3108].
  • the MC3 or MC5 -containing BHK cells are stimulated with potential MC3 or MC5 agonists, and the degree of stimulation of cAMP is measured using the FlashPlate® cAMP assay, cat. No SMP004, NENTM Life Science Products.
  • the cells are produced by transfecting the cDNA encoding MC3 or MC5 receptor into BHK570, and selecting for stable clones expressing the hMC3 receptor.
  • the cells are grown in DMEM, 10 % FCS, 1 mg/ml G418 and 1 % pen/strep.
  • the reaction is stopped with 100 ⁇ Detection Mix pro well (Detection Mix 1 1 ml Detection Buffer + 100 ⁇ ( ⁇ 2 ⁇ ) cAMP [ 125 l] Tracer).
  • the plates are then sealed with plastic, shaken for 30 minutes and allowed to stand overnight (or for 2h), and then counted in the Topcounter, 2 min/well (Note that in general, the assay procedure de- scribed in the kit-protocol is followed; however, the cAMP standards are diluted in 0.1 % HSA and 0.005% Tween 20, and not in stimulation buffer).
  • EC 5 o values are calculated by non-linear regression analysis of dose-response curves (6 points minimum) using the Windows program GraphPad Prism, GraphPad software, USA. Results are expressed in nM. E max values are calculated as % of NDP-a-MSH maximal stimulation in the hMC3cAMP assay (maximal NDP-a-MSH stimulation 100%).

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 TREATMENT OF OBESITY FIELD OF THE INVENTION
The present invention relates to novel peptides with improved solubility which are specific to one or more melanocortin receptors, to the use of said peptides in therapy, to methods of treatment comprising administration of said peptides to patients, and to the use of said peptides in the manufacture of medicaments.
BACKGROUND OF THE INVENTION
Obesity is a well known risk factor for the development of common diseases such as atherosclerosis, hypertension, type 2 diabetes, dyslipidaemia, coronary heart disease, gallbladder disease, osteoarthritis, premature death, certain types of cancer and various other malignancies. It also causes considerable problems through reduced motility and decreased quality of life. In the industrialized western world the prevalence of obesity has increased significantly in the past few decades. Only a few pharmacological treatments are available to date, namely Sibutramine (Abbot, acting via serotonergic and noradrenaline mechanisms), Orlistat (Roche and GlaxoSmithKline, works by reducing fat uptake from the gut). Because obesity represents a very high risk factor in serious and even fatal common diseases, its treatment should be a high public health priority and there is a need for pharmaceutical compounds useful in the treatment of obesity.
Pro-opiomelanocortin (POMC) is the precursor of the melanocortin family of pep- tides, which include o, β- and γ-melanocyte stimulating hormone (MSH) peptides and adrenocorticotropic hormone (ACTH), as well as other peptides such as β-endorphin. POMC is expressed in neurons of the central and peripheral nervous system and in the pituitary. Several of the melanocortin peptides, including ACTH and a-MSH, have been shown to have appetite-suppressing activity when administered to rats by intracerebroventricular (icv) injec- tion [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.
Five melanocortin receptor subtypes, MC1-5 receptors have been identified. MC1 , MC2 and MC5 receptors are mainly expressed in peripheral tissues, whereas MC3 and MC4 receptors are mainly centrally expressed. MC3 receptors are also expressed in several pe- ripheral tissues. In addition to being involved in energy homeostasis, MC3 receptors have also been suggested to be involved in several inflammatory diseases. It has been suggested that MC5 receptors are involved in exocrine secretion and in inflammation. MC4 receptors have been shown to be involved in the regulation of body weight and feeding behavior, as MC4 knock-out mice develop obesity [Huzar et al., Cell 88, 131-141 (1997)] and common variants near MC4 receptor have been found to be associated with fat mass, weight and risk of obesity [Loos et al. Nat Genet., 40(6):768-75 (2008)]. Furthermore, studies with mice showed that overexpression in the mouse brain of the melanocortin receptor antagonists agouti protein and agouti-related protein (AGRP), led to the development of obesity [Kleibig et al., PNAS 92, 4728-4732 (1995)]. Moreover, icv injection of a C-terminal fragment of AGRP increases feeding and antagonizes the inhibitory effect of α-MSH on food intake.
MC4 receptor agonists could serve as anorectic drugs and/or energy expenditure increasing drugs 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 activation of MC4 receptor. On the other hand, MC4 receptor antagonists may be useful in the treatment of cachexia or anorexia, of waisting in frail elderly patients, chronic pain, neuropathy and neurogenic inflammation.
A large number of patent applications disclose various classes of non-peptidic small molecules as melanocortin receptor modulators, of which 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 98/271 13. Hadley [Pigment Cell Res. (1991 ) 4:180-185] reported a prolonged effect of specific melanotropic peptides conjugated to fatty acids, the prolongation ef- fected by a transformation of the modulators from being reversibly acting to being irreversibly acting being caused by the conjugated fatty acids.
SUMMARY OF THE INVENTION
The present invention relates to novel peptides which are specific to one or more melanocortin receptors with improved water solubility at neutral pH, to the use of said peptides in therapy, to methods of treatment comprising administration of said peptides to patients, and to the use of said peptides in the manufacture of medicaments.
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, in a first embodiment (embodiment 1 ), the invention relates pep- tide agonists highly specific for melanocortin receptor wherein the compound is a peptide according to formula I:
R1-c[X1-His-D-Phe-Arg-Trp-X2]- Z1-Z2-Z3-Z4-Z5-Z6-R2 [I]
wherein
R1 represents Ci-3 alkanoyl; X1 represents Asp, Glu, Lys, Orn, Dab, Dap, Cys, homo-Cys or Penicillamine.
X2 represents Asp, Glu, Lys, Orn, Dab, Dap, Cys, homo-Cys or Penicillamine.
wherein X1 and X2 are joined, rendering the compound of formula I cyclic, either via a disulfide bridge, X1 and X2 being independently Cys, homo-Cys or Penicillamine, or via a lactam bridge, either from X1 being Asp or Glu and X2 being Lys, Orn, Dab or Dap, or from X1 being Lys, Orn, Dab or Dap and X2 being Asp or Glu.
Z1 represents Pro, D-Pro, Hyp or D-Hyp;
Z2 represents Pro, D-Pro, Hyp or D-Hyp;
Z3 represents Lys, Arg , His, D-Lys, D-Arg or D-His;
Z4 represents Glu, Asp, D-Glu, D-Asp, Ser or D-Ser;
Z5 represents Lys(bis carboxymethyl);
Z6 is absent or represents a peptide fragment comprising one to four amino acid residues derived from Met, D-Met, Leu, D-Leu, Nle, D-Nle, lie, D-lle, Val or D-Val;
and pharmaceutically acceptable salts, prodrugs and solvates thereof.
R2 represents -OR or -NR2, R being hydrogen or Ci-3 alkyl.
The invention further relates to the use of compounds of the invention in therapy, to pharmaceutical compositions comprising compounds of the invention, and to the use of compounds of the invention in the manufacture of medicaments. DESCRIPTION OF THE INVENTION
Among further embodiments of compounds of the present invention are the following:
2. A compound according to embodiment 1 , wherein X1 represents Asp or Glu.
3. A compound according to embodiments 1 -2, wherein X2 represents Lys, Orn, Dab or Dap;
4. A compound according to embodiments 1 -3, wherein X1 represents Glu.
5. A compound according to embodiments 1 -3, wherein X1 represents Asp.
6. A compound according to embodiments 1 -3, wherein X2 represents Orn or Lys.
7. A compound according to embodiments 1 -3, wherein X2 represents Orn.
8. A compound according to embodiments 1 -3, wherein X2 represents Lys.
9. A compound according to embodiments 1 -3, wherein X2 represents Dab.
10. A compound according to embodiments 1 -3, wherein X2 represents Dap.
1 1 . A compound according to embodiment 1 , wherein X1 represents Lys, Orn, Dab or Dap. 12. A compound according to embodiments 1 and 1 1 , wherein X2 represents Asp or
Glu.
13. A compound according to embodiments 1 and 1 1 , wherein X1 represents Lys or
Orn.
14. A compound according to embodiments 1 and 1 1 , wherein X1 represents Lys.
15. A compound according to embodiments 1 and 1 1 , wherein X1 represents Orn.
16. A compound according to embodiments 1 and 1 1 , wherein X1 represents Dab.
17. A compound according to embodiments 1 and 1 1 , wherein X1 represents Dap.
18. A compound according to embodiments 1 and 1 1 , wherein X2 represents Asp. 19. A compound according to embodiments 1 and 1 1 , wherein X2 represents Glu.
20. A compound according to embodiment 1 , wherein X1 represents Cys, homo-Cys or Penicillamine.
21 . A compound according to embodiments 1 and 20, wherein X2 represents Cys, homo-Cys or Penicillamine.
22. A compound according to embodiments 1 and 20, wherein X1 represents Cys.
23. A compound according to embodiments 1 and 20, wherein X1 represents homo- Cys.
24. A compound according to embodiments 1 and 20, wherein X1 represents Penicillamine.
25. A compound according to embodiments 1 and 20, wherein X2 represents Cys.
26. A compound according to embodiments 1 and 20, wherein X2 represents homo- Cys.
27. A compound according to embodiments 1 and 20, wherein X2 represents Penicillamine.
28. A compound according to embodiment 1 , selected from the group consisting of:
Figure imgf000005_0001
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Nle-NH2
Figure imgf000006_0001
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-NH2
Figure imgf000006_0002
Figure imgf000006_0003
Figure imgf000006_0004
-c[Lys-His-D-Phe-Arg-Trp-Asp]-Pro-Pro-Arg-Ser-Lys(biscarboxymethyl)-Leu-NH2
Figure imgf000007_0001
-c[Orn-His-D-Phe-Arg-Trp-Glu]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Leu-NH2
Figure imgf000007_0002
-c[Orn-His-D-Phe-Arg-Trp-Glu]-Pro-Pro-Arg-Asp-Lys(biscarboxymethyl)-Phe-NH2
Figure imgf000007_0003
-c[Orn-His-D-Phe-Arg-Trp-Glu]-Pro-Pro-Arg-Glu-Lys(biscarboxymethyl)-Val-NH2
Figure imgf000007_0004
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-Arg-Glu-Lys(biscarboxymethyl)-Nle-NH2 -c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Glu-Lys(biscarboxymethyl)-Val-NH2
Figure imgf000008_0001
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Glu-Lys(biscarboxymethyl)-Nle-NH2
Figure imgf000008_0002
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Val-NH2
Figure imgf000008_0003
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Phe-NH2
Figure imgf000008_0004
The present invention also encompasses combinations of two or more embodiments of compounds 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 relation 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 binding affinity of a compound with respect to MC1 , MC3, MC5 and MC4 may be determined by comparing the Ki from an MC1 , MC3 or MC5 binding assay as described below under "Assay IV" (MC1 ), "Assay VII" (MC3) and "Assay VIII" (MC5), respectively, with Ki from an MC4 binding assay as described below under "Assay V" (MC4). If the binding affinity of a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times greater () with respect to MC4 than with respect to MC1 , it is deemed to be a selective MC4 agonist with respect to MC1 . If the binding affinity of a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times greater (higher) with respect to MC4 than with respect to MC3, it is deemed to be a selective MC4 agonist with respect to MC3. If the binding affinity of a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times greater with respect to MC4 than with respect to MC5, it is deemed to be a selective MC4 agonist with respect to MC5. The agonistic potency of a compound with respect to MC3, MC4 and MC5 may be determined in functional assays as described in "Assay II" (MC3 and MC5), "Assay IX" (MC3) 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 respect to MC3, it is deemed to be a selective MC4 agonist with respect to MC3. 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 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 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 binding affinity determined for MC1 as described in "Assay IV" with the binding affinity for MC3 determined as described in "Assay VII". If the binding affinity of a compound is more than 10 times, such as more than 50 times, e.g. more than 100 times greater 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 affinity determined as described in "Assay VII and VIII". If the binding affinity of a compound is more than 10 times, such as more the 50 times, e.g. more than 100 times greater with re- spect to MC3 than with respect to MC5, it is deemed to be a selective MC3 agonist with re- spect to MC5. The MC4 selectivity of a compound with respect to MC3 and MC5 is determined as discussed above.
Compounds of the present invention modulate melanocortin receptors, and they are therefore 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 activation of MC4.
Among further aspects or embodiments of the present invention are the following:
29. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in delaying the progression from impaired glucose tolerance (IGT) to type 2 diabetes.
30. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in delaying the pro- gression from non-insulin-requiring type 2 diabetes to insulin-requiring type 2 diabetes.
31 . A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in treating obesity or preventing overweight.
32. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in regulating appetite.
33. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in inducing satiety.
34. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for preventing weight gain af- ter successful weight loss.
35. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in increasing energy expenditure.
36. A method of delaying the progression from IGT to type 2 diabetes, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
37. A method of delaying the progression from non-insulin-requiring type 2 diabetes to insulin-requiring type 2 diabetes, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
38. A method of treating obesity or preventing overweight, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodi- ments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
39. A method of regulating appetite, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
40. A method of inducing satiety, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
41 . A method of preventing weight gain after successfully having lost weight, comprising administering to a patient in need thereof an effective amount of a compound accord- ing to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
42. A method of increasing energy expenditure, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1- 28, optionally in combination with one or more additional therapeutically active compounds.
Among yet further aspects or embodiments of the present invention are the following:
43. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in treating a disease or state related to overweight or obesity.
44. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in treating bulimia.
45. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for treating binge-eating.
46. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in treating a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart dis-ease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea and risk of premature death. 47. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for providing neuronal protection, for having an effect on ischemic heart disease, cerebral ischemia or anti-inflammatory effects and for the treatment of autoimmune diseases, e.g. multiple sclerosis.
48. 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 according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
49. A method of treating bulimia, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
50. A method of treating binge-eating, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
51 . A method of treating a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, impaired glucose tolerance (IGT), dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea and risk of premature death, comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
52. A method for providing neuronal protection, for having an effect on ischemic heart disease, cerebral ischemia or anti-inflammatory effects and for the treatment of autoimmune diseases, e.g. multiple sclerosis., comprising administering to a patient in need thereof an effective amount of a compound according to any of embodiments 1 -28, optionally in combination with one or more additional therapeutically active compounds.
Compounds of the present invention may be suited for the treatment of diseases in obese or overweight patients. Accordingly, a yet further aspect or embodiment of the inven- tion relates to the following:
53. A compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds, for use in treating, in an obese patient, a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea, risk of premature death, neuronal protection, effect in ischemic heart disease or anti-inflammatory effects.
54. A method of treating, in an obese patient, a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, IGT, dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea, risk of premature death, neuronal protection, effect in ischemic heart disease or anti-inflammatory effects, comprising administering to an obese patient in need thereof an effective amount of a compound according to any of embodiments 1-28, optionally in combination with one or more additional therapeutically active compounds.
Yet further aspects or embodiments of the invention relate to:
55. A compound according to any of embodiments 29-35, 43-47 and 53, wherein said additional therapeutically active compound is selected from antidiabetic agents, anti- hyperlipidemic agents, antiobesity agents, antihypertensive agents and agents for the treat- ment of complications resulting from, or associated with, diabetes.
56. A method according to any of embodiments 36-42, 48-52 and 54, wherein said additional therapeutically active compound is selected from antidiabetic agents, antihyperlipi- demic agents, antiobesity agents, antihypertensive agents and agents for the treatment of complications resulting from, or associated with, diabetes.
57. A compound according to any of embodiments 1 -35, 143-47, 53 and 55, which is in a unit dosage form comprising from about 0.05mg to about 1000mg of said compound.
58. A method according to any of embodiments 36-42, 48-52, 54 and 56, wherein said compound according to any of embodiments 1-28 is administered to said patient in a unit dosage form comprising from about 0.05mg to about 1000mg of said compound.
59. A method according to any of embodiments 36-42, 48-52, 54 and 56, wherein said compound according to any of embodiments 1 -28 is administered to said patient, once daily.
60. A method according to any of embodiments 36-42, 48-52, 54 and 56, wherein said compound according to any of embodiments 1 -28 is administered to said patient once weekly.
61 . A method of activating MC4 in a subject, the method comprising administering to said subject an effective amount of a compound according to any of embodiments 1-28. 62. A method according to any of embodiments 36-42, 48-52, 54, 56 and 58-61 , wherein said compound according to any of embodiments 1 -28 is administered parenterally, orally, nasally, buccally or sublingually.
63. A method according to any of embodiments 36-42, 48-52, 54, 56 and 58-61 , wherein said compound according to any of embodiments 1-28 is administered parenterally or sublingually.
Another aspect or embodiment of the invention relates to:
64. A pharmaceutical composition comprising a compound according to any of em- bodiments 1-35, 43-47, 53 and 55.
65. A pharmaceutical composition according to embodiment 64, further comprising one or more additional therapeutically active compounds or substances.
66. A pharmaceutical composition according to any of embodiments 64-65, wherein the pH is neutral to weakly basic.
67. A pharmaceutical composition according to any of embodiments 64-65, wherein the pH is from about 7.0 to about 8.0.
68. A pharmaceutical composition according to any of embodiments 64-65, wherein the pH is about 7.5.
Yet another aspect or embodiment of the invention relates to the following:
69. A compound according to any of embodiments 1 -28 for use in therapy.
70. A pharmaceutical composition according to any of embodiments 64-68 for use in therapy.
71 . Use of a compound according to any of embodiments 1-28 in the manufacture of a medicament for delaying the progression from impaired glucose tolerance (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 diabetes, IGT, dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea or risk of premature death; or treating, in an obese patient, a disease or state selected from type 2 diabetes, IGT, dyspilidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, risk of premature death; for providing neuronal pro- tection, for having an effect on ischemic heart disease, cerebral ischemia or antiinflammatory effects and for the treatment of autoimmune diseases, e.g. multiple sclerosis.
Compounds of the invention that act as 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 agonists have antipyretic effects, and both have been suggested to be involved in peripheral nerve regeneration. MC4 agonists are also known to reduce stress response. In addition to treating drug abuse, treating or preventing hemorrhagic shock, and reducing stress response, compounds of the invention may also be of value in treating alcohol abuse, treating stroke, treating ischemia and protecting against neuronal damage.
As already indicated, in all of the therapeutic methods or indications disclosed above, the compound of the present invention may be administered alone. However, it may also be administered in combination with one or more additional therapeutically active agents, substances or compounds, 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, preferably from about 0.01 to about 10mg/kg body weight, more preferably from about 0.01 to about 5 mg/kg body weight per day, e.g. from about 0.05 to about 10 mg/kg body weight per day or from about 0.03 to about 5mg/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 techniques well known to those skilled in the art. A typical unit dosage form intended for oral administration one or more times per day, such as from one to three times per day, may suitably contain from about 0.05 to about 1000mg, preferably from about 0.1 to about 500mg, such as from about 0.5 to about 200mg of a compound of the invention.
Compounds of the invention comprise compounds that are believed to be well- suited to administration with longer intervals than, for example, once daily, thus, appropriately formulated compounds of the invention may be suitable for, e.g., twice-weekly or once- weekly administration by a suitable route of administration, such as one of the routes disclosed herein. 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, and suitable additional compounds or substances may be selected, for example, from antidiabetic 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, insulin derivatives or analogues, GLP-1 (glucagon like peptide-1 ) derivatives or analogues [such as those disclosed in WO 98/08871 (Novo Nordisk A/S), which is incorporated herein by reference, or other GLP-1 analogues such as exenatide (Byetta, Eli Lilly/Amylin; AVE0010, Sanofi-Aventis), taspoglutide (Roche), albiglutide (Syncria, GlaxoSmithKline), amylin, amylin analogues (e.g. Symlin™/Pramlintide) as well as orally active hypoglycemic agents.
Suitable orally active hypoglycemic agents include: metformin, imidazolines; sulfonylureas; biguanides; meglitinides; oxadiazolidinediones; thiazolidinediones; insulin sensitizers; oglucosidase inhibitors; 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) which are incorporated herein by reference; potassium channel openers such as ormitiglinide; potassium channel blockers such as nateglinide or BTS-67582; glucagon receptor antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), all of which are incorporated herein by reference; GLP-1 receptor agonists such as those disclosed in WO 00/42026 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference; amylin analogues (agonists on the amylin receptor); DPP- IV (dipeptidyl peptidase-IV) inhibitors; PTPase (protein tyrosine phosphatase) inhibitors; glu- cokinase activators, such as those described in WO 02/08209 to Hoffmann La Roche; inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis; glucose uptake modulators; GSK-3 (glycogen synthase kinase-3) inhibitors; compounds modifying lipid metabolism, such as antihyperlipidemic agents and antilipidemic agents; compounds lowering food intake; as well as PPAR (peroxisome proliferator-activated recep- tor) 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 insulin analogues; sulfonylureas, e.g. tolbutamide, chlorpropamide, tolazamide, gliben- clamide, glipizide, glimepiride, glicazide or glyburide; biguanides, e.g. metformin; and meglit- inides, e.g. repaglinide or senaglinide/nateglinide. Further examples of suitable additional therapeutically active substances include thiazolidinedione insulin sensitizers, e.g. troglitazone, ciglitazone, pioglitazone, rosiglitazone, isaglitazone, darglitazone, englitazone, CS-01 1/CI-287 or T 174, or the compounds disclosed in WO 97/41097 (DRF-2344), WO 97/41 1 19, WO 97/41 120, WO 00/41 121 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 Nord- isk A/S), the contents of all of which are incorporated herein by reference.
Still further examples of suitable additional therapeutically active substances include: oglucosidase 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. tolbutamide, glibenclamide, glipizide, glicazide, BTS-67582 or repag- linide;
Other suitable additional therapeutically active substances include antihyperlipi- demic 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 in- elude antiobesity agents and appetite-regulating agents. Such substances may be selected from the group consisting of CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y receptor 1 and/or 5) antagonists, MC3 (melanocortin receptor 3) agonists, MC3 antagonists, MC4 (melanocortin receptor 4) agonists, orexin receptor antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, urocortin agonists, neuromedin U analogues (agonists on the neuromedin U receptor subtypes 1 and 2), β3 adrenergic agonists such as CL-316243, AJ-9677, GW-0604, LY362884, LY377267 or AZ-40140, MC1 (melanocortin receptor 1 ) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists, serotonin reuptake inhibitors (e.g. fluoxetine, seroxat or cita- lopram), serotonin and norepinephrine reuptake inhibitors, 5HT (serotonin) agonists, 5HT6 agonists, 5HT2c agonists such as APD356 (US6953787), bombesin agonists, galanin antagonists, growth hormone, growth factors such as prolactin or placental lactogen, growth hormone releasing compounds, 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 analogues, GLP-1 analogues, ciliary neurotrophic factor, amylin analogues, peptide YY3-36 (PYY3-36) (Batterham et al, Nature 418, 650-654 (2002)), PYY3-36 analogues, NPY Y2 receptor agonists, NPY Y4 receptor agonists and substances acting as combined NPY Y2 and NPY Y4 agonists, FGF21 and analogues thereof, μ-opioid receptor antagonists, oxyntomodulin or analogues thereof.
Further suitable antiobesity agents are bupropion (antidepressant), topiramate (anti- convulsant), ecopipam (dopamine D1/D5 antagonist) and naltrexone (opioid antagonist), and combinations thereof. Combinations of these antiobesity agents would be e.g.: phenter- mine+topiramate, bupropion sustained release (SR)+naltrexone SR, zonisamide SR and bupropion SR. Among embodiments of suitable antiobesity agents for use in a method of the invention as additional therapeutically active substances in combination with a compound of the invention are leptin and analogues or derivatives of leptin.
Additional embodiments of suitable antiobesity agents are serotonin and norepinephrine reuptake 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 stimu- lating agents, e.g. dexamphetamine, amphetamine, phentermine, mazindol, phendi- metrazine, diethylpropion, fenfluramine or dexfenfluramine.
Other examples of suitable additional therapeutically active compounds include antihypertensive agents. Examples of antihypertensive agents are β-blockers such as alpre- nolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting en- zyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, ni- modipine, diltiazem and verapamil, and oblockers 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.
In the case, in particular, of administration of a compound of the invention, optionally in combination with one or more additional therapeutically active compounds or substances as disclosed above, for a purpose related to treatment or prevention of obesity or overweight, i.e. related to reduction or prevention of excess adiposity, it may be of relevance to employ such administration in combination with surgical intervention for the purpose of achieving weight loss or preventing weight gain, e.g. in combination with bariatric surgical intervention. Examples of frequently used bariatric surgical techniques include, but are not limited to, the following: vertical banded gastroplasty (also known as "stomach stapling"), wherein a part of the stomach is stapled to create a smaller pre-stomach pouch which serves as a new stom- ach; gastric banding, e.g. using an adjustable gastric band system (such as the Swedish Adjustable Gastric Band (SAGB), the LAP-BAND™ or the MIDband™), wherein a small pre- stomach pouch which is to serve as a new stomach is created using an elastomeric (e.g. silicone) band which can be adjusted in size by the patient ; and gastric bypass surgery, e.g. "Roux-en-Y" bypass wherein a small stomach pouch is created using a stapler device and is connected to the distal small intestine, the upper part of the small intestine being reattached in a Y-shaped configuration.
Another technique which is within the scope of the term "bariatric surgery" and variants thereof (e.g. "weight-loss surgery", "weight-loss surgical intervention" "weight-loss surgical procedure", "bariatric surgical intervention", "bariatric surgical procedure" and the like) as employed in the context of the present invention is gastric balloon surgery, wherein an inflatable device resembling a balloon is introduced into the stomach and then inflated, the purpose being to reduce the accessible volume within the stomach to create a sensation of satiety in the patient at an earlier stage than normal during food intake, and thereby cause a reduction in food intake by the patient.
Another technique which is within the scope of the term "bariatric surgery" is Endo-
Barrier™ technology, which is a proprietary platform of products for the treatment of type 2 diabetes and obesity. The EndoBarrier Gastrointestinal Liner works by creating a physical barrier between ingested food and the intestinal wall, which may change how hormonal signals that originate in the intestine are activated, mimicking the effects of gastric bypass pro- cedure. Weight loss is enhanced by combining the EndoBarrier Gastrointestinal Liner with the customizable EndoBarrier Flow Restrictor. Combination of the EndoBarrier Gastrointestinal Liner with the EndoBarrier Flow Restrictor was shown to affect multiple mechanisms of action.
All of the above-mentioned techniques are in principle reversible. Non-limiting ex- amples of additional, irreversible and consequently generally less frequently employed techniques of relevance in the present context include biliopancreatic diversion and sleeve gastrectomy (the latter of which may also be employed in conjunction with duodenal switch), both of which entail surgical resection of a substantial portion of the stomach.
The administration of a compound of the invention (optionally in combination with one or more additional therapeutically active compounds or substances as disclosed above) may take place for a period prior to carrying out the bariatric surgical intervention in question and/or for a period of time subsequent thereto. In many cases it may be preferable to begin administration of a compound of the invention after bariatric surgical intervention has taken place.
The treatment of obesity might be possible by using long-acting melanocortin 4 receptor agonists (MC4 agonists) comprising a peptide part and an albumin binding fatty acid or alkyltetrazole chain as described in e.g. WO2007/009894, WO2008/087186 and WO2008/087187. These compounds have more basic than acidic residues, resulting in good solubility at acidic pH, but poor solubility at neutral or weakly basic pH. Solubility at pH from 6 to 9 is considered to be an advantage, since this could improve local tolerance and make it possible to combine the MC4 agonist with other drugs, soluble only at neutral to weakly basic pH.
The compounds of the present invention have a novel chemical structure. The compounds of the present invention comprise a macrocyclic peptide part, a linear peptide part linked to the C-terminus of the macrocycle. The peptide backbone has some similarity with the hormone β-MSH.
The primary function of the macrocyclic peptide part is to bind to the MC4 receptor, resulting in activation of the MC4 receptor, with selectivity for MC4 over MC1 , MC3 and MC5 receptors. The linear peptide part is also important for receptor binding and selectivity and is therefore limited to certain amino acid sequences. The desired balance between MC4 potency and receptor selectivity is achieved by the covalent combination of macrocyclic peptide part, linear peptide part linked to the C-terminus of the macrocycle, according to the above- described embodiments of compounds of the invention.
The compounds of the present invention have high MC4 receptor potency and higher MC4 receptor selectivity in relation to previously disclosed peptides in the art. The compounds of the present invention can be a soluble MC4 receptor agonist, for example with solubility of at least 0.2 mmol/l, at least 0.5 mmol/l, at least 2 mmol/l, at least 4 mmol/l, at least 8 mmol/l, at least 10 mmol/l, or at least 15 mmol/l, at pH 7.5.
In the present context, if not stated otherwise, the terms "soluble", "solubility ", "solu- ble in aquous solution", "aqueous solubility", "water soluble", "water-soluble", "water solubil- ity"and "water-solubility", refer to the solubility of a compound in water or in an aqueous salt or aqueous buffer solution, for example a 10 mM phosphate solution, or in an aqueous solution containing other compounds, but no organic solvents.
The term "obesity" implies an excess of adipose tissue. When energy intake ex- ceeds 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 components to weight balance, and an abnormality on either side (intake or expenditure) can lead to obesity. In this context, obesity is best viewed as any degree of excess adipose tissue 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 adipose tissue. However, in the context of the present invention, 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.
The use of a prefix of the type "Cx-y" preceding the name of a radical, such as in Cx-yalkyl (e.g. C6-2oalkyl) is intended to indicate a radical of the designated type having from x to y carbon atoms.
The term "alkyl" as used herein refers to a straight-chain, branched and/or cyclic, saturated monovalent hydrocarbon radical.
The term "alkenyl" as used herein refers to a straight-chain, branched and/or cyclic, monovalent hydrocarbon radical comprising at least one carbon-carbon double bond.
The term "alkynyl" as used herein refers to a straight-chain, branched and/or cyclic, monovalent hydrocarbon radical comprising at least one carbon-carbon triple bond, and it may optionally also comprise one or more carbon-carbon double bonds.
The term "alkylene" as used herein refers to a straight-chain, branched and/or cyclic, saturated bivalent hydrocarbon radical.
The term "alkenylene" as used herein refers to a straight-chain, branched and/or cyclic, bivalent hydrocarbon radical comprising at least one carbon-carbon double bond.
The term "alkynylene" as used herein refers to a straight-chain, branched and/or cyclic, bivalent hydrocarbon radical comprising at least one carbon-carbon triple bond, and it may optionally also comprise one or more carbon-carbon double bonds. The term "alkoxy" as used herein is intended to indicate a radical of the formula - OR', wherein R' is alkyl as indicated above.
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 aro- matic. 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 table of the elements, which includes fluorine, chlorine, bromine and iodine (corresponding to fluoro, chloro, bromo and iodo substituents, respectively).
The term "tetrazol-5-yl" is intended to indicate 1 H-tetrazol-5-yl or 2H-tetrazol-5-yl. In the present context, common rules for peptide nomenclature based on the three letter amino acid code apply, unless exceptions are specifically indicated. Briefly, the central portion of the amino acid structure is represented by the three letter code (e.g. Ala, Lys) and L-configuration is assumed, unless D-configuration is specifically indicated by "D-" followed by the three letter code (e.g. D-Ala, D-Lys). A substituent at the amino group replaces one hydrogen atom and its name is placed before the three letter code, whereas a C-terminal substituent replaces the carboxylic hydroxy group and its name appears after the three letter code. For example, "acetyl-Gly-Gly-NH2" represents CH3-C(=0)-NH-CH2-C(=0)-NH-CH2- C(=0)-NH2. Unless indicated otherwise, amino acids with additional amino or carboxy groups in the side chains (such as Lys, Orn, Dap, Glu, Asp and others) are connected to their neighboring groups by amide bonds formed at the N-2 (a-nitrogen) atom and the C-1 (C=0) carbon atom.
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 co- valent bond.
In the present context, the term "agonist" is intended to indicate a substance (ligand) that activates 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 antagonist, partially inhibiting the effect of a full agonist.
Receptor neutral antagonists, which block the action of an agonist, but do not affect the receptor-constitutive activity. Receptor inverse agonists, which block the action of an agonist and at the same time attenuate 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 accept- able acid addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hy- droiodic, phosphoric, sulfuric and nitric acids, and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, tartaric, ascorbic, pamoic, bismethyl- ene-salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, p-toluenesulfonic acids and the like. Further examples 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 use 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 routine experimentation, by constructing a matrix of values and testing different points in the matrix, 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 management 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 condition from which the patient is suffering, such as administration of the active compound^) in question to alleviate symptoms or complications thereof, to delay the progression of the disease, 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 between a solute (in casu, a compound according to the present invention) and a solvent. Solvents may include, by way of example, water, ethanol, or acetic acid.
The amino acid abbreviations used in the present context have the following meanings:
Ala Alanine β-Ala
2 COOH
Asn Asparagine
Asp Aspartic acid β-Asp
Figure imgf000024_0001
a-nitrogen and β-carboxy group form the amide bonds to the two neighboring residues
Arg Arginine
Cit Citrulline
Cys Cysteine
Dab (S)-2,4-diaminobutyric acid
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
homoArg
Figure imgf000028_0001
homo-arginine
homoCys
Figure imgf000028_0002
homo-cysteine
homoLys
Figure imgf000028_0003
a-nitrogen and carboxy group form the amide bonds to the two neighboring residues homoLys(biscarboxymethyl)
Figure imgf000028_0004
a-nitrogen and carboxy-carbon atom C-1 form the to the two neighboring residues homoLys(BCMA)
Figure imgf000029_0001
a-nitrogen and carboxy-carbon atom C-1 form the amide bonds to the two neighboring residues
BCMA = [bis(carboxymethyl)amino]acetyl
homoSer
Figure imgf000029_0002
homo-serine
Hyp 4-hydroxyproline
lie Isoleucine
Leu Leucine
Lys Lysine
Figure imgf000030_0001
bonds
amide b|
Figure imgf000031_0001
Pro Proline
Ser Serine
Thr Threonine
Tyr Tyrosine
Trp Tryptophan
Val Valine
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.
PHARMACEUTICAL COMPOSITIONS
As already mentioned, one aspect of the present invention provides a pharmaceutical composition (formulation) comprising a compound of the present invention. Appropriate embodiments 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), stabilizers) and/or surfactant(s). In one embodiment of the invention the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water, and the term "aqueous formulation" in the present context may normally be taken to indicate a formulation comprising at least 50 % by weight (w/w) of water. Such a formulation is typically a solution or a suspension. 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 invention 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 dissolution.
In a further aspect, the invention relates to a pharmaceutical composition (formula- tion) comprising 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 pharmaceutical 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" I 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 packaged for storage, whether in a liquid form, in a frozen state, or in a dried form for later reconstitution into a liquid form or other form suitable for administration to a subject. By "dried form" is meant the product obtained when a liquid pharmaceutical composition or formulation is dried by freeze-drying (i.e., lyophilization; see, for example, Williams and Polli (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: 1 169-1206; and Mumenthaler et al. (1994) Pharm. Res. 1 1 : 12-20], or by air-drying [see, e.g., Carpenter and Crowe (1988) Cryobiology 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 pharmaceutical 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 storage 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 pre- sent 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 mixtures thereof) of a particular amino acid (e.g. methionine, histidine, arginine, lysine, isoleucine, 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 embodiment, 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 decreasing aggregate formation by the oligo- or polypeptide during storage of liquid pharmaceutical compositions of the invention. Suitable arginine analogues include, for example, aminoguanidine, ornithine and N-monoethyl-L-arginine. Suitable methionine ana- logues include 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 acting as the therapeutic agent is a peptide comprising at least one methionine residue susceptible to such oxidation. The term "inhibit" in this context refers to minimization of ac- cumulation of methionine-oxidized species over time. Inhibition of methionine oxidation results in increased 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, 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. [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 tetrafu notional 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 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, sublingual, buccal, in the mouth, oral, in the stomach and intestine, nasal, pulmonary (for example through the bronchioles and alveoli or a combination thereof), epidermal, der- mal, transdermal, vaginal, rectal, ocular (for example through the conjunctiva), uretal and parenteral.
Compositions of the present invention may be administered in various dosage forms, for example in the form of solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, rinses, cap- sules (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 situ- 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 compli- ance, or any combination thereof. Examples of carriers, drug delivery systems and advanced drug delivery systems include, but are not limited to: polymers, for example cellulose and derivatives; polysaccharides, for example dextran and derivatives, starch and derivatives; polyvinyl alcohol); acrylate and methacrylate polymers; polylactic and polyglycolic acid and block co-polymers thereof; polyethylene glycols; carrier proteins, for example albumin; gels, for example thermogelling systems, 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 behavior 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, sustained-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 sustained-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 subcu- taneous administration. Without limiting the scope of the invention, examples of useful controlled release systems and compositions are those containing hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles,
Methods for producing controlled-release systems useful for compositions of the present invention include, but are not limited to, crystallization, condensation, co- crystallization, precipitation, co-precipitation, emulsification, dispersion, high-pressure ho- mogenisation, encapsulation, 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 Pharmaceuti- cal Sciences, vol. 99: Protein Formulation and Delivery (MacNally, E.J., 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 can 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 transdermal administration (e.g. by needle-free injection or via a patch, such as an iontopho- retic patch) or transmucosal (e.g. buccal) administration.
The term "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability. The term "physical stability" in the context of a formulation containing an oligo- or polypeptide refers to the tendency 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 sur- faces 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 measurements 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 background. The turbidity of a formulation is characterized by a visual score ranking the de- gree of turbidity, for instance on a scale from 0 to 3 (in that a formulation showing no turbidity corresponds to a visual score 0, whilst a formulation showing visual turbidity in daylight corresponds to visual score 3). A formulation is normally classified physically unstable with respect 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 buried 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, spec- troscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenan- throline and the like. Other spectroscopic probes are metal complexes of amino acids, such as cobalt complexes of hydrophobic 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 chemical covalent changes in oligo- or polypeptide structure leading to formation of chemical degradation products with potentially lower biological potency and/or potentially increased immunogenicity 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 completely avoided and gradually increasing amounts of chemical degradation products may often 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 hydrolysed 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 substance are covalently bound to each other through transamidation and/or disulfide interactions, leading to formation of covalently bound dimer, oligomer or polymer degradation products (see, e.g., Stability of Protein Pharmaceuticals, Ahern. T.J. & 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 individual 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 construed as limiting the invention in any way.
The use of any and all examples, or exemplary language (including "for instance", "for example", "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 mat- ter recited in the claims appended hereto, as permitted by applicable law.
EXAMPLES
List of abbreviations employed
AcOH acetic acid
BCMA [bis(carboxymethyl)amino]acetyl
Bn benzyl
BSA bovine serum albumin
DBU 1 ,8-diazabicyclo[5.4.0]undec-7-ene
DCM dichloromethane
Dde 1 -(4,4-dimethyl-2,6-dioxocyclohex-1 -ylidene)ethyl
DIC /V,/V-diisopropylcarbodiimide
DIPEA ethyldiisopropylamine
DMAP 4-(dimethylamino)pyridine
DMF /V,/V-dimethylformamide
DMSO dimethylsulfoxide
EDC 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
EDT 1 ,2-ethanedithiol
EGTA glycol-bis(2-aminoethylether)-/V,/VJV',/V^etraacetic acid (ethyleneglycol tetraacetic acid)
eq equivalents
FCS fetal calf serum
Fmoc 9/-/-fluoren-9-ylmethyloxycarbonyl
Fmoc SPPS Fmoc-based solid-phase peptide synthesis
HBTU 2-(1 H-benzotriazol-1 -yl-)-1 , 1 ,3,3-tetramethyluronium hexafluorophosphate
HEPES 2-[4-(2-hydroxyethyl)-piperazin-1-yl]ethanesulfonic acid
HFIP 1 ,1 , 1 ,3,3,3-hexafluoro-2-propanol
HOAt 1 -hydroxy-7-aza-benzotriazole
HOBt 1 -hydroxybenzotriazole HSA human serum albumin
IBMX 3-isobutyl-1-methylxanthine
ivDde 1 -(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl
MC1 melanocortin receptor subtype 1 (also denoted melanocortin receptor 1 ) MC2 melanocortin receptor subtype 2 (also denoted melanocortin receptor 2)
MC3 melanocortin receptor subtype 3 (also denoted melanocortin receptor 3)
MC4 melanocortin receptor subtype 4 (also denoted melanocortin receptor 4)
MC5 melanocortin receptor subtype 5 (also denoted melanocortin receptor 5)
MeCN acetonitrile
MeOH methanol
min minutes
α-MSH a-form of melanocyte-stimulating hormone
Mtt 4-methyltrityl
MTX methotrexate
NEt3 triethylamine
NMP A/-methylpyrrolidin-2-one
OPhiPr 2-phenylisopropyloxy = 1-methyl-1-phenyl-ethyloxy
OSu ester 2,5-dioxo-pyrrolidin-1 -yl ester
PBS phosphate-buffered saline
PEI polyethyleneimine
PhiPr 2-phenylisopropyl = 1 -methyl-1 -phenyl-ethyl
PyBOP (benzotriazol-l-yloxy)trispyrrolidinophosphonium hexafluorophosphate
TFA trifluoroacetic acid
THF tetrahydrofuran
TIPS triisopropylsilane
TSTU 0-(/V-succinimidyl)-/\/,/\/,/\/',/\/'-tetramethyluronium tetrafluoroborate trityl triphenylmethyl
Trt triphenylmethyl
UPLC ultra performance liquid chromatography
All compounds of the present invention can be synthesized by those skilled in the art using standard coupling and deprotection steps. Non-standard procedures and syntheses of special building blocks are described below. A description of necessary tools and synthetic methods including standard abbreviations for peptide synthesis can be found in "Reagents for Peptide and High-Throughput Synthesis", 2004/5 Catalogue, Novabiochem. General procedures
Peptide synthesis on an Applied Biosvstems peptide synthesizer ABI-433A
The peptide is synthesized according to the Fmoc strategy on an Applied Biosystems 433 peptide synthesizer on a 0.25 mmol or 1.0 mmol scale using the manufacturer supplied FastMoc UV protocols which employ the Fmoc protected amino acid (4 equivalents), HOBt (4 equivalents), HBTU (4 equivalents) and DIPEA (8 equivalents) in NMP, and UV monitoring of the deprotection of the Fmoc protection group. Piperidine in NMP is used for deprotection of the Fmoc protected amino acids.
Cleavage from the resin and side-chain deprotection
After completed solid-phase peptide synthesis, the resin is extensively washed with DCM. The resin is then washed with a premixed solution of DCM-triisopropylsilane-water- mercaptoethanol (92.5:2.5:2.5:2.5). After filtration, a mixture of TFA-triisopropylsilane-water- mercaptoethanol (92.5:2.5:2.5:2.5; at least 40 ml per mmol of resin) is added, and the mixture agitated for 3 hours before the resin is drained and the filtrate is collected. The resin is washed with TFA-triisopropylsilane-water-mercaptoethanol (92.5:2.5:2.5:2.5) and the filtrate is collected. To the combined filtrates, ice-cold diethyl ether (10 x the volume of the cleavage mixture) is added and the resulting precipitate is filtered off, washed with diethyl ether and dried.
Purification and quantification
The crude peptide is dissolved in a suitable mixture of water and MeCN or N- methylformamide and purified by reversed-phase preparative HPLC (Waters Deltaprep 4000 or Gilson) on a column containing C18-silica gel. Elution is performed with an increasing gradient of MeCN in water containing 0.1 % TFA. Relevant fractions are checked by analytical HPLC or UPLC. Fractions containing the pure target peptide are mixed and concentrated under reduced pressure. The resulting solution is analyzed (HPLC, LCMS) and the product is quantified using a chemiluminescent nitrogen specific HPLC detector (Antek 8060 HPLC- CLND) or by measuring UV-absorption at 280 nm. The product is dispensed into glass vials. The vials are capped with Millipore glassfibre prefilters. Freeze-drying for three days affords the peptide trifluoroacetate as a white solid. Preparation of Fmoc-Lys(bis(ieri-butoxycarbonylmethyl))-OH
Figure imgf000046_0001
Fmoc-Lys(bis(tert-butoxycarbonylmet yl))-OH A solution of benzyl chloroformate (8.8 ml, 61.3 mmol) in DCM (50 mL) was added dropwise to a stirred solution of Fmoc-Lys(Boc)-OH (50 g, 53,6 mmol), DIPEA (27 ml, 78 mmol) and DMAP (650 mg, 5.3 mmol) in DCM (250 mL) at 0 °C. The mixture was stirred at 0 °C for 24 hrs; then it was washed with 5% aqueous citric acid and water (200 mL). The organic layer was dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was taken up in DCM (30 mL), filtered (S3) and purified by column chromatography (silica gel, hexanes/ethyl acetate 3:1 ). The fractions containing the product were evaporated in vacuo. The resulting solid was reevaporated from ethyl acetate to give Fmoc-Lys(Boc)-OBn as white amorphous powder.
Yield: 49.0 g (82%).
1H NMR spectrum (300 MHz, CDCI3): δ 7.79 (d, J=7.3 Hz, 2 H); 7.62 (d, J=7.3 Hz, 2 H); 7.48 - 7.29 (m, 9 H); 5.44 (d, 1 H); 5.21 (dd, 2 H); 4.62 - 4.33 (m, 3 H); 4.24 (t, 1 H); 3.20 - 2.97 (m, 2 H); 1.97 - 1.61 (m, 2 H); 1.57 - 1.38 (m, 1 1 H); 1.41 - 1.15 (m, 2 H). The above Fmoc-Lys(Boc)-OBn (31.32 g, 54 mmol) was dissolved in anhydrous DCM (60 ml_), and solution of hydrogen chloride in dioxane (2.1 M, 205 mmol, 55 ml.) was added. The reaction mixture was stirred at room temperature for 10 hrs before removal of the solvent under reduced pressure. The solid residue was dried on air. This crude product was used without further purification. LC/MS analysis proved a completion of the reaction.
The reaction was done in two batches.
Crude Fmoc-Lys-OBn HCI salt (50.8 g, 102 mmol) was dissolved in dry DMF (250 ml_), and DIPEA (87 ml, 510 mmol), and ferf-butyl bromoacetate (45 ml_, 306 mmol) were added to the solution. The mixture was stirred at room temperature for 3 hrs, and DMF was removed under reduced pressure (at 50 °C). The residue was suspended in water (500 ml.) and extracted with DCM (3 x 500 ml_). The organic layer was dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was purified by column chromatography (silica gel, gradient elution hexanes/ethyl acetate 9:1 to 7:3) to give Fmoc-Lys(bis(ferf- butoxycarbonylmethyl))-OBn as pale yellow oil. Chromatography of mixed fractions was repeated.
Yield: 54.24 g (77%).
1H NMR spectrum (300 MHz, CDCI3): δ 7.76 (d, J=7.2 Hz, 2 H); 7.60 (d, J=6.6 Hz, 2 H); 7.45 - 7.23 (m, 9 H); 5.51 (d, 2 H); 5.17 (dd, 2 H); 4.44 - 4.30 (m, 2 H); 4.20 - 3.95 (m, 2 H); 3.41 (s, 4 H); 2.65 -2.58 (m, 3 H), 1.96 -1.30 (m, 6 H), 1.45 (s, 18 H).
Fmoc-Lys(bis(ferf-butoxycarbonylmethyl))-OBn (54.24 g, 79 mmol) was dissolved in methanol (500 ml_). Palladium on carbon (5 wt%, 3.35 g) was added to the solution. The suspension was stirred under hydrogen atmosphere at room temperature. After 3 hrs, the mixture was filtered through Celite and the filtrate was concentrated. The crude product was purified by flash column chromatography (silica gel, DCM/methanol 95:5) to afford the title compound Fmoc-Lys(bis(ferf-butoxycarbonylmethyl))-OH as white solid.
Yield: 31.4 g (67%).
Melting point: 51-52 °C.
1H NMR spectrum (300 MHz, CDCI3): δ 7.76 (d, J=7.3 Hz, 2 H); 7.60 (d, J=6.6 Hz, 2 H); 7.39 (t, J=7.3 Hz, 2 H); 7.30 (t, J=7.4 Hz, 2 H); 5,67 (d, J=7.2 Hz, 1 H); 4.31 -4.53 (m, 3 H); 4.17- 4.26 (m, 1 H); 3.54 (s, 1 H); 2.64-2.91 (m, 2 H); 1 .44 (s, 18 H), 1.19-1 .99 (m, 6 H).
The following compounds were produced according to the general procedures described above: Example 1
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-Arg-Asp-Lys(biscarboxymethyl)-Nle-NH2
Figure imgf000048_0001
LCT-MS, method 500_2000W_POS, m/z:867,41 ((M+2)2+) calculated mass 1732,88g/mol
Example 2
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Nle-NH2
Figure imgf000048_0002
LCT-MS, method 500_2000W_POS, m/z:857,97((M+2)2+) calculated mass 1713,83g/mol Example 3
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-NH2
Figure imgf000048_0003
LCT-MS, method 500_2000W_POS, m/z:801 ,25((M+2)2+) calculated mass 1600,75g/mol Example 4
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-Arg-Asp-Lys(biscarboxymethyl)-NH2
Figure imgf000049_0001
LCT-MS, method 500_2000W_POS, m/z:810,76((M+2)2+) calculated mass 1619,79g/mol
The following compounds can be produced according to the general procedures above:
Example 5
-c[Lys-His-D-Phe-Arg-Trp-Asp]-Pro-Pro-His-Ser-Lys(biscarboxymethyl)-Leu-NH2
Figure imgf000049_0002
Example 6
Ac-c[Lys-His-D-Phe-Arg-Trp-Asp]-Pro-Pro-Arg-Ser-Lys(biscarboxymethyl)-Leu-NH2
Figure imgf000049_0003
Example 8
-c[Orn-His-D-Phe-Arg-Trp-Glu]-Pro-Pro-Arg-Asp-Lys(biscarboxymethyl)-Phe-NH2
Figure imgf000050_0001
Example 9
-c[Orn-His-D-Phe-Arg-Trp-Glu]-Pro-Pro-Arg-Glu-Lys(biscarboxymethyl)-Val-NH2
Figure imgf000050_0002
Example 10
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-Arg-Glu-Lys(biscarboxymethyl)-Nle-NH2
Figure imgf000050_0003
Example 11
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Glu-Lys(biscarboxymethyl)-Val-NH2
Figure imgf000050_0004
Example 12
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Glu-Lys(biscarboxymethyl)-Nle-NH2
Figure imgf000051_0001
Example 13
-c[Glu-His-D-Phe-Arg-Trp-Om]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Val-NH2
Figure imgf000051_0002
Example 14
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Phe-NH2
Figure imgf000051_0003
PHARMACOLOGICAL METHODS
Assay (I) - Experimental protocol for efficacy testing on appetite with MC4 analogues, using an ad libitum fed rat model.
Male Sprague Dawley rats from Taconic Europe are used for the experiments. The rats have a body weight of 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 180-200g. Immediately after arrival and two weeks before dosing the rats are housed in reversed light cycle (dark from 10 am to 10 pm two rats in each cage). One week before dosing, rats are moved to the FeedWin system, where the rats are placed in individual cages for acclimatisation. 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 that period the animals are handled at least 3 times. 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.
Immediately before dosing the rats are randomised to the various treatment groups (n = 5-7) by body weight. Rats are dosed in the morning before onset of dark at 10 am with a 1-3 mg/kg solution administered subcutaneously (sc). Data are recorded for 48 hours and illustrated per dose group as accumulated food intake as a function of time. At the end of the experimental session, the animals are euthanised. Results describe reduction of food intake of the test group as a percentages of the mean food intake of the vehicle group.
Rats (app. 250g) are dosed at day 1 of the experiment, according to the table below, in the morning before onset of light. After dosing water-, food-intake and activity are registered by the FeedWin system. Data is collected each 15 minutes for 48 hours.
The effects on food intake is measured using the FeedWin system (Ellegards Systems, Faaborg, Denmark) which contains 32 stations for individual and continuous registration of food and water intakes. One station is defined by 1 cage with a metal lid plus 2 scales, one for food-intake and one for water-intake. Food and water intake is estimated by measurements of the disappearance of preloaded amounts of food and water that are placed on the 2 scales on each side of the cage. Food intake data are recorded electronically. Movements of the animal during recordings of food intake are registered by passive infrared sensors (PIR) placed on top of the cage. These sensors detect body heat within a protected area. In addition two PIR cameras (one for food and one for water) register the time and number of episodes spent eating and drinking. The stations are linked to 2 PC's where a specially designed programme (FeedWin) collects, registers and processes all results.
SYSTEM 1 , scantainer 1 +2, cage 1A to 16A
SYSTEM 2, scantainer 3+4, cage 1 B to 16B
The individual data are recorded in Microsoft excel sheets. Outliers are excluded after applying the Grubbs statistical evaluation test for outliers and the result are presented graphically using the GraphPad Prism program. Results describe reduction of food intake as a percentage of the mean food intake of the vehicle group. Results for 24, and 48 hours, respectively can be given. 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 supernatant removed. The cells are washed twice with stimulation buffer (5mM HEPES, 0.1 % ovalbumin, 0.005% Tween™ 20 and 0.5mM IBMX, pH 7.4), and then resuspended in stimulation buffer to a final concentration of 1x106 or 2x10s cells/ml. 25 μΙ of cell suspension is added to the microtiter plates containing 25 μΙ of test compound or reference compound (all diluted in stimulation buffer). The plates are incubated for 30 minutes at room temperature (RT) on a plate-shaker set to a low rate of shaking. The reaction is stopped by adding 25 μΙ of acceptor beads with anti-cAMP, and 2 min later 50 μΙ 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.
EC5o 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 a-MSH, and inhibited by increasing the amount of potential antagonist. 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 supernatant removed. The cells are washed twice with stimulation buffer, and resuspended in stimulation buffer to a final concentration of 2x106 cells/ml (consumption thereof: 7 ml per 96-well microtiter plate). 50 μΙ of cell suspension is added to the Flash Plate containing 50 μΙ of test compound or reference compound (all diluted in PBS, 0.1 % HSA and 0.005% Tween). The mixture is shaken for 5 minutes and then allowed to stand for 25 minutes at RT. The re- action is stopped by addition of 100 μΙ Detection Mix per well (Detection Mix 1 1 ml Detection Buffer + 100 μΙ (~2μΟί) cAMP [125l] tracer). The plates are then sealed with plastic, shaken for 30 minutes, and allowed to stand overnight (or for 2 hours) and then counted in the Topcounter (2 min/well). The assay procedure and the buffers are generally as described in the Flash Plate kit-protocol (Flash Plate® cAMP assay (NEN™ Life Science Products, cat. No. SMP004)). However the cAMP standards are diluted in PBS with 0.1 % HSA and 0.005% Tween™ 20 and not in stimulation buffer.
EC5o 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 was performed on BHK cell membranes stably expressing the MC1 receptor. The assay was performed in a total volume of 250 μΙ: 25 μΙ of 125NDP-a-MSH (22 pM in final concentration), 25 μΙ of test compound/control and 200 μΙ of cell membrane (25μg/ml). Test compounds were dissolved in DMSO. Radioactively labeled ligand, membranes and test compounds were diluted in buffer: 25 mM HEPES, pH 7.4, 0.1 mM CaCI2, 1 mM MgS04, 1 mM EDTA, 0.1 % HSA and 0.005% Tween™ 20. Alternatively, HSA may be substituted with ovalbumin. The samples were incubated at 30°C for 90min. in Costar round-botton microtiter plates. Incubation was terminated by filtration on a Packard harvester filtermate. Rapid filtration through Packard Unifilter-96 GF/B filters pre-treated with polyetylenimine (PerkinElmer 6005277). The filters were washed with ice-cold 0.9%NaCI 8- 10 times. The plates were air-dried at app. 55°c for 30min, and 50μΙ Microscint 0 (Packard, cat. No. 6013616) was added to each well. The plates were counted in a Topcounter (1 min/well). The data were analysed by non-linear regression analysis of binding curves, using the Windows™ program GraphPad™ Prism (GraphPad Software, USA).
Table 1 . In vitro data on receptor binding
Figure imgf000055_0001
Assay (V) - Melanocortin receptor 4 (MC4) binding assay
In vitro 125NDP-g-MSH binding to recombinant BHK cells expressing human MC4 receptor (filtration assay).
The assay was performed in 5 ml minisorb vials (Sarstedt No. 55.526) or in 96-well filter- plates (Millipore MADVN 6550), and using BHK cells expressing the human MC4 receptor using BHK cells stably expressing the human MC4 receptor. The membranes were prepared from frozen or fresh cells that were homogenized in 20 mM HEPES pH 7.1 , 5 mM MgCI2 and 1 mg/ml bacitracin and centrifuged at 15000 rpm at 4°C, 10 min in a Sorvall RC 5B plus, SS- 34 rotor. The supernatant was discarded, and the pellets were re-suspended in buffer, homogenized and centrifuged two more times. The final pellets were resuspended in the buffer mentioned above, and the protein concentration was measured and adjusted with buffer to 14 to 17 mg/ml and the membrane preparation were kept at -80°C until assay. The assay was run directly on a dilution of this cell membrane suspension, without any further preparation. The BHK cell membranes are kept at -80°C until assay, and the assay is run directly on a dilution of this cell membrane suspension, without further preparation. The suspension is diluted to give maximally 10% specific binding, i.e. to approx. 50-100 fold dilution. The assay was performed in a total volume of 200 μΙ: 50 μΙ of cell suspension, 50 μΙ of 125NDP-a-MSH (« 79 pM in final concentration), 50 μΙ of test compound and 50 μΙ binding buffer (pH 7) mixed and incubated for 2 h at 25°C [binding buffer: 25 mM HEPES, pH 7.0, 1 mM CaCI2, 1 mM MgSC-4, 1 mM EGTA, 0.02% Bacitracin, 0.005% Tween™ 20 and 0.1 % HSA or, alternatively, 0.1 % ovalbumin (Sigma; catalogue No. A-5503)]. Test compounds were dissolved in DMSO and diluted in binding buffer. Radiolabeled ligand and membranes were diluted in binding buffer. The incubation was stopped by dilution with 2 X 100 μΙ ice-cold 0.9% NaCI.The radioactivity retained on the filters was counted using a Cobra II auto gamma counter.
The data were analysed by non-linear regression analysis of binding curves, using the Windows™ program GraphPad™ Prism (GraphPad Software, USA).
Assay (VI) - 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. Light:dark 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 recording 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 measurement). Correction for changes in 02 and C02 content in the inflowing air is made in each 10- 18 min cycle.
Data are calculated per metabolic weight [(kg body weight) 0 75] for oxygen consump- tion and carbon dioxide production, and per animal for heat. Oxygen consumption (V02) is regarded as the major energy expenditure parameter of interest. Assay (VII) - Melanocortin receptor 3 (MC3) binding assay
The MC3 receptor binding assay is performed on BHK cell membranes stably expressing the human MC3 receptor. The human MC3 receptor is cloned by PCR and subcloned into pcDNA3 expression vector. Cells stably expressing the human MC3 receptor are generated by transfecting the expression vector into BHK cells and using G418 to select for MC3 clones. The BHK MC3 clones are cultured in DMEM with glutamax, 10% FCS, 1 % pen/strep and 1 mg/ml G418 at 37°C and 5% C02.
The binding is performed on a membrane preparation prepared in the following way: The cells are rinsed with PBS and incubated with Versene for approximately 5 min before harvesting. The cells are flushed with PBS and the cell-suspension is centrifuged for 10 min at 2800xG. The pellet is resuspended in 20ml buffer (20mM Tris pH 7.2 + 5mM EDTA + 1 mg/ml Bacitracin (Sigma B-0125)) and homogenized with a glass-teflon homogenizer, 10 times and low speed. The cell suspension is centrifuged at 4°C, 4100xG for 20min. Pellet is resuspended in buffer and the membranes are diluted to a protein concentration of 1 mg/ml in buffer, aliquoted and kept at -80°C until use.
The assay is performed in a volume of 100μΙ. Mix in the following order 25μΙ test compound, 25 μΙ 125l-NDP-a-MSH (app. 60 000 cpm/well ~ 0.25nM in final concentration) and 50μΙ membranes (30μg/well) and incubate in Costar round-bottom wells microtiter plate, (catalogue number 3365). Test-compounds are dissolved in DMSO or H20. Radioligand, membranes and test compounds are diluted in buffer; (25 mM HEPES pH 7.4, 1 mM CaCI2, 5 mM MgS04, 0.1 % Ovalbumin (Sigma A-5503), 0.005% Tween-20 and 5% Hydroxypropyl-β- cyclodextrin 97%, (Acros organics, code 297561000). The assay mixture is incubated for 1 h at 20-25°C. Incubation is terminated by filtration on a Packard harvester filtermate 196. Rapid filtration through Packard Unifilter-96 GF/B filters pre-treated for 1 h with 0.5% poly- ethylenimine is carried out. The filters are washed with ice-cold 0.9% NaCI 8-10 times. The plate is air dried at 55°C for 30 min, and 50μΙ Microscint 0 (Packard) is added.The radioactivity retained on the filter is counted using a Packard TopCount.NXT.
Results; IC50 values are calculated by non-linear regression analysis of binding curves (6 points minimum) using the windows program GraphPad Prism, GraphPad software, USA. Ki- values were calculated according to the Cheng-Prusoff equation [Y-C. Cheng and W.H. Pru- soff, Biochem. Pharmacol. 22 (1973) pp. 3099-3108].
Assay (VIII) - Melanocortin receptor 5 (MC5) binding assay
The MC5 receptor binding assay is performed on BHK cell membranes stably expressing the human MC3 receptor. The human MC5 receptor is cloned by PCR and subcloned into pcDNA3 expression vector. Cells stably expressing the human MC5 receptor are generated by transfecting the expression vector into BHK cells and using G418 to select for MC5 clones. The BHK MC5 clones are cultured in DMEM with glutamax, 10% FCS, 1 % pen/strep and 1 mg/ml G418 at 37°C and 5% C02.
The binding is performed on a membrane preparation prepared in the following way:
The cells are rinsed with PBS and incubated with Versene for approximately 5 min before harvesting. The cells are flushed with PBS and the cell suspension is centrifuged for 10 min at 2800xG. The pellet is resuspended in 20ml buffer (20mM Tris pH 7.2 + 5mM EDTA + 1 mg/ml Bacitracin (Sigma B-0125)) and homogenized with a glass-teflon homogenizer, 10 times and low speed. The cell-suspension is centrifuged at 4°C, 4100xG for 20min. Pellet is resuspended in buffer and the membranes are diluted to a protein concentration of 1 mg/ml in buffer, aliquoted and kept at -80°C until use.
The assay is performed in a volume of 100 μΙ. Mix in the following order 25μΙ test- compound, 25 μΙ 125l-NDP-a-MSH (app. 60 000 cpm/well ~ 0.25nM in final concentration) and 50μΙ membranes (10μg/well) and incubate incubation in Costar round-bottom wells microtiter plate, catalogue number 3365: Test-compounds are dissolved in DMSO or H20. Radioligand, membranes and test-compounds are diluted in buffer; (25 mM HEPES pH 7.4, 1 mM CaCI2, 5 mM MgS04, 0.1 % Ovalbumin (Sigma A-5503) , 0.005% Tween-20 and 5% Hydroxypro- pyl-3-cyclodextrin, (97%, Acros organics, code 297561000). The assay mixture is incubated for 1 h at 20-25°C. Incubation is terminated by filtration on a Packard harvester filtermate 196. Rapid filtration through Packard Unifilter-96 GF/B filters pre-treated for 1 h with 0.5% poly- ethylenimine is carried out. The filters are washed with ice-cold 0.9% NaCI 8-10 times. The plate is air dried at 55°C for 30 min, and 50μΙ Microscint 0 (Packard) is added. The radioactivity retained on the filter is counted using a Packard TopCount.NXT.
Results: IC50 values are calculated by non-linear regression analysis of binding curves (6 points minimum) using the windows program GraphPad Prism, GraphPad software, USA. Ki- values were calculated according to the Cheng-Prusoff equation [Y-C. Cheng and W.H. Pru- soff, Biochem. Pharmacol. 22 (1973) pp. 3099-3108]. Assay (IX) - Melanocortin receptor 3 (MC3) or melanocortin receptor 5 (MC5) cAMP functional assay using the FlashPlate® cAMP detection kit
The MC3 or MC5 -containing BHK cells are stimulated with potential MC3 or MC5 agonists, and the degree of stimulation of cAMP is measured using the FlashPlate® cAMP assay, cat. No SMP004, NEN™ Life Science Products. BHK/hMC3 or BHK/hMC5 cells
The cells are produced by transfecting the cDNA encoding MC3 or MC5 receptor into BHK570, and selecting for stable clones expressing the hMC3 receptor.The cells are grown in DMEM, 10 % FCS, 1 mg/ml G418 and 1 % pen/strep.
Cells at approx. 80-90% confluence are washed with PBS, lifted from the plates with Versene and diluted in PBS. After centrifugation for 5 min at 1300 rpm the supernatant is removed, and the cells are resuspended in stimulation buffer to a final concentration of 2 x 106 cells/ml. 50 μΙ cell suspension is added to the Flashplate containing 50 μΙ of test-compound or reference compound (all dissolved in DMSO and diluted in 0.1 % HSA (Sigma A-1887) and 0.005% Tween 20). The mixture is shaken for 5 minutes and then allowed to stand for 25 minutes at room temperature. The reaction is stopped with 100 μΙ Detection Mix pro well (Detection Mix 1 1 ml Detection Buffer + 100 μΙ (~2 μθί) cAMP [125l] Tracer). The plates are then sealed with plastic, shaken for 30 minutes and allowed to stand overnight (or for 2h), and then counted in the Topcounter, 2 min/well (Note that in general, the assay procedure de- scribed in the kit-protocol is followed; however, the cAMP standards are diluted in 0.1 % HSA and 0.005% Tween 20, and not in stimulation buffer).
Results
EC5o values are calculated by non-linear regression analysis of dose-response curves (6 points minimum) using the Windows program GraphPad Prism, GraphPad software, USA. Results are expressed in nM. Emax values are calculated as % of NDP-a-MSH maximal stimulation in the hMC3cAMP assay (maximal NDP-a-MSH stimulation 100%).

Claims

1. A compound according to formula I:
R1-c[X1-His-D-Phe-Arg-Trp-X2]- Z1-Z2-Z3-Z4-Z5-Z6-R2 [I]
wherein
R1 represents Ci-3 alkanoyl;
X1 represents Asp, Glu, Lys, Orn, Dab, Dap, Cys, homo-Cys or Penicillamine.
X2 represents Asp, Glu, Lys, Orn, Dab, Dap, Cys, homo-Cys or Penicillamine.
wherein X1 and X2 are joined, rendering the compound of formula I cyclic, either via a disulfide bridge, X1 and X2 being independently Cys, homo-Cys or Penicillamine, or via a lactam bridge, either from X1 being Asp or Glu and X2 being Lys, Orn, Dab or Dap, or from X1 being Lys, Orn, Dab or Dap and X2 being Asp or Glu.
Z1 represents Pro, D-Pro, Hyp or D-Hyp;
Z2 represents Pro, D-Pro, Hyp or D-Hyp;
Z3 represents Lys, Arg , His, D-Lys, D-Arg or D-His;
Z4 represents Glu, Asp, D-Glu, D-Asp, Ser or D-Ser;
Z5 represents Lys(bis carboxymethyl);
Z6 is absent or represents a peptide fragment comprising one to four amino acid residues derived from Met, D-Met, Leu, D-Leu, Nle, D-Nle, lie, D-lle, Val or D-Val;
and pharmaceutically acceptable salts, prodrugs and solvates thereof.
R2 represents -OR or -NR2, R being hydrogen or Ci-3 alkyl.
2. A compound according to claim 1 , selected from the group consisting of: Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-Arg-Asp-Lys(biscarboxymethyl)-Nle-NH2
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Nle-NH2
Figure imgf000061_0001
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-NH2
Figure imgf000061_0002
Figure imgf000061_0003
Figure imgf000061_0004
-c[Lys-His-D-Phe-Arg-Trp-Asp]-Pro-Pro-Arg-Ser-Lys(biscarboxymethyl)-Leu-NH2
Figure imgf000062_0001
-c[Orn-His-D-Phe-Arg-Trp-Glu]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Leu-NH2
Figure imgf000062_0002
-c[Orn-His-D-Phe-Arg-Trp-Glu]-Pro-Pro-Arg-Asp-Lys(biscarboxymethyl)-Phe-NH2
Figure imgf000062_0003
Ac-c[Orn-His-D-Phe-Arg-Trp-Glu]-Pro-Pro-Arg-Glu-Lys(biscarboxymethyl)-Val-NH2
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-Arg-Glu-Lys(biscarboxymethyl)-Nle-NH2
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Glu-Lys(biscarboxymethyl)-Val-NH2
Figure imgf000063_0001
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Glu-Lys(biscarboxymethyl)-Nle-NH2
Figure imgf000063_0002
Ac-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Val-NH2
-c[Glu-His-D-Phe-Arg-Trp-Orn]-Pro-Pro-His-Asp-Lys(biscarboxymethyl)-Phe-NH2
Figure imgf000064_0001
3. A method of delaying the progression from IGT to type 2 diabetes, comprising administering to a patient in need thereof an effective amount of a compound according to any of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
4. A method of treating obesity or preventing overweight, comprising administering to a patient in need thereof an effective amount of a compound according to any of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
5. A method of regulating appetite, comprising administering to a patient in need thereof an effective amount of a compound according to any of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
6. A method of inducing satiety, comprising administering to a patient in need thereof an effective amount of a compound according to any of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
7. A method of preventing weight gain after successfully having lost weight, comprising administering to a patient in need thereof an effective amount of a compound according to any of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
8. 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 according to any of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
9. A method of treating bulimia, comprising administering to a patient in need thereof an effective amount of a compound according to any of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
10. A method of treating a disease or state selected from atherosclerosis, hypertension, 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 of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
1 1 . A method of treating, in an obese patient, a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, impaired glucose tolerance (IGT), dyslipide- mia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea or risk of premature death, neuronal protection, effect in ischemic heart disease or anti-inflammatory effects comprising administering to an obese patient in need thereof an effective amount of a compound according to any of claims 1 or 2, optionally in combination with one or more additional therapeutically active compounds.
12. A method according to any of claims 3-1 1 , wherein said additional therapeutically active compound is selected from antidiabetic agents, antihyperlipidemic agents, an- tiobesity agents, antihypertensive agents and agents for the treatment of complications resulting from, or associated with, diabetes.
13. A pharmaceutical composition comprising a compound according to any of claims 1 or 2 and one or more excipents.
14. A compound according to any of claims 1 or 2 for use in therapy.
15. The use of a compound according to any of claims 1 or 2 in the manufacture of a medicament for delaying the progression from impaired glucose tolerance (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 diabetes, IGT, dyslipidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea or risk of premature death; or treating, in an obese patient, a disease or state selected from atherosclerosis, hypertension, type 2 diabetes, IGT, dyspilidemia, coronary heart disease, gallbladder disease, gall stone, osteoarthritis, cancer, sexual dysfunction, hypthalamic amenorrhea or risk of premature death; for providing neuronal protection, for having an effect on ischemic heart disease or anti-inflammatory effects and for the treatment of autoimmune diseases, e.g. multiple sclerosis.
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