WO2011005611A1 - Neuromedin u receptor agonists and uses thereof - Google Patents

Neuromedin u receptor agonists and uses thereof Download PDF

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Publication number
WO2011005611A1
WO2011005611A1 PCT/US2010/040272 US2010040272W WO2011005611A1 WO 2011005611 A1 WO2011005611 A1 WO 2011005611A1 US 2010040272 W US2010040272 W US 2010040272W WO 2011005611 A1 WO2011005611 A1 WO 2011005611A1
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Prior art keywords
amino acid
ala
leu
peptide
phe
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PCT/US2010/040272
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French (fr)
Inventor
Antonello Pessi
Elisabetta Bianchi
Paolo Ingallinella
Donald J. Marsh
Andrea M. Peier
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Merck Sharp & Dohme Corp.
Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A.
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Publication of WO2011005611A1 publication Critical patent/WO2011005611A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the present invention relates to neuromedin U receptor agonists for use in the treatment of metabolic disorders such as obesity.
  • the present invention relates to neuromedin U receptor agonists that are analogs or derivatives of neuromedin U (NMU).
  • NMU Neuromedin U
  • NMU' s role in the regulation of energy homeostasis is supported by both pharmacologic and genetic data. Properties of NMU include inhibition of food intake and increase in energy expenditure seen when the substance is administered centrally (Howard et al, Nature 406: 70-74 (2000); Nakazato et al, Biochem. Biophys. Res. Comm. 277: 191-194 (2000); Ivanov et al, Endocrinol. 143: 3813-3821 (2002); and Wren et al, Endocrinol, 143: 4227-4234 (2002)). NMU-deficient mice develop obesity characterized by hyperphagia and reduced energy expenditure (Hanada et al, Nat. Med., 10: 1067-1073 (2004)), and transgenic mice
  • NMU neuropeptide-like protein
  • NMS 36-residue neuropeptide neuromedin S
  • Intracerebroventricular (icv) injection of NMS decreased 12 hour food intake during the dark period in rats. This anorexigenic effect was more potent and persistent than that observed with the same dose of NMU. NMS has also been disclosed in International applications
  • NMURl is predominantly expressed in the periphery, whereas NMUR2 is primarily expressed in the brain.
  • Pharmacologic experiments have served to better define NMU' s short- and long-term effects on energy homeostasis and to identify which NMU receptor(s) are involved in mediating these actions. It has been shown that acute administrations of NMU either centrally or peripherally reduce food intake in mice in a dose-dependent fashion.
  • the anorectic actions of centrally administered NMU are absent in NMUR2-deficient (Nmur2 '/' ) mice but are present in NMURl- deficient (Nmurl ' ⁇ ) mice.
  • the anorectic actions of peripherally administered NMU are absent in Nmurl '1' mice and present in NmurT ' mice.
  • acute peripheral administration of NMU dose-dependently increases core body temperature in mice, suggesting that NMURl may also modulate energy expenditure.
  • Chronic administration of NMU either centrally or peripherally reduces food intake, body weight and adiposity in mice, again in a dose- dependent fashion.
  • Nmur2 '/' transgenic mice body weight, body composition, body temperature and food intake are largely unaffected by chronic central administration of rat NMU- 23.
  • Nm url '1' transgenic mice body weight, body composition and food intake are largely unaffected by chronic peripheral administration of rat NMU-23.
  • NMURl- vs. NMUR2-mediated efficacy differ and appear to be independent of one another, but have a role in obesity
  • both NMURl- and NMUR2- selective agonists and NMURl /2 non-selective agonists may be useful for the treatment of obesity. Therefore, there is a need for neuromedin U receptor agonists useful in the treatment of metabolic disorders.
  • the present invention provides neuromedin U (NMU) receptor agonists comprising an NMU peptide or analog thereof conjugated to a functional group on a carrier protein.
  • NMU neuromedin U
  • Therapeutic applications of the neuromedin U receptor agonists include administering the neuromedin U receptor agonists to an individual to treat a metabolic disorder afflicting the individual.
  • Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, and type II diabetes.
  • Complications of diabetes such as retinopathy may be positively affected thereby as well.
  • Obesity is a comorbidity of and may well contribute to such disease states as diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis and certain forms of cancers.
  • Administration of one or more of the neuromedin U receptor agonists disclosed herein to effect weight loss in an individual may also be useful in preventing such diseases and as part of therapy for any one of the above-recited conditions, as well as others.
  • a method for treating a metabolic disease in an individual comprising administering to the individual one or more of the neuromedin U receptor agonists described above.
  • the metabolic disease may be selected from the group consisting of diabetes, metabolic syndrome, hyperglycemia, and obesity and may be administered via a route peripheral to the brain, such as an oral, mucosal, buccal, sublingual, nasal, rectal, subcutaneous, transdermal, intravenous, intramuscular, or intraperitoneal route.
  • the neuromedin U receptor agonists can be administered to an individual to effect a reduction in food intake by the individual, to effect a reduction in weight gain in the individual, to prevent weight gain in the individual, to effect weight loss in the individual, and/or to prevent weight regain in the individual.
  • the present invention provides an isolated neuromedin U receptor agonist conjugated to a carrier protein.
  • the neuromedin U receptor agonist has the formula (I)
  • amino acids 1 to 17 can be any amino acid or absent; wherein amino acid Xl ⁇ i 8 absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid X* 9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid ⁇ 20 is absent, Leu, GIy, sarcos
  • the peptide has the amino acid sequence ⁇ L ⁇ 2. ⁇ 3. ⁇ 4. ⁇ 5. ⁇ 6- ⁇ 7- ⁇ 8. ⁇ 9- ⁇ l 0- ⁇ l 1. ⁇ l2. ⁇ l 3_ ⁇ l 4. ⁇ 15. ⁇ l 6. ⁇ l 7. ⁇ l 8-Phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO: 2), wherein amino acids 1 to 17 can be any amino acid or absent,
  • the peptide comprises the amino acid sequence Phe- Arg- VaI-
  • amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group
  • amino acid Xl9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid
  • amino acid X20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid
  • amino acid ⁇ 21 is Phe, NMe-Phe
  • the peptide comprises the amino acid sequence ⁇ l-X2- X3. ⁇ 4. ⁇ 5_ ⁇ 6_ ⁇ 7. ⁇ 8 (SEQ ID NO:4) wherein amino acid Xl is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid X2 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X3 is absent, GIy, sarcosine (Sar), Leu or D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L- amino acid; amino acid ⁇ 4 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid X ⁇ is Arg, Lys,
  • the present invention further provides for the use of any one or more of the embodiments and aspects of the neuromedin U receptor agonist in the manufacture of a medicament for treatment of a metabolic disorder.
  • Disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, and type II diabetes. Complications of diabetes such as retinopathy may be positively affected thereby as well.
  • Obesity is a comorbidity of and may well contribute to such disease states as diabetes, hypertension, dyslipidemias,
  • the present invention provides a pharmaceutical composition comprising one or more of any of the above neuromedin U receptor agonists and a pharmaceutically acceptable carrier.
  • a method for treating a metabolic disorder in an individual comprising administering to the individual a therapeutically effective amount of a neuromedin U receptor agonist that has the formula (I) Zl-peptide-Z2 wherein the peptide has the amino acid sequence Xl- ⁇ 2- ⁇ 3 ⁇ 4_ ⁇ 5-. ⁇ 6_ ⁇ 7_ ⁇ 8- X 9- ⁇ l0. ⁇ l I_ ⁇ l2. ⁇ l3_ ⁇ l4. ⁇ l5. ⁇ l6- ⁇ l7. ⁇ l8. ⁇ l9- ⁇ 2 ⁇ - ⁇ 21. ⁇ 22- ⁇ 23. ⁇ 24. ⁇ 25 (SEQ ID NO:1), wherein amino acids 1 to 17 can be any amino acid or absent; wherein amino acid ⁇ !8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid ⁇ l9 is
  • the method is particularly useful for treating a metabolic disorder selected from the group consisting of obesity, metabolic syndrome or syndrome X, type II diabetes,
  • the peptide has the amino acid sequence Xl-X2- ⁇ 3-. ⁇ 4- ⁇ 5- ⁇ 6- ⁇ 7- ⁇ 8- ⁇ 9_ ⁇ l0_ ⁇ l L ⁇ l2- ⁇ l3. ⁇ l4 , ⁇ l5. ⁇ l6_ ⁇ l7. ⁇ l8.phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO:2), wherein amino acids 1 to 17 can be any amino acid or absent.
  • the peptide comprises the amino acid sequence Phe- Arg- VaI- Asp-Glu-GIu-Phe-Gln-Ser-Pro-Phe-AIa-Ser-Gln-Ser-Arg-Gly-Xl 8-Xl 9- ⁇ 20. ⁇ 21- ⁇ 22- ⁇ 23.
  • amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn 5 NIe 5 GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid Xl9 1S Ala, Trp 5 Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid X ⁇ l is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid X22 is Arg, Lys, Harg, Ala, or Leu; amino acid ⁇ 23 is Pro, Ser, Sar, Ala or Leu;
  • the peptide comprises the amino acid sequence ⁇ l ⁇ 2- ⁇ 3- ⁇ 4- ⁇ 5- ⁇ 6. ⁇ 7. ⁇ 8 (SEQ ID NO:4) wherein amino acid Xl is absent, Tyr or D-Tyr 5 Leu, Phe, VaI, GIn, NIe, GIu or D-GIu 5 Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid X ⁇ is AIa, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X3 is absent, GIy, sarcosine (Sar), Leu or D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L- amino acid; amino acid X4 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid X5 is Arg, Lys
  • the present invention further includes use of the compositions disclosed herein in the manufacture of a medicament for treatment of a metabolic disorder, which in further aspects can be obesity or type II diabetes.
  • pharmaceutical composition comprising a neuromedin U receptor agonist and a pharmaceutically acceptable carrier.
  • an "NMU peptide analog” is a peptide that has sufficient identity or homology to native human NMU having the amino acid sequence shown in SEQ ID NO:5 that it is capable of interacting with the NMURl and/or NMUR2 receptors as an agonist.
  • NMU peptide analogs can have one or more amino acid substitutions, modifications, or deletions at amino acid positions 1-25.
  • FIG. 1 Acute administration of palmitoylated NMU analogs significantly reduces food intake (A) and body weight (B) in diet-induced obese mice.
  • the present invention provides neuromedin U (NMU) receptor agonists comprising an NMU peptide or analog.
  • Therapeutic applications of the neuromedin U receptor agonists include administering the neuromedin U receptor agonists to an individual to treat a metabolic disorder afflicting the individual.
  • Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, and type II diabetes.
  • Complications of diabetes such as retinopathy may be positively affected thereby as well.
  • Obesity is a comorbidity of and may well contribute to such disease states as diabetes, hypertension, dyslipidemias,
  • a method for treating a metabolic disease in an individual comprising administering to the individual one or more of the neuromedin U receptor agonist s described above.
  • the metabolic disease may be selected from the group consisting of diabetes, metabolic syndrome,
  • hyperglycemia, and obesity may be administered via a route peripheral to the brain, such as an oral, mucosal, buccal, sublingual, nasal, rectal, subcutaneous, transdermal, intravenous, intramuscular, or intraperitoneal route.
  • the neuromedin U receptor agonists can be administered to an individual to effect a reduction in food intake by the individual, to effect a reduction in weight gain in the individual, to prevent weight gain in the individual, to effect weight loss in the individual, and/or to prevent weight regain in the individual.
  • the present invention provides an isolated neuromedin U receptor agonist conjugated to a carrier protein.
  • the neuromedin U receptor agonist has the formula (I)
  • amino acids 1 to 17 can be any amino acid or absent; wherein amino acid Xl8 j s absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid ⁇ l9 1S Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid ⁇ 20
  • the peptide has the amino acid sequence ⁇ l- ⁇ 2 , ⁇ 3_ ⁇ 4. ⁇ 5. ⁇ 6- ⁇ 7- ⁇ 8- ⁇ 9- ⁇ l0 . ⁇ l l- ⁇ l2- ⁇ l3- ⁇ l4- ⁇ l5- ⁇ l6- ⁇ l7_ ⁇ l8-Phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO: 2), wherein amino acids 1 to 17 can be any amino acid or absent.
  • the peptide comprises the amino acid sequence Phe-Arg-Val- Asp-Glu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-GIn-Ser-Arg-Gly ⁇ Xl 8_ ⁇ l9 ⁇ 2 ( L ⁇ 21. ⁇ 22. ⁇ 23.
  • amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, AIa, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid Xl9 j s Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid X21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; X22 js Arg, Lys, Harg, Ala, or Leu; amino acid ⁇ 23 is Pro, Ser, Sar, Ala or
  • the peptide comprises the amino acid sequence ⁇ l— ⁇ 2- ⁇ 3- ⁇ 4- ⁇ 5- ⁇ 6. ⁇ 7_ ⁇ 8 (SEQ ID NO:4) wherein amino acid Xl is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid ⁇ 2 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid ⁇ 3 is absent, GIy, sarcosine (Sar), Leu or D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L- amino acid; amino acid X ⁇ is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid X5 is Arg,
  • the present invention further provides for the use of any one or more of the embodiments and aspects of the neuromedin U receptor agonist in the manufacture of a medicament for treatment of a metabolic disorder.
  • Disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, and type II diabetes. Complications of diabetes such as retinopathy may be positively affected thereby as well.
  • Obesity is a comorbidity of and may well contribute to such disease states as diabetes, hypertension, dyslipidemias,
  • the present invention provides a pharmaceutical composition comprising one or more of any of the above neuromedin U receptor agonists and a pharmaceutically acceptable carrier.
  • a method for treating a metabolic disorder in an individual comprising administering to the individual a therapeutically effective amount of a neuromedin U receptor agonist that has the formula (I)
  • amino acids 1 to 17 can be any amino acid or absent; wherein amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn 3 NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid Xl9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X ⁇ O is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid ⁇ 21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; X22 is Arg, Lys, Harg, Ala, or Leu; amino acid ⁇ 23 is Pro, Ser, Sar, Al
  • the method is particularly useful for treating a metabolic disorder selected from the group consisting of obesity, metabolic syndrome or syndrome X, type II diabetes, complications of diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers.
  • a metabolic disorder selected from the group consisting of obesity, metabolic syndrome or syndrome X, type II diabetes, complications of diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers.
  • the peptide has the amino acid sequence ⁇ l- ⁇ 2. ⁇ 3_ ⁇ 4_ ⁇ 5_ ⁇ 6- ⁇ 7- ⁇ 8- ⁇ 9- ⁇ l ( L ⁇ l 1 _ ⁇ l 2. ⁇ l 3- ⁇ l4. ⁇ l 5. ⁇ l 6. ⁇ l 7_ ⁇ l 8-Phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID N0:2), wherein amino acids 1 to 17 can be any amino acid or absent.
  • the peptide comprises the amino acid sequence Phe- Arg- VaI- Asp-Glu-Glu-Phe-Gln-Ser-Pro ⁇ Phe-Ala-Ser-Gln-Ser-Arg-Gly-Xl 8-Xl 9- ⁇ 20_ ⁇ 21_ ⁇ 22_ ⁇ 23.
  • amino acid ⁇ l8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group
  • amino acid X ⁇ 9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid
  • amino acid X20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid
  • amino acid ⁇ 21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp
  • X22 is Arg, Lys, Harg, Ala, or Leu
  • amino acid X23 is Pro, Ser, Sar, Ala or Leu
  • amino acid X23 is Pro, Ser,
  • the peptide comprises the amino acid sequence ⁇ l-X2- ⁇ 3- ⁇ 4. ⁇ 5- ⁇ 6_ ⁇ 7- ⁇ 8 (SEQ ID NO:4) wherein amino acid Xl is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu 3 Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid ⁇ 2 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid ⁇ 3 is absent, GIy, sarcosine (Sar), Leu or D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L- amino acid; amino acid ⁇ 4 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid ⁇ 5 is Arg, Lys
  • neuromedin U receptor agonists of the present invention comprising the amino acid sequence Xl-X2-x3.x4.x5.x6-X7.x8.x9-Xl0.xn.xl2.xl3-Xl4.xl5.xl6.
  • Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO:2) wherein amino acids 1 to 16 can be any amino acid or absent are shown in Table 1.
  • the neuromedin U receptor agonists are protected at the C-terminus with an amino group.
  • neuromedin U receptor agonists of the present invention comprising the amino acid sequence Phe-Arg-Val-Asp-GIu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-Gln-Ser-Arg- Gly- ⁇ l8» ⁇ l9. ⁇ 20. ⁇ 21. ⁇ 22. ⁇ 23. ⁇ 24. ⁇ 25 (SEQ ID N0:3) or Xl ⁇ 2- ⁇ 3- ⁇ 4- ⁇ 5- ⁇ 6. ⁇ 7. ⁇ 8 (SEQ ID NO:4) wherein amino acid ⁇ l ⁇ or Xl is absent, Tyr, Ala, Leu, Phe, VaI, GIn, Asp, GIu , (D)-Glu,(D)-Tyr, (D)-Lys, homo-glutamic acid (hGlu), 3-methoxyphenyIalanine (MOF), 4- fluoro-phenylalanine (PhF), 4-nitro-phenylalanine (FNO), 4-carboxylmethoxy-pheny
  • Lipidated (either cholesteroylated or palmitoylated) neuromedin U receptor agonists were designed to have an improved in vivo pharmacological profile.
  • NMU36 and NMU37 are cholesteroylated peptides based on the native NMU sequence.
  • NMU36 has the structure Ac-CsFRVDEEFQSPFASQSRGYFLFRPRN- CONH 2 wherein the C5 is a cholest-5-en-3-yl (21R)-21-amino-17,22-dioxo-4,7,10,13-tetraoxa- 19-thia-16-azadocosan-l-oate attached to the thio group of cysteine (or Cys(oxa4-cholesterol)) as shown below.
  • NMU37 has the structure AC-CeFRVDEEFQSPFASQSRGYFLFRPRN-CONH 2 wherein the Cg is a cholest ⁇ 5-en-3-yl N-[43-(3- ⁇ [(2R)-2-amino-3-oxopropyl]sulfanyl ⁇ -2,5- dioxopyrrolidin- 1 -yl)-41 -oxo-4,7, 10,13,16,19,22,25,28,31 ,34,37-dodecaoxa-40- azatritetracontan-l-oyl]glycinate attached to the thio group of cysteine or Cys(oxai2-cholesterol)) as shown below.
  • NMU44 is a cholesteroylated peptide based on N-terminally trancated sequence 17-25, bearing an acetylated C ⁇ group at the N-terminus.
  • hydrophilic spacers between the cholesterol group and the cysteine moiety like those contained in C& and C7, was found to be important for the in vitro and in vivo activity of the cholesteroylated NMU derivatives.
  • NMU38 is a palmitoylated analog in which the palmitoyl group is linked at the N- terminus of the NMU peptide sequence.
  • NMU34 is a palmitoylated analog in which the palmitoyl group is linked at the N- terminus of the C-terminal nonamer of the NMU peptide sequence.
  • NMU39 and NMU40 are palmitoylated analogs in which a Ttds (l-amino-4,7,10- trioxa-13-tridecanamine succinimic acid) or a gamma glutamic residue ( ⁇ E) were respectively introduced as flexible spacers between the N-terminus of the NMU sequence and the palmitoyl group.
  • Ttds l-amino-4,7,10- trioxa-13-tridecanamine succinimic acid
  • ⁇ E gamma glutamic residue
  • NMU41 and NMU42 have respectively a Ttds group and a gamma glutamic acid residue at the N-terminus of the NMU peptide sequence. These analogs are control peptides with no palmitoyl but acetyl group at the N-terminus.
  • NMU58 is a PEGylated peptide in which a branched PEG of 40 kDa is linked at the N-terminus of the native human neuromedin U peptide.
  • the N-terminal group of the peptide was acylated with a branched (PEG)2 4OK N-hydroxysuccinimide analog (SUNBRIGHT GL2- 400GS2; NOF Corporation). This was designed to create a neuromedin U receptor agonist with improved pharmacological profile.
  • the peptide precursor, the wild type sequence of NMU was reacted with an N-hydroxysuccinimide derivative of a branched PEG of 40 kDa. PEGylation with this reagent occurs specifically at the N-terminal amino group of the peptide, as this is the only available amino group in the peptide.
  • NMU51 , NMU52, NMU53 and NMU54 are PEGylated peptides in which the
  • PEG moiety is linked at an internal position of the human NMU peptide sequence. These peptides were designed to create a neuromedin U receptor agonist with improved
  • PEGylation site was scanned every four residues by mutating the native residues into a Cys residue in order to allow site-specific derivatization of the thiol group with a branched (PEG) 2 4OkDa (C2).
  • PEG branched
  • C2 group was introduced at positions: 4 to obtain NMU51, 8 to obtain NMU52, 12 to obtain NMU53 and 16 to obtain NMU54.
  • NMU80, NMU81 and NMU33 are PEGylated peptides designed starting from the native NMU sequence and adding an acetylated cysteine residues at the N-terminus.
  • the cysteine thiolated group was derivatized with (a) linear (mPEG)40 kDa, through either 40 kDa methoxy ⁇ oly(ethylene glycol)maleimido-propionamide (Chirotech, Product Code 008-016) to obtain NMU80, or the iodoacetylated PEG reagent SUNBRIGHT ME-400IA (NOF Corporation) to obtain NMU81 ; or (b) linear (mPEG)5 kDa to obtain NMU33.
  • PEGylated peptides were designed based on the introduction of a spacer between the PEG moiety and the NMU peptide.
  • the spacer was introduced to reduce the impact on activity on the peptide sequence due to the addition of the PEG.
  • the spacer consists, for example, in five consecutive units of tranexamic acid (Txa)5,
  • NMUlOO and NMUlOl were designed starting from the full-length native NMU sequence.
  • the sequences were modified by introduction at the N-terminus of (Txa)s as spacer and an acetylated cysteine residue.
  • the cysteine thiolated group was derivatized with (a) N- ethylmaleimide to obtain NMUlOl, a control peptide for conjugation; or (b) (PEG)240 kDa to obtain NMUlOO.
  • NMU 108 and NMUl 18 were designed to obtain a PEGylated peptide based on the N-term ⁇ nally truncated sequence 17-25.
  • the peptide sequences were modified by
  • NMUl 18 a control peptide for conjugation.
  • the N-terminal group of the peptide NMUl 18 was acylated with a branched (PEG)2 40k N-hydroxysuccinimide analog (SUNBRIGHT GL2-400GS2, NOF Corporation) to obtain NMU108.
  • NMUl 16 and NMUl 19 were designed to obtain a PEGylated peptide based on the N ⁇ terminally truncated sequence 12-25. The peptide sequences were modified by
  • NMUl 16 a control peptide for conjugation.
  • the N-terminal group of the peptide NMUl 16 was acylated with a branched (PEG)2 40k N-hydroxysuccinimide analog (SUNBRJGHT GL2-400GS2, NOF Corporation) to obtain NMUl 19.
  • NMU130, NMU134, NMU129 and NMU137 the native NMU Phej 1 was changed to threonine, GIy 17 was changed to proline and Asn 2 s was changed to (D)-alanine.
  • the substitutions at Phe ⁇ , GIy 17, and Asn25 were made to increase in vivo stability while retaining functional activity, since these residues were found to be among the primary proteolytic cleavage sites.
  • NMU 129 and NMUl 37 were designed to obtain a PEGylated peptide based on the triple-mutant NMU peptide sequence as described above.
  • the N-terminal group of the peptide was (a) acetylated to obtain NMU 129, a control peptide for conjugation; or (b) acylated with a linear (PEG) 4OK N-hydroxysuccinimide analog (SUNBRIGHT ME-400HS; NOF Corporation) to obtain NMUl 37.
  • NMUl 30 and NMUl 34 were based on the amino acid sequence of NMU 129 and 137.
  • the peptide sequences were modified by introduction at the N-lerminus of (Txa)s as spacer to obtain NMUl 30, a control peptide for conjugation.
  • the N-terminal group of the peptide NMU130 was acylated with a linear (PEG) 4OK N-hydroxysuccinimide analog (SUNBRIGHT ME-400HS; NOF Corporation) to obtain NMU134.
  • NMU 138 and NMUl 39 have the same structure respectively of NMU 129 and NMUl 37 wherein the Arg24 has been substituted with a homo-arginine residue to increase in vivo stability.
  • the neuromedin U receptor agonist optionally includes a protecting group covalently joined to the N-terminal amino group.
  • a protecting group covalently joined to the N-terminal amino group of the neuromedin U receptor agonists reduces the reactivity of the amino terminus under in vivo conditions.
  • Amino protecting groups include -Cj.
  • the amino terminus protecting group is selected from the group consisting of acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl, and t-butyloxycarbonyl.
  • N-terminal amino acid is another modification that is contemplated for reducing the reactivity of the amino terminus under in vivo conditions.
  • compositions of the neuromedin U receptor agonists wherein the neuromedin U receptor agonist derivatives are linked to a polymer are also included within the scope of the present invention.
  • the polymer selected is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled as provided for in the present methods.
  • Included within the scope of polymers is a mixture of polymers.
  • the polymer will be pharmaceutically acceptable.
  • the polymer or mixture thereof may be selected from the group consisting of; for example, polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N- vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (for example, glycerol), and polyvinyl alcohol.
  • PEG polyethylene glycol
  • monomethoxy-polyethylene glycol dextran, cellulose, or other carbohydrate based polymers
  • poly-(N- vinyl pyrrolidone) polyethylene glycol propylene glycol homopolymers
  • a polypropylene oxide/ethylene oxide co-polymer for example, glycerol
  • polyoxyethylated polyols for example, glycerol
  • the neuromedin U receptor agonists are modified by PEGylation, cholesterylation, or palmitoylation.
  • the modification can be to any amino acid residue in the neuromedin U receptor agonist, however, in currently embodiments, the modification is to the N-terminal amino acid of the neuromedin U receptor agonist, either directly to the N-terminal amino acid or by way coupling to the thiol group of a cysteine residue added to the N-terminus or a linker added to the N-temiinus such as Ttds.
  • the N-terminus of the neuromedin U receptor agonist comprises a cysteine residue to which a protecting group is coupled to the N-terminal amino group of the cysteine residue and the cysteine thiolate group is derivatized with N-ethylmaleimide, PEG group, cholesterol group, or palmitoyl group.
  • an acetylated cysteine residue is added to the N-terminus of the neuromedin U receptor agonists, and the thiol group of the cysteine is derivatized with N-ethylmaleimide, PEG group, cholesterol group, or palmitoyl group.
  • PEG polyethylene glycol
  • Polyethylene glycol or PEG is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, including, but not limited to, mono-(Ci-io) alkoxy or aryloxy-polyethylene glycol.
  • Suitable PEG moieties include, for example, 40 kDa methoxy poly(ethylene glycol) propionaldehyde (Dow, Midland, Michigan); 60 kDa methoxy
  • poly(ethylene glycol) maleimido-propionamide (Dow, Midland, Michigan); 31 kDa alpha- methyl-w-(3-oxopropoxy), polyoxyethylene (NOF Corporation, Tokyo); mPEG2-NHS-40k (Nektar); mPEG2-MAL-40k (Nektar), SUNBRIGHT GL2-400MA ((PEG)240kDa) (NOF Corporation, Tokyo), SUNBRIGHT ME-200MA (PEG20kDa) (NOF Corporation, Tokyo).
  • the PEG groups are generally attached to the neuromedin U receptor agonists via acylation or alkylation through a reactive group on the PEG moiety (for example, a maleimide, an aldehyde, amino, thiol, or ester group) to a reactive group on the neuromedin U receptor agonist (for example, an aldehyde, amino, thiol, a maleimide, or ester group).
  • a reactive group on the PEG moiety for example, a maleimide, an aldehyde, amino, thiol, or ester group
  • a reactive group on the neuromedin U receptor agonist for example, an aldehyde, amino, thiol, a maleimide, or ester group.
  • the PEG molecule(s) may be covalently attached to any Lys, Cys, or
  • neuromedin U receptor agonists described herein can be PEGylated directly to any amino acid at the N-terminus by way of the N-terminal amino group.
  • a "linker arm" may be added to the neuromedin U receptor agonist to facilitate PEGylation.
  • PEGylation at the thiol side-chain of cysteine has been widely reported (See, e.g., Caliceti & Veronese, Adv. Drug Deliv. Rev. 55: 1261-77 (2003)). If there is no cysteine residue in the peptide, a cysteine residue can be introduced through substitution or by adding a cysteine to the N-terminal amino acid.
  • Those neuromedin U receptor agonists, which have been PEGylated have been PEGylated through the side chains of a cysteine residue added to the N-terminal amino acid.
  • the PEG molecule(s) may be covalently attached to an amide group in the C-terminus of the neuromedin U receptor agonist. In general, there is at least one PEG molecule covalently attached to the neuromedin U receptor agonist. In particular aspects, the PEG molecule is branched while in other aspects, the PEG molecule may be linear. In particular aspects, the PEG molecule is between 1 kDa and 100 kDa in molecular weight. In further aspects, the PEG molecule is selected from 10, 20, 30, 40, 50, 60, and 80 kDa. In further still aspects, it is selected from 20, 40, or 60 kDa.
  • the neuromedin U receptor agonists contain mPEG-cysteine.
  • the mPEG in mPEG-cysteine can have various molecular weights. The range of the molecular weight is preferably 5 kDa to 200 kDa, more preferably 5 kDa to 100 kDa, and further preferably 20 kDa to 60 kDA.
  • the mPEG can be linear or branched.
  • the neuromedin U receptor agonists are PEGylated through the side chains of a cysteine added to the N-terminal amino acid.
  • the agonists preferably contain mPEG-cysteine.
  • the mPEG in mPEG-cysteine can have various molecular weights. The range of the molecular weight is preferably 5kDa to 20OkDa, more preferably 5kDa to 10OkDa, and further preferably 2OkDa to 6OkDA.
  • the mPEG can be linear or branched.
  • a useful strategy for the PEGylation of synthetic neuromedin U receptor agonists consists of combining, through forming a conjugate linkage in solution, a peptide, and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other.
  • the neuromedin U receptor agonists can be easily prepared with conventional solid phase synthesis.
  • the neuromedin U receptor agonist is "preactivated” with an appropriate functional group at a specific site.
  • the precursors are purified and fully characterized prior to reacting with the PEG moiety.
  • Conjugation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC.
  • the PEGylated neuromedin U receptor agonist can be easily purified by cation exchange chromatography or preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.
  • the sites of PEGylation on the neuromedin U receptor agonists of the present invention were chosen taking into account the structure of NMU and its interactions with the NMU receptors.
  • the PEGylation is preferably site-specific.
  • PEGylation at the N-terminal amino group of the peptide NMU is possible since this is the only available amino group in the sequence.
  • the N-terminal group of the peptide was acylated with a branched ((PEG)240kDa N-hydroxysuccinimide analog (for example, SUNBRIGHT GL2-400GS2, NOF Corporation).
  • the neuromedin U receptor agonist can comprise other non-sequence modifications, for example, glycosylation, lipidation, acetylation, phosphorylation,
  • the neuromedin U receptor agonist herein utilize naturally-occurring amino acids or D isoforms of naturally occurring amino acids, substitutions with non-naturally occurring amino acids (for example., methionine sulfoxide, methionine methylsulfonium, norleucine, epsilon-aminocaproic acid, 4-aminobutanoic acid, tetrahydroisoquinoline-3-carboxylic acid, 8-aminocaprylic acid, 4 aminobutyric acid,
  • the neuromedin U receptor agonists comprise a fusion protein that having a First moiety, which is a neuromedin U receptor agonist, and a second moiety, which is a heterologous peptide.
  • the neuromedin U receptor agonist may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified neuromedin U receptor agonist and/or having other desirable properties.
  • a protecting group covalently joined to the C-terminal carboxy group reduces the reactivity of the carboxy terminus under in vivo conditions.
  • carboxylic acid groups of the peptide may be provided in the form of a salt of a pharmacologically-acceptable cation or esterified to form a Cl -6 ester, or converted to an amide of formula NRR.2 wherein R and R2 are each independently H or C ⁇ -6 alkyl, or combined to form a heterocyclic ring, such as a 5 -or 6-membered ring.
  • the carboxy terminus protecting group is preferably attached to the ⁇ - carbonyl group of the last amino acid.
  • Carboxy terminus protecting groups include, but are not limited to, amide, methylamide, and ethylamide.
  • Amino groups of the peptide may be in the form of a pharmacologically-acceptable acid addition salt, such as the HCl, HBr 5 acetic, benzoic, toluene sulfonic, maleic, tartaric, and other organic salts, or may be modified to Ci-g alkyl or dialkyl amino or further converted to an amide.
  • a pharmacologically-acceptable acid addition salt such as the HCl, HBr 5 acetic, benzoic, toluene sulfonic, maleic, tartaric, and other organic salts
  • Hydroxyl groups of the neuromedin U receptor agonist side chain may be converted to C ⁇ -6 alkoxy or to a C ⁇ - ⁇ ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chain may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C 1-6 alkyl, Ci_6 alitoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the neuromedin U receptor agonist side chains can be extended to homologous C2-4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • cyclic structures into the peptides of this invention to select and provide conformational constraints to the structure that result in enhanced stability.
  • a carboxyl-terminal or amino-terminal cysteine residue can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, thereby generating a cyclic peptide.
  • Other peptide cyclizing methods include the formation of thioethers and carboxyl-and amino-terminal amides and esters.
  • Another method to provide conformational constraints to the structure that result in enhanced stability relies on the substitution of one or more amino acids with iV-alkyl-amino acids.
  • Polysaccharide polymers are another type of water soluble polymer that may be used for protein modification.
  • Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by ⁇ 1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kDa to about 70 kDa.
  • Dextran is a suitable water soluble polymer for use as a vehicle by itself or in combination with another vehicle ⁇ See, for example, WO96/11953 and WO96/05309). The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456. Dextran of about 1 kDa to about 20 kDa is preferred when dextran is used as a vehicle in accordance with the present invention,
  • the linker is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. However, in certain embodiments, the linker may itself provide improved properties to the compositions of the present invention.
  • the linker is preferably made up of amino acids linked together by peptide bonds.
  • the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art.
  • the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine.
  • a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine.
  • linkers include are polyglycines (particularly (Gly)4, (Gly)s), poly(Gly-Ala), and polyalanines.
  • Other specific examples of linkers are (Gly)3Lys(Gly)4; (Gly)3AsnGlySer(Gly)2; (Gly)3Cys(Gly)4; and GlyProAsnGlyGly.
  • Non-peptide linkers can also be used.
  • These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (for example, Ci-g) lower acyl, halogen (for example, Cl, Br), CN, NH2, phenyl, and the like.
  • An exemplary non-peptide linker is a PEG linker, wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD.
  • the peptide linkers may be altered to form derivatives in the same manner as described above.
  • Other linkers include Ttds (N-(13-amino-4,7,10-trioxa-tridecayl)-succinamic acid).
  • the present invention includes diastereomers as well as their racemic and resolved enantiomerically pure forms.
  • the neuromedin U receptor agonists can contain D-amino acids, L-amino acids, or a combination thereof.
  • the amino acids are in the L-form with particular amino acids in D-form.
  • E ⁇ Glu Glutamic Acid
  • H ⁇ His ⁇ Histidine H ⁇ His ⁇ Histidine
  • the neuromedinU receptor agonists can be linked, conjugated, or fused to a carrier molecule such as albumin, transferrin, or an antibody or antibody fragment such as the Fab or Fc fragment.
  • a carrier molecule such as albumin, transferrin, or an antibody or antibody fragment such as the Fab or Fc fragment.
  • the neuromedin U receptor agonists can be linked via a linker moiety to a catalytic antibody such as disclosed in U.S. Pub. Application Nos. US20030175921 , US200301960676, and US20030129188, which describes linking of various peptides to the catalytic site of an aldolase catalytic antibody.
  • the neuromedin U receptor agonists can be conjugated to an Fc fragment via linker moiety or fused to the Fc fragment of an antibody in the form of a fusion protein in a manner similar to the GLP-I-Fc fusion proteins disclosed in International Applications WO2002/046227 and WO2005/007809.
  • the neuromedin U receptor agonist can be linked via a linker moiety to serum albumin, similar to the linking of GLP-I to albumin as disclosed in International application WO2000/069911 or U. S, Pub application US20070093417.
  • the neuromedin U receptor agonist can fused to a carrier molecule such as transferrin, for example similar to the transferrin-GLP-1 fusion proteins disclosed in U.S. Patent No. 7,176,278 or albumin-GLP-1 fusion proteins described in U. S. Patent No. 7,141,547.
  • Conjugation of peptides to carrier proteins is known in the art and has been used to covalently link a wide variety of peptides to a carrier protein.
  • a carrier protein For example, Poznonsky e ⁇ at., FEBS Letts 239: 18-22 (1998) conjugated human growth hormone to serum albumin. The resulting conjugate had reduced renal clearance and altered plasma clearance but retained its biological activity.
  • Paige et al., Pharma. Res. 12: 1883-1888 (1995) describe conjugating GCSF to serum albumin using a bifunctional polyethylene glycol linker. The conjugate had reduced renal clearance and increased serum stability but retained its biological activity.
  • Serum albumin comprising the present invention can be produced recombinantly and can include various modifications such as amino acid substitutions, deletions, or insertions, deglycosylation, particular predominant glycosylatiort structures, and can further include fusions to heterologous proteins, polypeptides, and peptides.
  • International Published Application No. WO92/00763 describes coupling antigen- binding fragments of IgA or IgM to serum albumin and thereby restoring antigen affinity of the fragments to levels comparable to intact IgA or IgM.
  • WO2004/081013 also discloses conjugating peptides and drags to IgG molecules.
  • U.S. Patent Nos. 6,593,295; 6329,336; 7,256,253; 6,849,714; and 6,849,714 describe covalently linking peptides to blood component proteins such as albumin or antibodies ex vivo and observing that the conjugates were resistant to protease digestion and thus had an extended half-life.
  • the patents disclose that a wide variety of peptides, including neuromedins, can be conjugated to the blood components using reactive groups such as maleimido and succinimidyl reactive groups.
  • 20070207952 describes covalently linking peptides and other macromolecules to carrier proteins such as albumin and in the case of anti-HIY antivirals, observing that the conjugates have superior pharmacological and, in particular, pharmacokinetic properties, and can have a prolong half-life in vivo.
  • Fc fragments are produced when an immunoglobulin (Ig) molecule is digested with papain, and is a region of an immunoglobulin molecule except for the variable region (VL) and the constant regions (CL) of the light chain and the variable region (VH) and the constant region 1 (CHI) of the heavy chain.
  • Ig immunoglobulin
  • VL variable region
  • CL constant regions
  • CHI constant region 1
  • Fc fragments comprising the present invention can also be produced recombinantly and can further include modifications such as amino acid substitutions, deletions, or additions, PEGylation, deglycosylation, particular predominant glycosylation structures, and can further include fusions to heterologous proteins, polypeptides, and peptides.
  • conjugation of the peptide to the carrier protein is effected by a covalent chemical linkage that excludes embodiments in which the peptide is covalently attached to the carrier protein by means of one or more amino acids in a peptide linkage, for example, a fusion protein.
  • the present invention does not include fusion proteins comprising the NMU peptide and the carrier protein.
  • compositions comprising a therapeutically effective amount of one or more of the neuromedin U receptor agonists disclosed herein for the treatment of a metabolic disorder in an individual.
  • Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, type II diabetes, complications of diabetes such as retinopathy, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers.
  • the obesity-related disorders herein are associated with, caused by, or result from obesity.
  • “Obesity” is a condition in which there is an excess of body fat.
  • the operational definition of obesity is based on the Body Mass Index (BMI), calculated as body weight per height in meters squared (kg/m.2).
  • BMI Body Mass Index
  • "Obesity” refers to a condition whereby an otherwise healthy subject has a Body Mass Index (BMI) greater than or equal to 30 kg/m2, or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg/m2.
  • An “obese subject” is an otherwise healthy subject with a Body Mass Index (BMI) greater than or equal to 30 kg/m2 or a subject with at least one co-morbidity with a BMI greater than or equal to 27 kg/m.2.
  • a "subject at risk for obesity” is an otherwise healthy subject with a BMI of 25 kg/m2 to less than 30 kg/m2 or a subject with at least one co-morbidity with a BMI of 25 kg/m2 to less than 27 kg/m2.
  • BMI Body Mass Index
  • “obesity” refers to a condition whereby a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg/m2.
  • an “obese subject” refers to a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m2.
  • a "subject at risk of obesity” is a subject with a BMI of greater than 23 kg/m2 to less than 25 kg/m2.
  • obesity is meant to encompass all of the above definitions of obesity.
  • Obesity-induced or obesity-related co-morbidities include, but are not limited to, diabetes, non-insulin dependent diabetes mellitus - type 2, impaired glucose tolerance, impaired fasting glucose, insulin resistance syndrome, dyslipidemia, hypertension, hyperuricacidemia, gout, coronary artery disease, myocardial infarction, angina pectoris, sleep apnea syndrome, Pickwickian syndrome, fatty liver; cerebral infarction, cerebral thrombosis, transient ischemic attack, orthopedic disorders, arthritis deformans, lumbodynia, emmenlopathy, and infertility.
  • co-morbidities include: hypertension, hyperlipidemia, dyslipidemia, glucose intolerance, cardiovascular disease, sleep apnea, diabetes mellitus, and other obesity-related conditions.
  • Treatment refers to the administration of the compounds of the present invention to reduce or maintain the body weight of an obese subject
  • One outcome of treatment may be reducing the body weight of an obese subject relative to that subject's body weight immediately before the administration of the compounds of the present invention.
  • Another outcome of treatment may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy.
  • Another outcome of treatment may be decreasing the occurrence of and/or the severity of obesity-related diseases.
  • the treatment may suitably result in a reduction in food or calorie intake by the subject, including a reduction in total food intake, or a reduction of intake of specific components of the diet such as carbohydrates or fats; and/or the inhibition of nutrient absorption; and/or the inhibition of the reduction of metabolic rate; and in weight reduction in patients in need thereof.
  • the treatment may also result in an alteration of metabolic rate, such as an increase in metabolic rate, rather than or in addition to an inhibition of the reduction of metabolic rate; and/or in minimization of the metabolic resistance that normally results from weight loss.
  • Prevention refers to the administration of the compounds of the present invention to reduce or maintain the body weight of a subject at risk of obesity.
  • One outcome of prevention may be reducing the body weight of a subject at risk of obesity relative to that subject's body weight immediately before the administration of the compounds of the present invention.
  • Another outcome of prevention may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy.
  • Another outcome of prevention may be preventing obesity from occurring if the treatment is administered prior to the onset of obesity in a subject at risk of obesity.
  • Another outcome of prevention may be decreasing the occurrence and/or severity of obesity-related disorders if the treatment is administered prior to the onset of obesity in a subject at risk of obesity.
  • Such treatment may prevent the occurrence, progression or severity of obesity-related disorders, such as, but not limited to, arteriosclerosis, Type II diabetes, polycystic ovarian disease, cardiovascular diseases, osteoarthritis,
  • dermatological disorders hypertension, insulin resistance, hypercholesterolemia,
  • hypertriglyceridemia and cholelithiasis.
  • the obesity-related disorders herein are associated with, caused by, or result from obesity.
  • obesity-related disorders include overeating and bulimia, hypertension, diabetes, elevated plasma insulin concentrations and insulin resistance, dyslipidemias, hyperlipidemia, endometrial, breast, prostate and colon cancer, osteoarthritis, obstructive sleep apnea, cholelithiasis, gallstones, heart disease, abnormal heart rhythms and arrythmias, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovarian disease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich's syndrome, GH-deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, e.g, children with acute lymphoblastic leukemia.
  • metabolic syndrome also known as syndrome
  • hypogonadism in males and hirsutism in females gastrointestinal motility disorders, such as obesity-related gastro-esophageal reflux, respiratory disorders, such as obesity-hypoventilation syndrome (Pickwickian syndrome), cardiovascular disorders, inflammation, such as systemic inflammation of the vasculature, arteriosclerosis, hypercholesterolemia, hyperuricaemia, lower back pain, gallbladder disease, gout, and kidney cancer.
  • the compounds of the present invention are also usefol for reducing the risk of secondary outcomes of obesity, such as reducing the risk of left ventricular hypertrophy.
  • diabetes includes both insulin-dependent diabetes mellitus (IDDM, also known as type I diabetes) and non-insulin-dependent diabetes mellitus (NIDDM, also known as Type II diabetes).
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin-dependent diabetes mellitus
  • Type I diabetes or insulin-dependent diabetes
  • Type II diabetes is the result of an absolute deficiency of insulin, the hormone which regulates glucose utilization.
  • Type II diabetes, or insulin-independent diabetes i.e., non-insulin-dependent diabetes mellitus
  • Most of the Type II diabetics are also obese.
  • the compounds of the present invention are useful for treating both Type I and Type II diabetes.
  • the compounds are especially effective for treating Type II diabetes.
  • the compounds of the present invention are also useful for treating and/or preventing gestational diabetes mellitus.
  • the neuromedin U receptor agonists disclosed herein may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • Such compositions comprise a therapeutically-effective amount of the neuromedin U receptor agonist and a pharmaceutically acceptable carrier.
  • Such a composition may also be comprised of (in addition to neuromedin U receptor agonist and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • Compositions comprising the neuromedin U receptor agonists can be administered, if desired, in the form of salts provided the salts are pharmaceutically acceptable. Salts may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry.
  • compositions comprising formula I are also useful for treating or preventing obesity and obesity-related disorders in cats and dogs.
  • mamal includes companion animals such as cats and dogs.
  • treating includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
  • salts refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids.
  • Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromelhamine, and the like.
  • basic ion exchange resins such as
  • pharmaceutically acceptable salt further includes all acceptable salts such as acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N- methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycolly
  • hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, isothionate, triethiodide, lactate, panoate, valerate, and the like which can be used as a dosage form for modifying the solubility or hydrolysis characteristics or can be used in sustained release or pro-drug
  • the term "pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s), approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered and includes, but is not limited to such sterile liquids as water and oils. The characteristics of the carrier will depend on the route of administration.
  • the neuromedin U receptor agonist may be in multimers (for example, heterodimers or homodimers) or complexes with itself or other peptides. As a result,
  • compositions of the invention may comprise one or more neuromedin U receptor agonists in such multimeric or complexed form.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously.
  • the amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • parenteral means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous.
  • the pharmacological composition can comprise one or more neuromedin U receptor agonists; one or more neuromedin U receptor agonists and one or more other agents for treating a metabolic disorder; or the pharmacological composition comprising the one or more neuromedin U receptor agonists can be used concurrently with a pharmacological composition comprising an agent for treating a metabolic disorder.
  • Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, type II diabetes, complications of diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers.
  • the pharmacological composition comprises another agent for treating a metabolic disorder or the treatment includes a second pharmacological composition comprising an agent for treating a metabolic disorder
  • the agent includes, but is not limited to, insulin, analogs, and derivatives; cannabinoid (CBl) receptor antagonists; glucagon like peptide 1 (GLP- 1) receptor agonists; glucagon receptor agonists and antagonists; glucose-dependent
  • GIP insulinotropic peptide
  • Ob receptor agonists e.g., leptin and analogs thereof
  • FGF-21 and analogs thereof amylin and analogs thereof
  • GPRl 19 receptor agonists GPR40 agonists
  • GPRl 16 receptor agonists serotonin 5-HT2C receptor agonists
  • melanocortin-4 receptor (MC4R) agonists M4R
  • histamine H3 receptor antagonists thyroid hormone receptor agonists
  • PPAR ⁇ receptor agonists agouti- related protein or analog; angiopoietin-like protein 6 (Angptl ⁇ ) proteins, peptides, analogs, and derivatives; GPR105 (P2YR14) antagonists; tetra
  • atorvastatin sitagliptin; metformin; orlistat; Qnexa; topiramate; naltrexone; bupriopion;
  • Suitable agents of use in combination with a composition of the present invention or in a treatment in combination with a composition of the present invention includes, but are not limited to:
  • anti-diabetic agents such as (1) PPAR ⁇ agonists such as glitazones (e.g. ciglitazone; darglitazone; englitazone; isaglitazone (MCC-555); pioglitazone (ACTOS);
  • glitazones e.g. ciglitazone; darglitazone; englitazone; isaglitazone (MCC-555); pioglitazone (ACTOS);
  • rosiglitazone (AVANDIA); troglitazone; rivoglitazone, BRL49653; CLX-0921 ; 5-BTZD, GW- 0207, LG- 100641 5 R483, and LY-300512, and the like and compounds disclosed in WO97/10813, 97/27857, 97/28115, 97/28137, 97/27847, 03/000685, and 03/027112 and
  • SPPARMS selective PPAR gamma modulators
  • Tl 31 Amgen
  • FK614 Flujisawa
  • netoglitazone and metaglidasen
  • biguanides such as buformm; metformin; and phenformin, and the like
  • PPP-IB protein tyrosine phosphatase- IB inhibitors
  • salbostatin CKD-711; MDL-25,637; MDL-73,945; and MOR 14, and the like; (7) alpha-amylase inhibitors such as tendamistat, trestatin, and Al-3688, and the like; (8) insulin secreatagogues such as linogliride nateglinide, mitiglinide (GLUFAST), IDl 101 A-4166, and the like; (9) fatty acid oxidation inhibitors, such as clomoxir, and etomoxir, and the like; (10) A2 antagonists, such as m ⁇ daglizole; isaglidole; deriglidole; idazoxan; earoxan; and fluparoxan, and the like; (11) insulin or insulin mimetics, such as biota, LP-100, novarapid, insulin detemir, insulin lispro, insulin glargine, insulin zinc suspension (lente and ultralente); Lys-Pro
  • retinoid modulators such as those disclosed in WO 03/000249
  • GSK 3beta/GSK 3 inhibitors such as 4-[2-(2-bromophenyl)-4-(4-fluorophenyl- lH-imidazol-5-yl]pyridine ; CT21022, CT20026, CT-98023, SB-216763, SB410111, SB-675236, CP-70949, XD4241 and those compounds disclosed in WO 03/037869, 03/03877, 03/037891, 03/024447, 05/000192, 05/019218 and the like; (19) glycogen phosphorylase (HGLPa) inhibitors, such as AVE 5688, PSN 357, GPi-879, those disclosed in WO 03/037864, WO 03/091213, WO 04/092158, WO 05/013975, WO 05/01;
  • GPRl 19 also called RUP3 ; SNORF 25
  • RUP3 also called RUP3
  • SNORF 25 such as RUP3 , HGPRBMY26, PFI 007, SNORF 25
  • adenosine receptor 2B antagonists such as ATL-618, AT1-802, E3080, and the like
  • carnitine palmitoyl transferase inhibitors such as ST 1327, and ST 1326, and the like
  • Fructose 1 ,6-bis ⁇ hospohatase inhibitors such as CS-917, MB7803, and the like
  • glucagon antagonists such as AT77077, BAY 694326, GW 4123X, NN2501, and those disclosed in WO 03/064404, WO 05/00781 , US 2004/0209928, US 2004/029943, and the like
  • lipi d lowering agents such as (1) bile acid sequestrants such as, cholestyramine, colesevelem, colestipol, dialkylaminoalkyl derivatives of a cross-linked dextran; Colestid®; LoCholest®; and Questran®, and the like; (2) HMG-CoA reductase inhibitors such as atorvastatin, itavastatin, pitavastatin, fluvastatin, lovastatin, pravastatin, rivastatin,
  • bile acid sequestrants such as, cholestyramine, colesevelem, colestipol, dialkylaminoalkyl derivatives of a cross-linked dextran; Colestid®; LoCholest®; and Questran®, and the like
  • HMG-CoA reductase inhibitors such as atorvastatin, itavastatin, pitavastatin, fluvastatin, lovastatin, pravastatin,
  • rosuvastatin simvastatin, rosuvastatin (ZD-4522), and the like, particularly simvastatin; (3) HMG-CoA synthase inhibitors; (4) cholesterol absorption inhibitors such as FMVP4 (Forbes Medi-Tech), KT6-971 (Kotobuki Pharmaceutical), FM-VAl 2 (Forbes Medi-Tech), FM-VP-24 (Forbes Medi-Tech), stanol esters, beta-sitosterol, sterol glycosides such as tiqueside; and azetidinones such as ezetimibe, and those disclosed in WO 04/005247 and the like; (5) acyl coenzyme A -cholesterol acyl transferase (ACAT) inhibitors such as avasimibe, eflucimibe, pactimibe (KY505), SMP 797 (Sumitomo), SM32504 (Sumitomo), and those disclosed in WO 03/09
  • PHA384640E Pfizer
  • S8921 Shionogi
  • AZD7806 AstrZeneca
  • AKl 05 Asah Kasei
  • Lp-PLA2 inhibitors such as SB480848 (GlaxoSmithldine), 659032 (GlaxoSmithkline), 6771 16 (GlaxoSmithkline), and the like
  • other agents which affect lipic composition including ETC1001/ESP31015 (Pfizer), ESP-55016 (Pfizer), AGIl 067 (AtheroGenics), AC3056 (Amylin), AZD4619 (AstrZeneca);
  • anti-hypertensive agents such as (1) diuretics, such as thiazides, including chlorthalidone, chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide, and hydrochlorothiazide; loop diuretics, such as bumetanide, ethacrynic acid, furosemide, and torsemide; potassium sparing agents, such as amiloride, and triamterene; and aldosterone antagonists, such as spironolactone, epirenone, and the like; (2) beta-adrenergic blockers such as acebutolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, carteolol, carvedilol, celiprolol, esrnolol, indenolol, metaprolol, nadolol, nebi
  • tenocapril trandolapril, and zofenopril, and the like; (5) neutral endopeptidase inhibitors such as omapatrilat, cadoxatril and ecadotril, fosidotril, sampatrilat, AVE7688, ER4030, and the like; (6) endothelin antagonists such as tezosentan, A308165, and YM62899, and the like; (7) vasodilators such as hydralazine, clonidine, minoxidil, and nicotinyl alcohol, and the like; (8) angiotensin II receptor antagonists such as candesartan, eprosartan, irbesartan, losartan, pratosartan, tasosartan, telmisartan, valsartan, and EXP-3137, FI6828K, and RNH6270, and the
  • anti-obesity agents such as (1) 5HT (serotonin) transporter inhibitors, such as paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline, and imipramine, and those disclosed in WO 03/00663, as well as serotonin/noradrenaline re uptake inhibitors such as sibutramine (MERIDIA/REDUCTIL) and dopamine uptake inhibitor/Norepenephrine uptake inhibitors such as radafaxine hydrochloride, 353162 (GlaxoSmithMine), and the like; (2) NE (norepinephrine) transporter inhibitors, such as GW 320659, despiramine, talsupram, and nomifensine; (3) CBl (carmabinoid-1 receptor) antagonist/inverse agonists, such as rimonabant (ACCOMPLIA Sanofi Synthelabo), SR-147778 (Sanofi Synthelabo), AVE
  • H3 ghrelin agonists/antagonists, such as BVT81-97 (BioVitrum), RC 1291 (Rejuvenon), SRD-04677 (Sumitomo), unacylated ghrelin (TheraTechnologies), and those disclosed in WO 01/87335, WO 02/08250, WO 05/012331, and the like;
  • H3 (histamine H3) antagonist/inverse agonists such as thioperamide, 3-(lH-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), clobenpropit, iodophenpropit, imoproxifan, GT2394 (Gl
  • MCHlR melanin-concentrating hormone 1 receptor
  • T-226296 Takeda
  • T71 Takeda/Amgen
  • AMGN- 608450 AMGN-503796
  • Amgen 856464
  • A798 Abbott
  • ATC0175/AR224349 Arena Pharmaceuticals
  • GW803430 GaxoSmithkine
  • NBI- IA Neurorocrine Biosciences
  • NGX-I Neurogen
  • SNP-7941 Synaptic
  • SNAP9847 Synaptic
  • T-226293 Schering Plough
  • TPI- 1361-17 Saitama Medical School/University of California Irvine
  • JP 13226269, JP 1437059, JP2004315511, and the like (7) MCH2R (melanin concentrating hormone 2R) agonist/antagonists; (8) NPYl (neuropeptide Y Yl) antagonists, such as BMS205749, BIBP3226, J-115814, BIBO 3304, LY-357897, CP-671906, and GI-264879A; and those disclosed in U.S. Patent No.
  • Neuropeptide Y Y5 Neuropeptide Y Y5
  • antagonists such as 152,804, S2367 (Shionogi), E-6999 (Esteve), GW- 56918OA, GW-594884A (GlaxoSmithklme), GW-587081X, GW-548118X; FR 235,208;
  • leptin such as recombinant human leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl human leptin (Amgen); (11) leptin derivatives, such as those disclosed in Patent Nos.
  • opioid antagonists such as nalmefene (Revex ®), 3-methoxynaltrexone, naloxone, and naltrexone; and those disclosed in WO
  • Patent No. 6358951 U.S. Patent Application Nos. 2002/049196 and 2002/022637; and WO 01/56592, and WO 02/32888; (19) 5HT2c (serotonin receptor 2c) agonists, such as APD3546/AR10A (Arena Pharmaceuticals), ATH88651 (Athersys), ATH88740 (Athersys), BVT933 (Biovitrum/GSK), DPCA37215 (BMS), IK264; LY448100 (Lilly), PNU 22394; WAY 470 (Wyeth), WAY629 (Wyeth), WAY161503 (Biovitrum), R-1065, VR1065 (Vernalis/Roche) YM 348; and those disclosed in U.S. Patent No. 3,914,250; and PCT Publications 01/66548, 02/36596, 02/48124, 02/10169, 02/44152;
  • Mc3r melanocortin 3 receptor
  • Mc4r melanocortin 4 receptor agonists, such as CHIR86036 (Chiron), CHIR915 (Chiron); ME-10142 (Melacure), ME-10145 (Melacure), HS-131 (Melacure),
  • NBI72432 Neurosciences
  • NNC 70-619 Novo Nordisk
  • TTP2435 Transtech
  • WO98/04526 WO98/32753, WO 01/74782, WO 02/32897, WO 03/014113, WO 03/016276, WO 03/016307, WO 03/024948, WO 03/024953, WO 03/037881, WO 04/108674, and the like;
  • DGATl diacylglycerol acyltransferase 1 inhibitors
  • DGAT2 diacylglycerol acyltransferase 2inhibitors
  • PDE phosphodiesterase
  • inhibitors such as theophylline, pentoxifylline, zaprinast, sildenafil, amrinone, milrinone, cilostamide, rolipram, and cilomilast, as well as those described in WO 03/037432, WO 03/0378
  • UCP-I uncoupling protein 1
  • activators such as phytanic acid, 4-[(E)- 2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-l-propenyl]benzoic acid (TTNPB), and retinoic acid; and those disclosed in WO 99/00123; (35) acyl-estrogens, such as oleoyl-estrone, disclosed in del Mar-Grasa, M. et al., Obesity Research, 9:202-9 (2001); (36) glucocorticoid receptor antagonists, such as CP472555 (Pfizer), KB 3305, and those disclosed in WO
  • dehydrogenase type 1) inhibitors such as BVT 3498 (AMG 331), BVT 2733, 3-(l-adamantyl)-4- ethyl-5-(ethylthio)-4H-l,2,4-triazole, 3-(l-adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H- 1,2,4-triazoIe, S-adamantanyM ⁇ J ⁇ lOJ ⁇ a-decahydro-l ⁇ -triazolo ⁇ - a][l l]annulene, and those compounds disclosed in WO 01/90091, 01/90090, 01/90092,
  • lipase inhibitors such as tetrahydrolipstatin (orlistat/XENICAL), ATL962 (Alizyme/Takeda),
  • GT389255 (Genzyme/Peptimmune)Triton WR1339, RHC80267, lipstatin, teasaponin, and diethyl ⁇ mbelliferyl phosphate, FL-386, WAY- 121898, Bay-N-3176, valilactone, esteracin, ebelactone A, ebelactone B, and RHC 80267, and those disclosed in WO 01/77094, WO
  • SB271046 (GlaxoSmithkline), RO-046790 (Roche), and the like; (53) fatty acid transport protein 4 (FATP4); (54) acetyl-CoA carboxylase (ACC) inhibitors such as CP640186, CP610431, CP640188 (Pfizer); (55) C-terminal growth hormone fragments such as AOD9604 (Monash Univ/Metabolic Pharmaceuticals), and the like; (56) oxyntomodulin; (57) neuropeptide FF receptor antagonists such as those disclosed in WO 04/083218, and the like; (58) amylin agonists such as Symlin/pramlintide/AC137 (Amylin); (59) Hoodia and trichocaulon extracts; (60) BVT74713 and other gut lipid appetite suppressants; (61) dopamine agonists such as bupropion (WELLBUTRIN/GlaxoSmithkline); (62) zonisamide (ZONEGRAN/Dain
  • Examples of specific compounds that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention further include specific CBl antagonists/inverse agonists include those described in WO03/077847, including: N-[3-(4-chlorophenyl)-2(5)-phenyl- 1 (5)-methylpropyl]-2-(4- trifluoromethyl-2-pyrimidyloxy)-2-methylpropaiiamide, JV- [3 -(4-chlorophenyl)-2-(3- cyanophenyl)-l-methylpropyl]-2-(5-trifluoromethyl-2-pyridyloxy)-2-methylpropanamide J JV-[3- (4-chlorophenyl)-2-(5 -chloro- 3 -pyridyl)- 1 -methylpropyl] -2-(5-trifluoromethyl-2-pyridyloxy)-2- methylpropanamide, and pharmaceutical
  • WO05/00 ⁇ 809 which includes the following: 3- ⁇ l-[bis(4-chloro ⁇ henyl)methyl]azetidin-3- ylidene ⁇ -3 ⁇ (3 ,5-difluorophenyl)-2,2-dimethylpropanenitrile J 1 - ⁇ 1 -[ 1 ⁇ (4- chlorophenyl)pentyl]azetidin-3-yl ⁇ - 1 -(3,5-difluorophenyl)-2-methylpropan-2-ol.
  • Specific ACC- 1/2 inhibitors that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: r-[(4 J 8-dimethoxyquinolin-2-yl)carbonyl]-6-(l/i-tetrazol-5-yl)spiro[chroman-2 J 4 1 - ⁇ iperidin]-4-one; (5- ⁇ 1 '-[(4,8-dimethoxyquinolin-2-yl)carbonyl]-4-oxospiro[chroman-2,4 1 - piperidin]-6-yl ⁇ -2H-tetrazol-2-yl)methyl pivalate; 5- ⁇ 1 '-[(8-cycIopropyl-4-methoxyquinolin-2- yl)carbonyl]-4-oxospiro[chroman-2,4'-piperidin]-6-yl ⁇ nicotinic acid; 1 '-(8-methoxy-4- mo ⁇
  • Specific MCHlR antagonist compounds that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: 1 - ⁇ 4-[(l -ethylazetidin-3-yl)oxy]phenyl ⁇ -4-[(4-fluorobenzyI)oxy]pyridin- 2( 1 H)-one, 4- [(4-fluorobenzyl)oxy] - 1 - ⁇ 4- [( 1 -isopropylazetidin-3-yl)oxy]phenyl ⁇ pyridin-2( 1 H)- one, l-[4-(azetidin-3-yloxy)phenylJ-4-[(5-chloropyridin-2-yl)methoxy]pyridin-2(lH)-one, 4-[(5- chloro ⁇ yridin-2-yl)methoxy] - 1 - ⁇ 4-[( 1 -ethylazetidin-3-y
  • a specific DP-IV inhibitor that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention is 7- [(3R)-3-amino-4-(2 J 4,5-trifluorophenyl)butanoyl]-3-(trifiuoromethyl)-5,6,7,8-tetrahydro4 f 2 > 4- triazolo[4,3-a] ⁇ yrazine, or a pharmaceutically acceptable salt thereof.
  • Specific CCKlR agonists that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: 3 -(4- ⁇ [ 1 -(3 -ethoxyphenyl)-2-(4-methylphenyl)- 1 H -imidazol-4-yl] carbonyl ⁇ - 1 - piperazinyl)- 1 -naphthoic acid; 3 -(4- ⁇ [ 1 -(3-ethoxyphenyl)-2-(2-fluoro-4-methyl ⁇ henyl)- 1 H - imidazol-4-yl]carbonyl ⁇ - 1 -piperazinyl)- 1 -naphthoic acid; 3-(4- ⁇ [ 1 -(3 ⁇ ethoxy ⁇ henyl)-2-(4- fluorophenyl)- 1 H -imidazol-4-yl]carbonyl ⁇ - 1 -piperaz
  • Specific MC4R agonists that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: l) (5 J S)-l' ⁇ [(3 J R,4 ⁇ )-l-fer?-butyl-3-(2,3,4-trifluorophenyl)piperidin-4-yl]carbonyl ⁇ -3- chloro-2-methyI-5-[l-methyl-l-(l-methyl-lH-l,2 5 4-triazol-5-yl)ethyl]-5H-spiro[faro[3,4- &]pyridine-7,4'-piperidine]; 2) (5i?)-l'- ⁇ [(3i?
  • Methods of administrating the pharmacological compositions comprising the one or more neuromedin U receptor agonists to an individual include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, and the like), ocular, and the like and can be administered together with other biologically-active agents.
  • Administration can be systemic or local, hi addition, it may be advantageous to administer the composition into the central nervous system by any suitable route, including intraventricular and intrathecal injection.
  • Intraventricular injection may be facilitated by an intraventricular catheter attached to a reservoir (for example, an Ommaya reservoir).
  • Pulmonary administration may also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. It may also be desirable to administer the one or more neuromedin U receptor agonists locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant.
  • neuromedin U receptor agonists including, but not limited to, encapsulation in liposomes, microparticles, microcapsules; minicells; polymers; capsules; tablets; and the like.
  • the neuromedin U receptor agonist may be delivered in a vesicle, in particular a liposome.
  • the neuromedin U receptor agonist is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,837,028 and U.S. Patent No. 4,737,323.
  • the neuromedin U receptor agonist can be delivered in a controlled release system including, but not limited to: a delivery pump (See, for example, Saudek, et ah, New Engl. J. Med.
  • the controlled release system can be placed in proximity of the therapeutic target (for example, the brain), thus requiring only a fraction of the systemic dose. See, for example, Goodson, In: Medical Applications of Controlled Release, 1984. (CRC Press, Bocca Raton, FIa.).
  • compositions comprising the neuromedin U receptor agonist which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques by those of average skill within the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the attending physician will decide the amount of the composition with which to treat each individual patient. Initially, the attending physician will administer low doses of the composition and observe the patient's response.
  • the daily dose range lies within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases.
  • suitable dosage ranges for intravenous administration of the compositions comprising the neuromedin U receptor agonist are generally about 5-500 micrograms ( ⁇ g) of active compound per kilogram (Kg) body weight.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient. Ultimately the attending physician will decide on the appropriate duration of therapy using compositions comprising the neuromedin U receptor agonist of the present invention. Dosage will also vary according to the age, weight and response of the individual patient.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions and neuromedin U receptor agonists.
  • Optionally associated with such containers may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • This example shows the synthesis of NMU receptor Agonists.
  • the neuromedin U receptor agonists (See Table 1) were synthesized by solid phase using Fmoc/tBu chemistry on a peptide synthesizer ABI433A (Applied Biosystems). For each peptide 0.75 g of a resin aminomethylated polystirene LL (100-200 mesh, 0.41 mmol/g) (Novabiochem) resin derivatized with a modified Rink linker p- [(R 5 S)-Ot- [9H-Fluoren-9-yl- methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid (Rink, Tetrahedron Lett.
  • the peptides were synthesized by solid phase using Fmoc/t-Bu chemistry on a peptide multisynthesizer Simphony (Protein Technologies Inc.) or APEX396 (AAPTEC).
  • Simphony Protein Technologies Inc.
  • APEX396 AAPTEC
  • a resin aminomethylated polystirene LL 100-200 mesh, 0.41 mmol/g
  • Novabiochem Novabiochem
  • Rink linker p-[(R,S)- ⁇ -[9H- Fluoren-9-yl-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid
  • the amino acids were activated with equimolar amounts of HBTU (2-(lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate), solution 0.5 M in DMF, and a 2-fold molar excess of DIEA (N,N-diisopro ⁇ ylethylamine) solution 2 M in NMP.
  • HBTU 2-(lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate
  • the side chain protecting groups were: tert-butyl for Asp, GIu, (D)GIu, GIa, hGlu, CbF, Ser and Tyr; trityl for Asn, Cys and GIn; 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl for Arg and Harg; the alpha-carboxyl protecting group of gamma-Glu was tert-butyl
  • the N-terminal acetylation reaction was performed at the end of the peptide assembly by reaction with a 10-fold excess of acetic anhydride in DMF.
  • the N-terminal palmltoylation reaction (for NMU34, 38, 39 and 40) was performed at the end of the peptide assembly by reaction with a four-fold excess of activated palmitic acid over the resin free amino groups.
  • the palmitic acici was activated with equimolar amounts of DIPC (1,3-D ⁇ isopropylcarbodiimide) and HOBt (Hydroxybertzotriazole) in DMF.
  • the dry peptide-resins were individually treated with 20 raL of the cleavage mixture, 88% TFA, 5% phenol, 2% triisopropylsilane and 5% water (Sole and Barany, J. Org. Chem. 57: 5399-5403 (1992)) for 2.5 hours at room temperature. Each resin was filtered and the solution was added to cold methyl-t-butyl ether in order to precipitate the peptide. After centrifugation, the peptide pellets were washed with fresh cold methyl-t-butyl ether to remove the organic scavengers. The process was repeated twice. Final pellets were dried, resuspended in H20, 20% acetonitrile, and lyophilized.
  • the crude peptides were purified by reverse-phase HPLC using Waters RCM Delta-PakTM C 4 or C 18 cartridges (40 x 200 mm, 15 ⁇ m) or ReproSil-Pur C 4 (50 x 150 mm, 10 ⁇ m) ( Dr. Maisch GmbH) or Phenomenex Proteo C 12 (50 x 150 mm, 10 ⁇ m) and using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, flow rate 80 mL/min, or Waters X- Bridge C ⁇ (19 x 150 mm, 10 ⁇ m), flow rate 30 mL/min.
  • Analytical HPLC was performed on a Phenomenex Jupiter C4 column (150 x 4.6 mm, 5 ⁇ m) or ReproSil-Pur 300 C4 column (150 x 4.6 mm, 5 ⁇ m) ( Dr. Maisch GmbH) or Beckman Ultrasphere Cl g column (250 x 4.6 mm, 5 ⁇ m), flow rate one mL/min, using H 2 O, 0.1% TFA (A) and CH3CN, 0.1% TFA (B) as solvents.
  • the peptides were characterized on an Alliance Waters Chromatograph, with a ACE C-4 (300 A), 3 ⁇ m column, 150 x 4.6 mm, (CPS analitica p/n ACE-213-1546), at 45 0 C.
  • the purified peptides were characterized by electrospray mass spectrometry on a Micromass LCZ platform.
  • NMUlOl The synthesis of peptide NMUlOl was performed by dissolving the thiol containing NMU peptide precursor in sodium phosphate 0.2M , urea 8M, EDTA 4mM, pH 6.5. A three molar excess of N-ethylmaleimide was added. After one hour incubation, the peptide was purified by HPLC.
  • PEGylation reactions were run under conditions permitting amide bond (NMU58, 108, 1 19, 134, 137 and 139) or thioether bond formation (NMU51, 52, 53, 54, 80, 81, 33, 100).
  • the PEGylated NMU peptides were then isolated using cation exchange chromatography (FXC) followed by size exclusion chromatography (SEC) or reverse-phase (RP) HPLC.
  • FXC cation exchange chromatography
  • SEC size exclusion chromatography
  • RP reverse-phase
  • Cation exchange chromatography IXC was carried out on MacroCap SP (GE Healthcare) column (26 x 120 mm) with a linear gradient of NaCl (0-0.6M) in 3.5 column volumes in formic acid 0.05%, flow rate loading one mL/min, gradient elution 6 mL/min.
  • NMU58, NMUl 08, NMUl 19, NMU 134, NMUl 37 and NMUl 39 peptides were synthesized from the NMU peptide precursors to produce derivatives with PEG covalently attached via an amide bond. Synthesis of N MU 58
  • IXC immunosorbent assay for polystyrene
  • SEC size exclusion chromatography
  • IXC chromatography
  • NMUl 34 20 mg of peptide precursor (2.9 ⁇ moles) were dissolved at 10 mg/mL concentration in 8M urea, 0.2 M HEPES 5 pH 7.3. Then 810 mg of SUNBRIGHT ME-400HS (NOF Corporation) (18.9 ⁇ moles) dissolved at 100 mg/mL concentration in water (1:3.5 mole/mole ratio of peptide to PEG) were added to this solution. After 4 hours incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange chromatography (IXC). The IXC purified PEGylated-peptide was further purified by size exclusion chromatography (SEC) and characterized by RP-HPLC and MALDI-Tof.
  • SEC size exclusion chromatography
  • IXC immunosorbent assay for polystyrene
  • SEC size exclusion chromatography
  • the IXC purified PEGylated-peptide was further purified by size exclusion chromatography (SEC) and characterized by RP-HPLC and MALDI-Tof.
  • NMU51, NMU52, NMU53, NMU54, NMU80, NMU81, NMU33 and NMUlOO peptides were synthesized from the thiol-containing NMU peptide precursors to produce derivatives with PEG covalently attached via a thioether bond. Synthesis ofNMUSl, NMU52, NMU53 and NMU54
  • peptide precursor 10 mg were dissolved at 20 mg/mL concentration in 0.2 M TRIS, pH 8, urea 8M, 4 roM EDTA. Then 200 mg of SUNBRIGHT ME- 400IA (NOF Corporation) (4.8 ⁇ moles) dissolved at 100 mg/mL concentration in water (1 :1 * 5 mole/mole ratio of peptide to PEG) were added to this solution. After one hour incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange chromatography (IXC). The IXC purified PEGylated-peptide was further purified by RP-HPLC and characterized by RP-HPLC and MALDI-Tof.
  • IXC cation exchange chromatography
  • Derivatizations with cholesterol were run under conditions permitting thioether bond formation.
  • the cholesteroylated neuromedin U receptor agonists were then purified by RP- HPLC and characterized by electrospray mass spectrometry.
  • the cholesteroylated peptide was purified by reverse-phase HPLC on a Waters RCM Delta-PakTM C4 cartridge (40 x 100 mm, 15 ⁇ m), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and the following gradient: 55%-55% B (in five minutes) - 75% B (in 20 minutes) - 80% B (in two minutes), flow rate 80 mL/min.
  • the purified product was characterized by RP-HPLC and electrospray mass spectrometry.
  • the cholesteroylated peptide was purified by reverse-phase HPLC on a Waters RCM Delta-PakTM C4 cartridge (40 x 100 mm, 15 ⁇ m), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and the following gradient: 45%-45% B (in five minutes) - 65% B (in 20 minutes) - 80% B (in two minutes), flow rate 80 mL/min.
  • the purified product was characterized by RP-HPLC and electrospray mass spectrometry.
  • the cholesteroylated peptide was purified by reverse-phase HPLC on Waters RCM Delta-PakTM C4 cartridges (25 x 200 mm, 15 ⁇ m), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and the following gradient: 70% ⁇ 70% B (in five minutes) - 90% B (in 20 minutes) - 90% B (in two minutes), flow rate 30 mL/min.
  • the purified product was characterized by RP-HPLC and electrospray mass spectrometry.
  • This example shows the in vitro activity of NMU analogs on NMU receptors.
  • the NMU receptors signal primarily through G ⁇ q/j ⁇ proteins; therefore FLIPR, a calcium mobilization assay, was used to measure functional activity using ceil lines expressing the human and mouse NMU receptors.
  • FLIPR assay Stable cell lines expressing human and or rodent NMURl or human NMUR2 receptors were plated at a density of 12,000 cells per well overnight on poly- lysine coated 384- well black- walled plates. The following day, the media was removed from the plates and the cells were subsequently loaded with Fluo-3 (Molecular Probes), a calcium sensitive dye, diluted in FLIPR buffer (IX Hank's buffered saline containing 20 mM HEPES 5 0.1% BSA, 2.5 mM probenecid (Sigma) and 1.6 mM TR40). All reagents are from Invitrogen unless otherwise noted.
  • NMU8-P3- 11 through NMU8-P3-47 Modification of the N terminus of NMU with PEGylation, cholesteroylation, or palmitoylation results in a rightward shift in the in vitro potency. Modification of P3 residue in the NMU8 sequence showed trends to a NMURl -selective peptide since there was little or no activity at the NMUR2 receptor (NMU8-P3- 11 through NMU8-P3-47).
  • NMURl knockout mice were generated using standard homologous recombination techniques. Nmurl mice were subsequently transferred to Taconic Farms where they were either maintained on a 75% C57BL/6 x 25% 129S6/SvEv mixed genetic background or backcrossed ten generations to C57BL/6.
  • NMUR2 knockout (Nmur2-/-) mice were licensed from Deltagen Inc., San Mateo, CA and subsequently transferred to Taconic Farms where they were either maintained on a 75% C57BL/6 x 25% 129/OlaHsd mixed genetic background or backcrossed for ten generations to C57BL/6. Mice were individually housed in Tecniplast cages in a conventional SPF facility.
  • mice were initially maintained on a regular chow diet and then early in their life were switched to a high fat diet (D 12492: 60 % kcal from fat; Research Diets, Inc., New Brunswick, NJ) with ad libitum access to water in a 12-hour light/12-hour dark cycle.
  • D 12492 60 % kcal from fat; Research Diets, Inc., New Brunswick, NJ
  • Ad libitum fed male diet-induced obese mice were weighed and dosed either i.p. or s.c. about 30 minutes prior to the onset of the dark phase of the light cycle and provided with a preweighed aliquot of high fat diet D 12492 which was then weighed 2 hours and 18 hours (day 1), 42 hours (day 2), 66 hours (day 3), and 90 hours (day 4) after the onset of the initial dark phase. Mice were weighed at the 18, 42, 66, and 90 hour time points. Data showed the outcome of the feeding study (all values are reported as mean ⁇ SEM and data was analyzed using a two- tailed unpaired Student's t test; p values ⁇ 0.05 were reported as significant and are denoted with an asterisk).
  • Figures IA and IB show that acute peripheral administration of palmitoylated NMU analogs, significantly reduced food intake in diet-induced obese mice for two days post dose.
  • Figure 2 illustrates the finding that the anorectic effects of NMU38, a palmitoylated NMU analog, are mediated by the contribution of both the NMURl and NMUR2 receptors.
  • Acute administration of NMU38 was highly efficacious in wild-type animals but the anorectic effect was diminished in both Nmurl- and Nmur2-deficient animals on day 1 post dose.
  • the effects on food intake were completely gone on day 2 post dose in the Nmurl -deficient mice.
  • the data indicate that NMU38 evokes reductions in food intake on day 2 primarily through the NMURl receptor.
  • Figure 3 illustrate the finding that cholesteroylated NMU analogs can reduce food intake for up to two days following a single administration.
  • Figure 2 illustrates the finding that the anorectic effects of NMU37, a cholesleroylated NMU analog, are mediated by the contribution of both the NMURl and NMUR2 receptors.
  • Acute administration of NMU38 was highly efficacious in wild-type animals but the anorectic effect was diminished in both Nmurl - and Nmur2-deficient animals on day 1 post dose. The effects on food intake were completely gone on day 2 post dose in both the Nmurl -and Nmur2-deficient mice.
  • Figure 5 illustrates the finding that the PEGylated NMU analogs, NMU80 and NMU81 , can reduce food intake for up to three days following a single subcutaneous dose.
  • NMU80 reduced food intake for two days while NMU81 evoked significant reductions in food intake for three days post dose.

Abstract

Neuromedin U receptor agonists for use in the treatment of metabolic disorders such as obesity are described. In particular, described are neuromedin U receptor agonists that are analogs or derivatives of neuromedin U (NMU).

Description

TITLE OF THE INVENTION
NEUROMEDIN U RECEPTOR AGONISTS AND USES THEREOF
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to neuromedin U receptor agonists for use in the treatment of metabolic disorders such as obesity. In particular, the present invention relates to neuromedin U receptor agonists that are analogs or derivatives of neuromedin U (NMU). (2) Description of Related Art
Neuromedin U (NMU) was originally isolated from porcine spinal cord based upon its ability to contract rat uterine smooth muscle and has since been implicated in a variety of other physiological processes, including stress, nociception, inflammation, cardiovascular function and energy homeostasis. Characterization of NMU has identified three peptides with similar bioactivity, full length NMU, (a 25-mer (NMU-25)) in humans, pigs, and dogs, a 23-mer (NMU-23) in rats and mice, and an 8-mer (NMU-8). NMU-8 is derived from cleavage of full- length NMU and shares an identical C-terminus with the full-length precursor. NMU-8 is highly conserved among vertebrates, containing seven C-terminal residues that are identical across all species that have been examined; these residues are critical for bioactivity (Brighton et al, Pharmacol. Rev. 56: 231-248 (2004)).
NMU' s role in the regulation of energy homeostasis is supported by both pharmacologic and genetic data. Properties of NMU include inhibition of food intake and increase in energy expenditure seen when the substance is administered centrally (Howard et al, Nature 406: 70-74 (2000); Nakazato et al, Biochem. Biophys. Res. Comm. 277: 191-194 (2000); Ivanov et al, Endocrinol. 143: 3813-3821 (2002); and Wren et al, Endocrinol, 143: 4227-4234 (2002)). NMU-deficient mice develop obesity characterized by hyperphagia and reduced energy expenditure (Hanada et al, Nat. Med., 10: 1067-1073 (2004)), and transgenic mice
overexpressing NMU are lean and hypophagic (Kowalski etal, J. Endocrinol.185: 151-164 (2005)). The internal energy status of an animal affects expression and release of NMU as well (Wren et al, ibid).
Mori et al (EMBO J. 24: 325-335 (2005)) recently identified a 36-residue neuropeptide neuromedin S (NMS), which was purified from rat brain extracts. The restricted expression of NMS in the SCN core and the ability of NMS to shift the phase of the circadian rhythm demonstrated that NMS is important for the regulation of circadian rhythm. NMS is a candidate for a nonphotic entrainment factor of circadian rhythm. Ida et al (Endocrinol. 146: 4217-4223 (2005)) provided evidence suggesting NMS is an anorexigenic hormone.
Intracerebroventricular (icv) injection of NMS decreased 12 hour food intake during the dark period in rats. This anorexigenic effect was more potent and persistent than that observed with the same dose of NMU. NMS has also been disclosed in International applications
WO2006/086769, WO2007/0221 123, and WO2007/075439.
Two high affinity NMU and NMS receptors, NMURl (Intl. Patent Appl. No. PCT/US99/15941) and NMUR2 (U. S. Patent No. 7163799), have been identified. NMURl is predominantly expressed in the periphery, whereas NMUR2 is primarily expressed in the brain. Pharmacologic experiments have served to better define NMU' s short- and long-term effects on energy homeostasis and to identify which NMU receptor(s) are involved in mediating these actions. It has been shown that acute administrations of NMU either centrally or peripherally reduce food intake in mice in a dose-dependent fashion. The anorectic actions of centrally administered NMU are absent in NMUR2-deficient (Nmur2'/') mice but are present in NMURl- deficient (Nmurl'^) mice. In contrast, the anorectic actions of peripherally administered NMU are absent in Nmurl'1' mice and present in NmurT ' mice. Additionally, acute peripheral administration of NMU dose-dependently increases core body temperature in mice, suggesting that NMURl may also modulate energy expenditure. Chronic administration of NMU either centrally or peripherally reduces food intake, body weight and adiposity in mice, again in a dose- dependent fashion. In Nmur2'/' transgenic mice, body weight, body composition, body temperature and food intake are largely unaffected by chronic central administration of rat NMU- 23. In Nm url'1' transgenic mice, body weight, body composition and food intake are largely unaffected by chronic peripheral administration of rat NMU-23.
Because the sites of action for NMURl- vs. NMUR2-mediated efficacy differ and appear to be independent of one another, but have a role in obesity, it is suggested that both NMURl- and NMUR2- selective agonists and NMURl /2 non-selective agonists may be useful for the treatment of obesity. Therefore, there is a need for neuromedin U receptor agonists useful in the treatment of metabolic disorders.
BRIEF SUMMARY OF THE INVENTION
The present invention provides neuromedin U (NMU) receptor agonists comprising an NMU peptide or analog thereof conjugated to a functional group on a carrier protein.
Therapeutic applications of the neuromedin U receptor agonists include administering the neuromedin U receptor agonists to an individual to treat a metabolic disorder afflicting the individual. Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, and type II diabetes. Complications of diabetes such as retinopathy may be positively affected thereby as well. Obesity is a comorbidity of and may well contribute to such disease states as diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis and certain forms of cancers. Administration of one or more of the neuromedin U receptor agonists disclosed herein to effect weight loss in an individual may also be useful in preventing such diseases and as part of therapy for any one of the above-recited conditions, as well as others. In other embodiments, there is provided a method for treating a metabolic disease in an individual comprising administering to the individual one or more of the neuromedin U receptor agonists described above. The metabolic disease may be selected from the group consisting of diabetes, metabolic syndrome, hyperglycemia, and obesity and may be administered via a route peripheral to the brain, such as an oral, mucosal, buccal, sublingual, nasal, rectal, subcutaneous, transdermal, intravenous, intramuscular, or intraperitoneal route. Finally, the neuromedin U receptor agonists can be administered to an individual to effect a reduction in food intake by the individual, to effect a reduction in weight gain in the individual, to prevent weight gain in the individual, to effect weight loss in the individual, and/or to prevent weight regain in the individual.
Accordingly, the present invention provides an isolated neuromedin U receptor agonist conjugated to a carrier protein. In general, the neuromedin U receptor agonist has the formula (I)
Zl-peptide-Z2 wherein the peptide has the amino acid sequence χLχ2-.χ3-χ4_χ5-χ6-χ7-χ8- X9.χlθ.χl Lxl2.xl3-Xl4-Xl5-xl6-xl7-xl8.xl9-x20-x21.x22.x23.x24.x25 (SEQ ID NO:1), wherein amino acids 1 to 17 can be any amino acid or absent; wherein amino acid Xl^i8 absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid X* 9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid χ20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid χ21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; χ22 is Arg, Lys, Harg, Ala, or Leu; amino acid χ23 is Pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid χ25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala; Pr is a carrier protein; iX is optionally a protecting group that, if present, is joined to the N-terminus amino group; and Z^ is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group.
In particular aspects, the peptide has the amino acid sequence χLχ2.χ3.χ4.χ5. χ6-χ7-χ8.χ9-χl 0-χl 1.χl2.χl 3_χl 4.χ 15.χl 6.χl 7.χl 8-Phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO: 2), wherein amino acids 1 to 17 can be any amino acid or absent,
In another aspect, the peptide comprises the amino acid sequence Phe- Arg- VaI-
Asp-Glu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-Gln-Ser-Arg-Gly-Xl 8_χl 9.χ20.χ21 _χ22-χ23_ χ24.χ25 (SEQ ID NO:3) wherein amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid Xl9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid χ21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; X22 is Arg, Lys, Harg, Ala, or Leu; amino acid χ23 is Pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid X25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala.
In a further still aspect, the peptide comprises the amino acid sequence χl-X2- X3.χ4.χ5_χ6_χ7.χ8 (SEQ ID NO:4) wherein amino acid Xl is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid X2 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X3 is absent, GIy, sarcosine (Sar), Leu or D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L- amino acid; amino acid χ4 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid X^ is Arg, Lys, Harg, Ala, or Leu; amino acid X^ is Pro, Ser, Sar, Ala or Leu; amino acid X? is Arg, Harg or Lys; and amino acid X8 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala.
The present invention further provides for the use of any one or more of the embodiments and aspects of the neuromedin U receptor agonist in the manufacture of a medicament for treatment of a metabolic disorder. Disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, and type II diabetes. Complications of diabetes such as retinopathy may be positively affected thereby as well. Obesity is a comorbidity of and may well contribute to such disease states as diabetes, hypertension, dyslipidemias,
cardiovascular disease, gallstones, osteoarthritis and certain forms of cancers. Thus, the present invention provides a pharmaceutical composition comprising one or more of any of the above neuromedin U receptor agonists and a pharmaceutically acceptable carrier.
Therefore, further provided is a method for treating a metabolic disorder in an individual comprising administering to the individual a therapeutically effective amount of a neuromedin U receptor agonist that has the formula (I) Zl-peptide-Z2 wherein the peptide has the amino acid sequence Xl-χ2-χ3~χ4_χ5-.χ6_χ7_χ8- X9-χl0.χl I_χl2.χl3_χl4.χl5.χl6-χl7.χl8.χl9-χ2θ-χ21.χ22-χ23.χ24.χ25 (SEQ ID NO:1), wherein amino acids 1 to 17 can be any amino acid or absent; wherein amino acid χ!8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid χl9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid χ20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid χ21 is Phe, NMe-Phe5 an aliphatic amino acid, an aromatic amino acid, Ala or Trp; χ22 is Arg, Lys, Harg, Ala, or Leu; amino acid X23 is Pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid χ25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala; Zl is optionally a protecting group that, if present, is joined to the N-terminus amino group; and Z2 is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group. .
The method is particularly useful for treating a metabolic disorder selected from the group consisting of obesity, metabolic syndrome or syndrome X, type II diabetes,
complications of diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers.
In particular aspects, the peptide has the amino acid sequence Xl-X2-χ3-.χ4-χ5- χ6-χ7-χ8-χ9_χl0_χl Lχl2-χl3.χl4,χl5.χl6_χl7.χl8.phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO:2), wherein amino acids 1 to 17 can be any amino acid or absent.
In another aspect, the peptide comprises the amino acid sequence Phe- Arg- VaI- Asp-Glu-GIu-Phe-Gln-Ser-Pro-Phe-AIa-Ser-Gln-Ser-Arg-Gly-Xl 8-Xl 9-χ20.χ21-χ22-χ23. χ24.χ25 (SEQ ID NO:3) wherein amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn5 NIe5 GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid Xl9 1S Ala, Trp5 Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid X^l is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; X22 is Arg, Lys, Harg, Ala, or Leu; amino acid χ23 is Pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid X25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala.
In a further still aspect, the peptide comprises the amino acid sequence χl~χ2- χ3-χ4-χ5-χ6.χ7.χ8 (SEQ ID NO:4) wherein amino acid Xl is absent, Tyr or D-Tyr5 Leu, Phe, VaI, GIn, NIe, GIu or D-GIu5 Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid X^ is AIa, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X3 is absent, GIy, sarcosine (Sar), Leu or D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L- amino acid; amino acid X4 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid X5 is Arg, Lys, Harg, Ala, or Leu; amino acid X6 is Pro, Ser, Sar, Ala or Leu; amino acid χ7 is Arg, Harg or Lys; and amino acid X^ is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala.
The present invention further includes use of the compositions disclosed herein in the manufacture of a medicament for treatment of a metabolic disorder, which in further aspects can be obesity or type II diabetes. Accordingly, further provided are pharmaceutical composition comprising a neuromedin U receptor agonist and a pharmaceutically acceptable carrier. As used herein, an "NMU peptide analog" is a peptide that has sufficient identity or homology to native human NMU having the amino acid sequence shown in SEQ ID NO:5 that it is capable of interacting with the NMURl and/or NMUR2 receptors as an agonist. Thus, NMU peptide analogs can have one or more amino acid substitutions, modifications, or deletions at amino acid positions 1-25.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Acute administration of palmitoylated NMU analogs significantly reduces food intake (A) and body weight (B) in diet-induced obese mice.
Figure 2. Acute administration of palmitoylated NMU analog, NMU38, significantly reduces food intake in Nmurl- and Nmur2-deficient mice.
Figure 3. Acute administration of cholesteroylated analogs significantly reduces food intake (A) and body weight (B) in diet-induced obese mice.
Figure 4. Acute administration of cholesteroylated NMU analog, NMU37, significantly reduces food intake in Nmurl - and Nmur2-deficient mice.
Figure 5. Acute administration of PEGylated analogs NMU80 andNMU81, significantly reduce food intake (A) and body weight (B) in diet-induced obese mice.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides neuromedin U (NMU) receptor agonists comprising an NMU peptide or analog. Therapeutic applications of the neuromedin U receptor agonists include administering the neuromedin U receptor agonists to an individual to treat a metabolic disorder afflicting the individual. Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, and type II diabetes. Complications of diabetes such as retinopathy may be positively affected thereby as well. Obesity is a comorbidity of and may well contribute to such disease states as diabetes, hypertension, dyslipidemias,
cardiovascular disease, gallstones, osteoarthritis and certain forms of cancers. Administration of one or more of the neuromedin U receptor agonists disclosed herein to effect weight loss in an individual may also be useful in preventing such diseases and as part of therapy for any one of the above-recited conditions, as well as others. In other embodiments, there is provided a method for treating a metabolic disease in an individual comprising administering to the individual one or more of the neuromedin U receptor agonist s described above. The metabolic disease may be selected from the group consisting of diabetes, metabolic syndrome,
hyperglycemia, and obesity and may be administered via a route peripheral to the brain, such as an oral, mucosal, buccal, sublingual, nasal, rectal, subcutaneous, transdermal, intravenous, intramuscular, or intraperitoneal route. Finally, the neuromedin U receptor agonists can be administered to an individual to effect a reduction in food intake by the individual, to effect a reduction in weight gain in the individual, to prevent weight gain in the individual, to effect weight loss in the individual, and/or to prevent weight regain in the individual.
Accordingly, the present invention provides an isolated neuromedin U receptor agonist conjugated to a carrier protein. In general, the neuromedin U receptor agonist has the formula (I)
Zl-peptide-Z2 wherein the peptide has the amino acid sequence Xl-X2-χ3-.χ4-χ5_χ6-χ7_χ8- X9.χlθ.χl Lxl2.xl3.xl4.xl5.xl6.xl7.xl8.xl9.x20.x21-x22-x23.x24.x25 (SEQ ID NO:1), wherein amino acids 1 to 17 can be any amino acid or absent; wherein amino acid Xl8 js absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid χl9 1S Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid χ20 |s absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid χ21 is Phe, NMe~Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; χ22 is Arg, Lys, Harg, Ala, or Leu; amino acid X23 is Pro, Ser, Sar, Ala or Leu; amino acid χ24 [s Arg, Harg or Lys; and amino acid X25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala; Zl is an optionally present protecting group; and Z2 is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group.
In particular aspects, the peptide has the amino acid sequence χl-χ2,χ3_χ4.χ5. χ6-χ7-χ8-χ9-χl0.χl l-χl2-χl3-χl4-χl5-χl6-χl7_χl8-Phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO: 2), wherein amino acids 1 to 17 can be any amino acid or absent.
In another aspect, the peptide comprises the amino acid sequence Phe-Arg-Val- Asp-Glu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-GIn-Ser-Arg-Gly~Xl 8_χl9~χ2(Lχ21.χ22.χ23. χ24.χ25 (SEQ ID NO:3) wherein amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, AIa, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid Xl9 js Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid X21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; X22 js Arg, Lys, Harg, Ala, or Leu; amino acid χ23 is Pro, Ser, Sar, Ala or Leu; amino acid X24 is Arg, Harg or Lys; and amino acid X25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala.
In a further still aspect, the peptide comprises the amino acid sequence χl— χ2- χ3-χ4-χ5-χ6.χ7_χ8 (SEQ ID NO:4) wherein amino acid Xl is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid χ2 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid χ3 is absent, GIy, sarcosine (Sar), Leu or D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L- amino acid; amino acid X^ is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid X5 is Arg, Lys, Harg, Ala, or Leu; amino acid Xβ is Pro, Ser, Sar, Ala or Leu; amino acid X? is Arg, Harg or Lys; and amino acid X^ is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala.
The present invention further provides for the use of any one or more of the embodiments and aspects of the neuromedin U receptor agonist in the manufacture of a medicament for treatment of a metabolic disorder. Disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, and type II diabetes. Complications of diabetes such as retinopathy may be positively affected thereby as well. Obesity is a comorbidity of and may well contribute to such disease states as diabetes, hypertension, dyslipidemias,
cardiovascular disease, gallstones, osteoarthritis and certain forms of cancers. Thus, the present invention provides a pharmaceutical composition comprising one or more of any of the above neuromedin U receptor agonists and a pharmaceutically acceptable carrier.
Therefore, further provided is a method for treating a metabolic disorder in an individual comprising administering to the individual a therapeutically effective amount of a neuromedin U receptor agonist that has the formula (I)
Zl-ρeptide-Z2 wherein the peptide has the amino acid sequence Xl-χ2.χ3-χ4_χ5.χ6-.χ7..χ8. χ9-χlθ-χl 1-X12-X13-X14-X15-X16.X17-X18.X19-X20.X21.X22.X23-X24.X25 (SEQ ID
NO:1), wherein amino acids 1 to 17 can be any amino acid or absent; wherein amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn3 NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid Xl9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X^O is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid χ21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; X22 is Arg, Lys, Harg, Ala, or Leu; amino acid χ23 is Pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid χ25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala; Zl is an optionally present protecting group; and Z^ is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group.
The method is particularly useful for treating a metabolic disorder selected from the group consisting of obesity, metabolic syndrome or syndrome X, type II diabetes, complications of diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers. In particular aspects, the peptide has the amino acid sequence χl-χ2.χ3_χ4_χ5_ χ6-χ7-χ8-χ9-χl(Lχl 1 _χl 2.χl 3-χl4.χl 5.χl 6.χl 7_χl 8-Phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID N0:2), wherein amino acids 1 to 17 can be any amino acid or absent.
In another aspect, the peptide comprises the amino acid sequence Phe- Arg- VaI- Asp-Glu-Glu-Phe-Gln-Ser-Pro~Phe-Ala-Ser-Gln-Ser-Arg-Gly-Xl 8-Xl 9-χ20_χ21_χ22_χ23. χ24.χ25 (SEQ ID N0:3) wherein amino acid χl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid XΪ9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid χ21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; X22 is Arg, Lys, Harg, Ala, or Leu; amino acid X23 is Pro, Ser, Sar, Ala or Leu; amino acid X24 is Arg, Harg or Lys; and amino acid χ25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala.
In a further still aspect, the peptide comprises the amino acid sequence χl-X2- χ3-χ4.χ5-χ6_χ7-χ8 (SEQ ID NO:4) wherein amino acid Xl is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu3 Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid χ2 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid χ3 is absent, GIy, sarcosine (Sar), Leu or D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L- amino acid; amino acid χ4 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid χ5 is Arg, Lys, Harg, Ala, or Leu; amino acid χ6 is Pro, Ser, Sar, Ala or Leu; amino acid χ7 is Arg, Harg or Lys; and amino acid χ8 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala.
Examples of neuromedin U receptor agonists of the present invention comprising the amino acid sequence Xl-X2-x3.x4.x5.x6-X7.x8.x9-Xl0.xn.xl2.xl3-Xl4.xl5.xl6. Gly-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO:2) wherein amino acids 1 to 16 can be any amino acid or absent are shown in Table 1. The neuromedin U receptor agonists are protected at the C-terminus with an amino group.
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Examples of neuromedin U receptor agonists of the present invention comprising the amino acid sequence Phe-Arg-Val-Asp-GIu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-Gln-Ser-Arg- Gly-χl8»χl9.χ20.χ21.χ22.χ23.χ24.χ25 (SEQ ID N0:3) or Xl~χ2-χ3-χ4-χ5-χ6.χ7.χ8 (SEQ ID NO:4) wherein amino acid χlδ or Xl is absent, Tyr, Ala, Leu, Phe, VaI, GIn, Asp, GIu , (D)-Glu,(D)-Tyr, (D)-Lys, homo-glutamic acid (hGlu), 3-methoxyphenyIalanine (MOF), 4- fluoro-phenylalanine (PhF), 4-nitro-phenylalanine (FNO), 4-carboxylmethoxy-phenylalanine (CbF), β-cyclopropyl-alanine (Cpa) or NIe; Xl9 or χ2 is Phe, tyr, GIn, Trp, 3,3-diphenylalanine (Dif), styrylalanine (Sty), 2-naphthyl-alanine (Nap), 3-methoxy-phenylalamne (MOF), 4-amino- phenylalanine (AmF), β-(2-thienyl)-alanine (Tha), 3-(4-thiazoyl)-alanine (Thz), β-cyclopropyl- alanine (Cpa), Nva or NIe; χ20 Or χ3 is Leu, GIy5 (D)-IIe5 (D)-Tyrs (D)-VaI, (D)- Arg, 1-amino- 1-cyclopropane-carboxylic acid (Acp), 1-amino-cyclohexane-carboxylic acid (Acx), (D)-Nva, (D)-2-aminobutyric acid ((D)- Abu), (D)-NIe, He3 VaI, Lys, Arg, GIn, β-cyclopropyl-alanine (Cpa), β-cyclohexyl-lalanine (Cha), Nva, 2-aminobutyric acid (Abu), propargyl-glycϊne (Pra) or NIe; χ21 or χ4 is Phe, propargyl-glycine (Pra), Trp, l,2,3,4-tetrahydroisoquinoHne-3-carboxylic acid (Tic), Tyr, 4-fluoro-phenylalanine (PhF), 4-nitro-phenylalanine (FNO), 4-triϊTttoromethyl- phenylalanine (TFF), 4-methyl-phenylalanine (MeF), 4-amino-phenylalanine (AmF), β- cyclohexyHalanine (Cha), β-cyclopropyl-alanine (Cρa)5 allyl-glycine (AIg) or Me; χ22 or χ5 is Arg, Lys, homo-arginine (Harg), ornithine (Orn), γ-diaminobutyric acid (Dab); χ23 or Xό is Pro, thiazolidine-4-carboxylic acid (SPro) or Ser; χ24 or X7 is Arg or homo-arginine (Harg); X25 or X8 is absent, Asn, Ala, NIe, (D)-NIe5 G5 (D)-AIa, 2-amino-4,4-difluorobutyric acid (AbuF2), β- diaminopropionic acid (Dap), 3-aminoethyl-benzoic acid (Mamb) or 2-hydroxy-ethylamine. Examples of peptides having the above amino acid sequences are shown in Table 2. In general, these peptides are either (NMURl or NMUR2) subtype-specific or bispecific.
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0002
Lipidated (either cholesteroylated or palmitoylated) neuromedin U receptor agonists were designed to have an improved in vivo pharmacological profile.
Peptides NMU36 and NMU37 are cholesteroylated peptides based on the native NMU sequence. NMU36 has the structure Ac-CsFRVDEEFQSPFASQSRGYFLFRPRN- CONH2 wherein the C5 is a cholest-5-en-3-yl (21R)-21-amino-17,22-dioxo-4,7,10,13-tetraoxa- 19-thia-16-azadocosan-l-oate attached to the thio group of cysteine (or Cys(oxa4-cholesterol)) as shown below.
Figure imgf000016_0001
NMU37 has the structure AC-CeFRVDEEFQSPFASQSRGYFLFRPRN-CONH2 wherein the Cg is a cholest~5-en-3-yl N-[43-(3-{[(2R)-2-amino-3-oxopropyl]sulfanyl}-2,5- dioxopyrrolidin- 1 -yl)-41 -oxo-4,7, 10,13,16,19,22,25,28,31 ,34,37-dodecaoxa-40- azatritetracontan-l-oyl]glycinate attached to the thio group of cysteine or Cys(oxai2-cholesterol)) as shown below.
Figure imgf000017_0001
NMU44 is a cholesteroylated peptide based on N-terminally trancated sequence 17-25, bearing an acetylated Cβ group at the N-terminus.
The presence of hydrophilic spacers between the cholesterol group and the cysteine moiety, like those contained in C& and C7, was found to be important for the in vitro and in vivo activity of the cholesteroylated NMU derivatives.
NMU38 is a palmitoylated analog in which the palmitoyl group is linked at the N- terminus of the NMU peptide sequence.
NMU34 is a palmitoylated analog in which the palmitoyl group is linked at the N- terminus of the C-terminal nonamer of the NMU peptide sequence.
NMU39 and NMU40 are palmitoylated analogs in which a Ttds (l-amino-4,7,10- trioxa-13-tridecanamine succinimic acid) or a gamma glutamic residue (γE) were respectively introduced as flexible spacers between the N-terminus of the NMU sequence and the palmitoyl group.
NMU41 and NMU42 have respectively a Ttds group and a gamma glutamic acid residue at the N-terminus of the NMU peptide sequence. These analogs are control peptides with no palmitoyl but acetyl group at the N-terminus.
NMU58 is a PEGylated peptide in which a branched PEG of 40 kDa is linked at the N-terminus of the native human neuromedin U peptide. The N-terminal group of the peptide was acylated with a branched (PEG)2 4OK N-hydroxysuccinimide analog (SUNBRIGHT GL2- 400GS2; NOF Corporation). This was designed to create a neuromedin U receptor agonist with improved pharmacological profile. The peptide precursor, the wild type sequence of NMU, was reacted with an N-hydroxysuccinimide derivative of a branched PEG of 40 kDa. PEGylation with this reagent occurs specifically at the N-terminal amino group of the peptide, as this is the only available amino group in the peptide.
NMU51 , NMU52, NMU53 and NMU54 are PEGylated peptides in which the
PEG moiety is linked at an internal position of the human NMU peptide sequence. These peptides were designed to create a neuromedin U receptor agonist with improved
pharmacological profile. PEGylation site was scanned every four residues by mutating the native residues into a Cys residue in order to allow site-specific derivatization of the thiol group with a branched (PEG)24OkDa (C2). In particular, the C2 group was introduced at positions: 4 to obtain NMU51, 8 to obtain NMU52, 12 to obtain NMU53 and 16 to obtain NMU54.
NMU80, NMU81 and NMU33 are PEGylated peptides designed starting from the native NMU sequence and adding an acetylated cysteine residues at the N-terminus. The cysteine thiolated group was derivatized with (a) linear (mPEG)40 kDa, through either 40 kDa methoxy ρoly(ethylene glycol)maleimido-propionamide (Chirotech, Product Code 008-016) to obtain NMU80, or the iodoacetylated PEG reagent SUNBRIGHT ME-400IA (NOF Corporation) to obtain NMU81 ; or (b) linear (mPEG)5 kDa to obtain NMU33.
PEGylated peptides were designed based on the introduction of a spacer between the PEG moiety and the NMU peptide. The spacer was introduced to reduce the impact on activity on the peptide sequence due to the addition of the PEG. The spacer consists, for example, in five consecutive units of tranexamic acid (Txa)5,
NMUlOO and NMUlOl were designed starting from the full-length native NMU sequence. The sequences were modified by introduction at the N-terminus of (Txa)s as spacer and an acetylated cysteine residue. The cysteine thiolated group was derivatized with (a) N- ethylmaleimide to obtain NMUlOl, a control peptide for conjugation; or (b) (PEG)240 kDa to obtain NMUlOO.
NMU 108 and NMUl 18 were designed to obtain a PEGylated peptide based on the N-termϊnally truncated sequence 17-25. The peptide sequences were modified by
introduction at the N-terminus of (Txa)s as spacer to obtain NMUl 18, a control peptide for conjugation. The N-terminal group of the peptide NMUl 18 was acylated with a branched (PEG)2 40k N-hydroxysuccinimide analog (SUNBRIGHT GL2-400GS2, NOF Corporation) to obtain NMU108. NMUl 16 and NMUl 19 were designed to obtain a PEGylated peptide based on the N~terminally truncated sequence 12-25. The peptide sequences were modified by
introduction at the N-terminus of (Txa)5 as spacer to obtain NMUl 16, a control peptide for conjugation. The N-terminal group of the peptide NMUl 16 was acylated with a branched (PEG)2 40k N-hydroxysuccinimide analog (SUNBRJGHT GL2-400GS2, NOF Corporation) to obtain NMUl 19.
In NMU130, NMU134, NMU129 and NMU137 the native NMU Phej 1 was changed to threonine, GIy 17 was changed to proline and Asn2s was changed to (D)-alanine. The substitutions at Pheπ, GIy 17, and Asn25 were made to increase in vivo stability while retaining functional activity, since these residues were found to be among the primary proteolytic cleavage sites.
NMU 129 and NMUl 37 were designed to obtain a PEGylated peptide based on the triple-mutant NMU peptide sequence as described above. The N-terminal group of the peptide was (a) acetylated to obtain NMU 129, a control peptide for conjugation; or (b) acylated with a linear (PEG) 4OK N-hydroxysuccinimide analog (SUNBRIGHT ME-400HS; NOF Corporation) to obtain NMUl 37.
NMUl 30 and NMUl 34 were based on the amino acid sequence of NMU 129 and 137. The peptide sequences were modified by introduction at the N-lerminus of (Txa)s as spacer to obtain NMUl 30, a control peptide for conjugation. The N-terminal group of the peptide NMU130 was acylated with a linear (PEG) 4OK N-hydroxysuccinimide analog (SUNBRIGHT ME-400HS; NOF Corporation) to obtain NMU134.
NMU 138 and NMUl 39 have the same structure respectively of NMU 129 and NMUl 37 wherein the Arg24 has been substituted with a homo-arginine residue to increase in vivo stability.
In particular aspects, the neuromedin U receptor agonist optionally includes a protecting group covalently joined to the N-terminal amino group. A protecting group covalently joined to the N-terminal amino group of the neuromedin U receptor agonists reduces the reactivity of the amino terminus under in vivo conditions. Amino protecting groups include -Cj. io alkyl, -Q-io substituted alkyl, -C2-10 alkenyl, -C2-10 substituted alkenyl, aryl, -Ci -6 alkyl aryl, -C(O)-(CH2)l-6-COOH, -C(O)-Ci -6 alkyl, -C(O)-aryl, -C(O)-O-Ci -6 alkyl, or -C(O)-O- aryl. In particular embodiments, the amino terminus protecting group is selected from the group consisting of acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl, and t-butyloxycarbonyl.
Deamination of the N-terminal amino acid is another modification that is contemplated for reducing the reactivity of the amino terminus under in vivo conditions.
Chemically modified compositions of the neuromedin U receptor agonists wherein the neuromedin U receptor agonist derivatives are linked to a polymer are also included within the scope of the present invention. The polymer selected is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled as provided for in the present methods. Included within the scope of polymers is a mixture of polymers. Preferably, for therapeutic use of the end- product preparation, the polymer will be pharmaceutically acceptable.
The polymer or mixture thereof may be selected from the group consisting of; for example, polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N- vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (for example, glycerol), and polyvinyl alcohol.
In further still embodiments, the neuromedin U receptor agonists are modified by PEGylation, cholesterylation, or palmitoylation. The modification can be to any amino acid residue in the neuromedin U receptor agonist, however, in currently embodiments, the modification is to the N-terminal amino acid of the neuromedin U receptor agonist, either directly to the N-terminal amino acid or by way coupling to the thiol group of a cysteine residue added to the N-terminus or a linker added to the N-temiinus such as Ttds. In further embodiments, the N-terminus of the neuromedin U receptor agonist comprises a cysteine residue to which a protecting group is coupled to the N-terminal amino group of the cysteine residue and the cysteine thiolate group is derivatized with N-ethylmaleimide, PEG group, cholesterol group, or palmitoyl group. In further still embodiments, an acetylated cysteine residue is added to the N-terminus of the neuromedin U receptor agonists, and the thiol group of the cysteine is derivatized with N-ethylmaleimide, PEG group, cholesterol group, or palmitoyl group.
It is well known that the properties of certain proteins can be modulated by attachment of polyethylene glycol (PEG) polymers, which increases the hydrodynamic volume of the protein and thereby slows its clearance by kidney filtration. (See, for example, Clark et al, J. Biol. Chem. 271: 21969-21977 (1996)). Therefore, it is envisioned that the core peptide residues can be PEGylated to provide enhanced therapeutic benefits such as, for example, increased efficacy by extending half-life in vivo. Thus, PEGylating the neuromedin U receptor agonists will improve the pharmacokinetics and pharmacodynamics of the neuromedin U receptor agonists.
Peptide PEGylation methods are well known in the literature and described in the following references, each of which is incorporated herein by reference: Lu et al, Int. J. Pept. Protein Res.43: 127-38 (1994); Lu et al, Pept. Res. 6: 140-6 (1993); Felix et al, Int. J. Pept. Protein Res. 46: 253-64 (1995); Gaertner et al, Bioconjug. Chem. 7: 38-44 (1996); Tsutsumi et al, Thromb. Haemost 77: 168-73 (1997); Francis et al, Int. J. Hematol. 68: 1-18 (1998);
Roberts et al, J. Pharm. Sci. 87: 1440-45 (1998); and Tan et al, Protein Expr. Purif. 12: 45-52 (1998). Polyethylene glycol or PEG is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, including, but not limited to, mono-(Ci-io) alkoxy or aryloxy-polyethylene glycol. Suitable PEG moieties include, for example, 40 kDa methoxy poly(ethylene glycol) propionaldehyde (Dow, Midland, Michigan); 60 kDa methoxy
poly(ethylene glycol) propionaldehyde (Dow, Midland, Michigan); 4OkDa methoxy
poly(ethylene glycol) maleimido-propionamide (Dow, Midland, Michigan); 31 kDa alpha- methyl-w-(3-oxopropoxy), polyoxyethylene (NOF Corporation, Tokyo); mPEG2-NHS-40k (Nektar); mPEG2-MAL-40k (Nektar), SUNBRIGHT GL2-400MA ((PEG)240kDa) (NOF Corporation, Tokyo), SUNBRIGHT ME-200MA (PEG20kDa) (NOF Corporation, Tokyo). The PEG groups are generally attached to the neuromedin U receptor agonists via acylation or alkylation through a reactive group on the PEG moiety (for example, a maleimide, an aldehyde, amino, thiol, or ester group) to a reactive group on the neuromedin U receptor agonist (for example, an aldehyde, amino, thiol, a maleimide, or ester group).
The PEG molecule(s) may be covalently attached to any Lys, Cys, or
K(CO(CH2)2SH) residues at any position in the neuromedin U receptor agonist. The
neuromedin U receptor agonists described herein can be PEGylated directly to any amino acid at the N-terminus by way of the N-terminal amino group. A "linker arm" may be added to the neuromedin U receptor agonist to facilitate PEGylation. PEGylation at the thiol side-chain of cysteine has been widely reported (See, e.g., Caliceti & Veronese, Adv. Drug Deliv. Rev. 55: 1261-77 (2003)). If there is no cysteine residue in the peptide, a cysteine residue can be introduced through substitution or by adding a cysteine to the N-terminal amino acid. Those neuromedin U receptor agonists, which have been PEGylated, have been PEGylated through the side chains of a cysteine residue added to the N-terminal amino acid.
hi some aspects, the PEG molecule(s) may be covalently attached to an amide group in the C-terminus of the neuromedin U receptor agonist. In general, there is at least one PEG molecule covalently attached to the neuromedin U receptor agonist. In particular aspects, the PEG molecule is branched while in other aspects, the PEG molecule may be linear. In particular aspects, the PEG molecule is between 1 kDa and 100 kDa in molecular weight. In further aspects, the PEG molecule is selected from 10, 20, 30, 40, 50, 60, and 80 kDa. In further still aspects, it is selected from 20, 40, or 60 kDa. Where there are two PEG molecules covalently attached to the neuromedin U receptor agonist of the present invention, each is 1 to 40 kDa and in particular aspects, they have molecular weights of 20 and 20 kDa, 10 and 30 IcDa, 30 and 30 kDa, 20 and 40 kDa, or 40 and 40 kDa. In particular aspects, the neuromedin U receptor agonists contain mPEG-cysteine. The mPEG in mPEG-cysteine can have various molecular weights. The range of the molecular weight is preferably 5 kDa to 200 kDa, more preferably 5 kDa to 100 kDa, and further preferably 20 kDa to 60 kDA. The mPEG can be linear or branched.
Currently, it is preferable that the neuromedin U receptor agonists are PEGylated through the side chains of a cysteine added to the N-terminal amino acid. Currently, the agonists preferably contain mPEG-cysteine. The mPEG in mPEG-cysteine can have various molecular weights. The range of the molecular weight is preferably 5kDa to 20OkDa, more preferably 5kDa to 10OkDa, and further preferably 2OkDa to 6OkDA. The mPEG can be linear or branched.
A useful strategy for the PEGylation of synthetic neuromedin U receptor agonists consists of combining, through forming a conjugate linkage in solution, a peptide, and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The neuromedin U receptor agonists can be easily prepared with conventional solid phase synthesis. The neuromedin U receptor agonist is "preactivated" with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Conjugation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated neuromedin U receptor agonist can be easily purified by cation exchange chromatography or preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.
The sites of PEGylation on the neuromedin U receptor agonists of the present invention were chosen taking into account the structure of NMU and its interactions with the NMU receptors. Hence, the PEGylation is preferably site-specific. PEGylation at the N-terminal amino group of the peptide NMU is possible since this is the only available amino group in the sequence. For instance the N-terminal group of the peptide was acylated with a branched ((PEG)240kDa N-hydroxysuccinimide analog (for example, SUNBRIGHT GL2-400GS2, NOF Corporation).
The neuromedin U receptor agonist can comprise other non-sequence modifications, for example, glycosylation, lipidation, acetylation, phosphorylation,
carboxylation, methylation, or any other manipulation or modification, such as conjugation with a labeling component. While, in particular aspects, the neuromedin U receptor agonist herein utilize naturally-occurring amino acids or D isoforms of naturally occurring amino acids, substitutions with non-naturally occurring amino acids (for example., methionine sulfoxide, methionine methylsulfonium, norleucine, epsilon-aminocaproic acid, 4-aminobutanoic acid, tetrahydroisoquinoline-3-carboxylic acid, 8-aminocaprylic acid, 4 aminobutyric acid,
Lys(N(epsilon)-trifiuoroacetyl) or synthetic analogs, for example, o-aminoisobutyric acid, p or y- amino acids, and cyclic analogs. In further still aspects, the neuromedin U receptor agonists comprise a fusion protein that having a First moiety, which is a neuromedin U receptor agonist, and a second moiety, which is a heterologous peptide.
The neuromedin U receptor agonist may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified neuromedin U receptor agonist and/or having other desirable properties. A protecting group covalently joined to the C-terminal carboxy group reduces the reactivity of the carboxy terminus under in vivo conditions. For example, carboxylic acid groups of the peptide, whether carboxyl- terminal or side chain, may be provided in the form of a salt of a pharmacologically-acceptable cation or esterified to form a Cl -6 ester, or converted to an amide of formula NRR.2 wherein R and R2 are each independently H or C\-6 alkyl, or combined to form a heterocyclic ring, such as a 5 -or 6-membered ring. The carboxy terminus protecting group is preferably attached to the α- carbonyl group of the last amino acid. Carboxy terminus protecting groups include, but are not limited to, amide, methylamide, and ethylamide. Amino groups of the peptide, whether N- terminal or side chain, may be in the form of a pharmacologically-acceptable acid addition salt, such as the HCl, HBr5 acetic, benzoic, toluene sulfonic, maleic, tartaric, and other organic salts, or may be modified to Ci-g alkyl or dialkyl amino or further converted to an amide.
Hydroxyl groups of the neuromedin U receptor agonist side chain may be converted to Cχ-6 alkoxy or to a C\-β ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chain may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C 1-6 alkyl, Ci_6 alitoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the neuromedin U receptor agonist side chains can be extended to homologous C2-4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this invention to select and provide conformational constraints to the structure that result in enhanced stability. For example, a carboxyl-terminal or amino-terminal cysteine residue can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, thereby generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl-and amino-terminal amides and esters. Another method to provide conformational constraints to the structure that result in enhanced stability relies on the substitution of one or more amino acids with iV-alkyl-amino acids.
Polysaccharide polymers are another type of water soluble polymer that may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α 1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kDa to about 70 kDa. Dextran is a suitable water soluble polymer for use as a vehicle by itself or in combination with another vehicle {See, for example, WO96/11953 and WO96/05309). The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456. Dextran of about 1 kDa to about 20 kDa is preferred when dextran is used as a vehicle in accordance with the present invention,
As described above, the presence of a "linker" group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. However, in certain embodiments, the linker may itself provide improved properties to the compositions of the present invention. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, in particular embodiments, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. hi a further embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, linkers include are polyglycines (particularly (Gly)4, (Gly)s), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are (Gly)3Lys(Gly)4; (Gly)3AsnGlySer(Gly)2; (Gly)3Cys(Gly)4; and GlyProAsnGlyGly.
Non-peptide linkers can also be used. For example, alkyl linkers such as-NH- (CH2) s-C(O)-, wherein s = 2-20 could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (for example, Ci-g) lower acyl, halogen (for example, Cl, Br), CN, NH2, phenyl, and the like. An exemplary non-peptide linker is a PEG linker, wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be altered to form derivatives in the same manner as described above. Other linkers include Ttds (N-(13-amino-4,7,10-trioxa-tridecayl)-succinamic acid).
The present invention includes diastereomers as well as their racemic and resolved enantiomerically pure forms. The neuromedin U receptor agonists can contain D-amino acids, L-amino acids, or a combination thereof. In general, the amino acids are in the L-form with particular amino acids in D-form. As is known in the art, individual amino acids can be represented as follows: A=Ala=Alanine; C=Cys=Cysteine; D~Asp-Aspartic Acid;
E^Glu=Glutamic Acid; F^Phe^Phenylalanine; G=Gly=Glycine; H^His^Histidine;
I-Ile-Isoleucine;
Figure imgf000024_0001
N=Asn=Asparagine; P-Pro=Proline; Q=Gln=Glutamine; R=Arg=Arginine; S^Ser=Serine; T-Thr^Threonine;
V-Val-Valine; W=Trp=Tryptophan; and Y=Tyr=Tyrosine.
The neuromedinU receptor agonists can be linked, conjugated, or fused to a carrier molecule such as albumin, transferrin, or an antibody or antibody fragment such as the Fab or Fc fragment. For example, the neuromedin U receptor agonists can be linked via a linker moiety to a catalytic antibody such as disclosed in U.S. Pub. Application Nos. US20030175921 , US200301960676, and US20030129188, which describes linking of various peptides to the catalytic site of an aldolase catalytic antibody. The neuromedin U receptor agonists can be conjugated to an Fc fragment via linker moiety or fused to the Fc fragment of an antibody in the form of a fusion protein in a manner similar to the GLP-I-Fc fusion proteins disclosed in International Applications WO2002/046227 and WO2005/007809. The neuromedin U receptor agonist can be linked via a linker moiety to serum albumin, similar to the linking of GLP-I to albumin as disclosed in International application WO2000/069911 or U. S, Pub application US20070093417. The neuromedin U receptor agonist can fused to a carrier molecule such as transferrin, for example similar to the transferrin-GLP-1 fusion proteins disclosed in U.S. Patent No. 7,176,278 or albumin-GLP-1 fusion proteins described in U. S. Patent No. 7,141,547.
Conjugation of peptides to carrier proteins is known in the art and has been used to covalently link a wide variety of peptides to a carrier protein. For example, Poznonsky eϊ at., FEBS Letts 239: 18-22 (1998) conjugated human growth hormone to serum albumin. The resulting conjugate had reduced renal clearance and altered plasma clearance but retained its biological activity. Paige et al., Pharma. Res. 12: 1883-1888 (1995) describe conjugating GCSF to serum albumin using a bifunctional polyethylene glycol linker. The conjugate had reduced renal clearance and increased serum stability but retained its biological activity. Serum albumin comprising the present invention can be produced recombinantly and can include various modifications such as amino acid substitutions, deletions, or insertions, deglycosylation, particular predominant glycosylatiort structures, and can further include fusions to heterologous proteins, polypeptides, and peptides. International Published Application No. WO92/00763 describes coupling antigen- binding fragments of IgA or IgM to serum albumin and thereby restoring antigen affinity of the fragments to levels comparable to intact IgA or IgM. WO2004/081013 also discloses conjugating peptides and drags to IgG molecules.
U.S. Patent Nos. 6,593,295; 6329,336; 7,256,253; 6,849,714; and 6,849,714 describe covalently linking peptides to blood component proteins such as albumin or antibodies ex vivo and observing that the conjugates were resistant to protease digestion and thus had an extended half-life. The patents disclose that a wide variety of peptides, including neuromedins, can be conjugated to the blood components using reactive groups such as maleimido and succinimidyl reactive groups. U.S. Published Application No. 20070207952 describes covalently linking peptides and other macromolecules to carrier proteins such as albumin and in the case of anti-HIY antivirals, observing that the conjugates have superior pharmacological and, in particular, pharmacokinetic properties, and can have a prolong half-life in vivo.
International Published Application Nos. WO2006/107120, WO2004/047337, WO2004/04336, and WO2005/047334 disclose conjugating various drugs and peptides to Fc fragments. Fc fragments are produced when an immunoglobulin (Ig) molecule is digested with papain, and is a region of an immunoglobulin molecule except for the variable region (VL) and the constant regions (CL) of the light chain and the variable region (VH) and the constant region 1 (CHI) of the heavy chain. Fc fragments comprising the present invention can also be produced recombinantly and can further include modifications such as amino acid substitutions, deletions, or additions, PEGylation, deglycosylation, particular predominant glycosylation structures, and can further include fusions to heterologous proteins, polypeptides, and peptides.
As used herein, conjugation of the peptide to the carrier protein is effected by a covalent chemical linkage that excludes embodiments in which the peptide is covalently attached to the carrier protein by means of one or more amino acids in a peptide linkage, for example, a fusion protein. Thus, the present invention does not include fusion proteins comprising the NMU peptide and the carrier protein.
Further provided are pharmaceutical compositions comprising a therapeutically effective amount of one or more of the neuromedin U receptor agonists disclosed herein for the treatment of a metabolic disorder in an individual. Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, type II diabetes, complications of diabetes such as retinopathy, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers. The obesity-related disorders herein are associated with, caused by, or result from obesity.
"Obesity" is a condition in which there is an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), calculated as body weight per height in meters squared (kg/m.2). "Obesity" refers to a condition whereby an otherwise healthy subject has a Body Mass Index (BMI) greater than or equal to 30 kg/m2, or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg/m2. An "obese subject" is an otherwise healthy subject with a Body Mass Index (BMI) greater than or equal to 30 kg/m2 or a subject with at least one co-morbidity with a BMI greater than or equal to 27 kg/m.2. A "subject at risk for obesity" is an otherwise healthy subject with a BMI of 25 kg/m2 to less than 30 kg/m2 or a subject with at least one co-morbidity with a BMI of 25 kg/m2 to less than 27 kg/m2.
The increased risks associated with obesity occur at a lower Body Mass Index (BMI) in Asians. In Asian countries, including Japan, "obesity" refers to a condition whereby a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg/m2. In Asian countries, including Japan, an "obese subject" refers to a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m2. In Asian countries, a "subject at risk of obesity" is a subject with a BMI of greater than 23 kg/m2 to less than 25 kg/m2.
As used herein, the term "obesity" is meant to encompass all of the above definitions of obesity.
Obesity-induced or obesity-related co-morbidities include, but are not limited to, diabetes, non-insulin dependent diabetes mellitus - type 2, impaired glucose tolerance, impaired fasting glucose, insulin resistance syndrome, dyslipidemia, hypertension, hyperuricacidemia, gout, coronary artery disease, myocardial infarction, angina pectoris, sleep apnea syndrome, Pickwickian syndrome, fatty liver; cerebral infarction, cerebral thrombosis, transient ischemic attack, orthopedic disorders, arthritis deformans, lumbodynia, emmenlopathy, and infertility. In particular, co-morbidities include: hypertension, hyperlipidemia, dyslipidemia, glucose intolerance, cardiovascular disease, sleep apnea, diabetes mellitus, and other obesity-related conditions.
"Treatment" (of obesity and obesity-related disorders) refers to the administration of the compounds of the present invention to reduce or maintain the body weight of an obese subject One outcome of treatment may be reducing the body weight of an obese subject relative to that subject's body weight immediately before the administration of the compounds of the present invention. Another outcome of treatment may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of treatment may be decreasing the occurrence of and/or the severity of obesity-related diseases. The treatment may suitably result in a reduction in food or calorie intake by the subject, including a reduction in total food intake, or a reduction of intake of specific components of the diet such as carbohydrates or fats; and/or the inhibition of nutrient absorption; and/or the inhibition of the reduction of metabolic rate; and in weight reduction in patients in need thereof. The treatment may also result in an alteration of metabolic rate, such as an increase in metabolic rate, rather than or in addition to an inhibition of the reduction of metabolic rate; and/or in minimization of the metabolic resistance that normally results from weight loss.
"Prevention" (of obesity and obesity-related disorders) refers to the administration of the compounds of the present invention to reduce or maintain the body weight of a subject at risk of obesity. One outcome of prevention may be reducing the body weight of a subject at risk of obesity relative to that subject's body weight immediately before the administration of the compounds of the present invention. Another outcome of prevention may be preventing body weight regain of body weight previously lost as a result of diet, exercise, or pharmacotherapy. Another outcome of prevention may be preventing obesity from occurring if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another outcome of prevention may be decreasing the occurrence and/or severity of obesity-related disorders if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Moreover, if treatment is commenced in already obese subjects, such treatment may prevent the occurrence, progression or severity of obesity-related disorders, such as, but not limited to, arteriosclerosis, Type II diabetes, polycystic ovarian disease, cardiovascular diseases, osteoarthritis,
dermatological disorders, hypertension, insulin resistance, hypercholesterolemia,
hypertriglyceridemia, and cholelithiasis.
The obesity-related disorders herein are associated with, caused by, or result from obesity. Examples of obesity-related disorders include overeating and bulimia, hypertension, diabetes, elevated plasma insulin concentrations and insulin resistance, dyslipidemias, hyperlipidemia, endometrial, breast, prostate and colon cancer, osteoarthritis, obstructive sleep apnea, cholelithiasis, gallstones, heart disease, abnormal heart rhythms and arrythmias, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovarian disease, craniopharyngioma, the Prader-Willi Syndrome, Frohlich's syndrome, GH-deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, e.g, children with acute lymphoblastic leukemia. Further examples of obesity-related disorders are metabolic syndrome, also known as syndrome X, insulin resistance syndrome, sexual and reproductive dysfunction, such as infertility,
hypogonadism in males and hirsutism in females, gastrointestinal motility disorders, such as obesity-related gastro-esophageal reflux, respiratory disorders, such as obesity-hypoventilation syndrome (Pickwickian syndrome), cardiovascular disorders, inflammation, such as systemic inflammation of the vasculature, arteriosclerosis, hypercholesterolemia, hyperuricaemia, lower back pain, gallbladder disease, gout, and kidney cancer. The compounds of the present invention are also usefol for reducing the risk of secondary outcomes of obesity, such as reducing the risk of left ventricular hypertrophy.
The term "diabetes," as used herein, includes both insulin-dependent diabetes mellitus (IDDM, also known as type I diabetes) and non-insulin-dependent diabetes mellitus (NIDDM, also known as Type II diabetes). Type I diabetes, or insulin-dependent diabetes, is the result of an absolute deficiency of insulin, the hormone which regulates glucose utilization. Type II diabetes, or insulin-independent diabetes (i.e., non-insulin-dependent diabetes mellitus), often occurs in the face of normal, or even elevated levels of insulin and appears to be the result of the inability of tissues to respond appropriately to insulin. Most of the Type II diabetics are also obese. The compounds of the present invention are useful for treating both Type I and Type II diabetes. The compounds are especially effective for treating Type II diabetes. The compounds of the present invention are also useful for treating and/or preventing gestational diabetes mellitus.
The neuromedin U receptor agonists disclosed herein may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such compositions comprise a therapeutically-effective amount of the neuromedin U receptor agonist and a pharmaceutically acceptable carrier. Such a composition may also be comprised of (in addition to neuromedin U receptor agonist and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Compositions comprising the neuromedin U receptor agonists can be administered, if desired, in the form of salts provided the salts are pharmaceutically acceptable. Salts may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry.
The term "individual" is meant to include humans and companion or domesticated animals such as dogs, cats, horses, and the like. Therefore, the compositions comprising formula I are also useful for treating or preventing obesity and obesity-related disorders in cats and dogs. As such, the term "mammal" includes companion animals such as cats and dogs.
As used herein, the term "treating" includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromelhamine, and the like. The term "pharmaceutically acceptable salt" further includes all acceptable salts such as acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N- methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycollylarsanilale, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide, tannate,
hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, isothionate, triethiodide, lactate, panoate, valerate, and the like which can be used as a dosage form for modifying the solubility or hydrolysis characteristics or can be used in sustained release or pro-drug
formulations. It will be understood that, as used herein, references to the neuromedin U receptor agonists of the general formula (I) are meant to also include the pharmaceutically acceptable salts.
As utilized herein, the term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s), approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered and includes, but is not limited to such sterile liquids as water and oils. The characteristics of the carrier will depend on the route of administration. The neuromedin U receptor agonist may be in multimers (for example, heterodimers or homodimers) or complexes with itself or other peptides. As a result,
pharmaceutical compositions of the invention may comprise one or more neuromedin U receptor agonists in such multimeric or complexed form.
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or
amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially, or simultaneously. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. The term, "parenteral" means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous.
The pharmacological composition can comprise one or more neuromedin U receptor agonists; one or more neuromedin U receptor agonists and one or more other agents for treating a metabolic disorder; or the pharmacological composition comprising the one or more neuromedin U receptor agonists can be used concurrently with a pharmacological composition comprising an agent for treating a metabolic disorder. Such disorders include, but are not limited to, obesity, metabolic syndrome or syndrome X, type II diabetes, complications of diabetes, hypertension, dyslipidemias, cardiovascular disease, gallstones, osteoarthritis, and certain forms of cancers.
When the pharmacological composition comprises another agent for treating a metabolic disorder or the treatment includes a second pharmacological composition comprising an agent for treating a metabolic disorder, the agent includes, but is not limited to, insulin, analogs, and derivatives; cannabinoid (CBl) receptor antagonists; glucagon like peptide 1 (GLP- 1) receptor agonists; glucagon receptor agonists and antagonists; glucose-dependent
insulinotropic peptide (GIP) receptor agonists; lipase inhibitors; Ob receptor agonists (e.g., leptin and analogs thereof); FGF-21 and analogs thereof; amylin and analogs thereof; GPRl 19 receptor agonists; GPR40 agonists; GPRl 16 receptor agonists; serotonin 5-HT2C receptor agonists; melanocortin-4 receptor (MC4R) agonists; PP ARa receptor agonists; histamine H3 receptor antagonists; thyroid hormone receptor agonists; cholecystokinin (CCK) receptor agonists; α2- adrenergic receptor agonists; β 3 -adrenergic receptor agonists; PPARγ receptor agonists; agouti- related protein or analog; angiopoietin-like protein 6 (Angptlό) proteins, peptides, analogs, and derivatives; GPR105 (P2YR14) antagonists; tetrahydrolipstatin; 2-4-dinitrophenol; acarbose,; sibutramine; phentamine; fat absorption blockers; simvastatin; mevastatin; ezetimibe;
atorvastatin; sitagliptin; metformin; orlistat; Qnexa; topiramate; naltrexone; bupriopion;
phentermine; losartan; losartan with hydrochlorothiazide; and the like.
Examples of suitable agents of use in combination with a composition of the present invention or in a treatment in combination with a composition of the present invention, includes, but are not limited to:
(a) anti-diabetic agents such as (1) PPARγ agonists such as glitazones (e.g. ciglitazone; darglitazone; englitazone; isaglitazone (MCC-555); pioglitazone (ACTOS);
rosiglitazone (AVANDIA); troglitazone; rivoglitazone, BRL49653; CLX-0921 ; 5-BTZD, GW- 0207, LG- 1006415 R483, and LY-300512, and the like and compounds disclosed in WO97/10813, 97/27857, 97/28115, 97/28137, 97/27847, 03/000685, and 03/027112 and
SPPARMS (selective PPAR gamma modulators) such as Tl 31 (Amgen), FK614 (Fujisawa), netoglitazone, and metaglidasen; (2) biguanides such as buformm; metformin; and phenformin, and the like; (3) protein tyrosine phosphatase- IB (PTP-IB) inhibitors such as ISIS 113715, A- 401674, A-364504, IDD-3, IDD 2846, KP-40046, KR61639, MC52445, MC52453, Ql, OC- 060062, OC-86839, OC29796, TTP-277BC1, and those agents disclosed in WO 04/041799, 04/050646, 02/26707, 02/26743, 04/092146, 03/048140, 04/089918, 03/002569, 04/065387, 04/127570, and US 2004/167183; (4) sulfonylureas such as acetohexamide; chlorpropamide; diabinese; glibenclamide; glipizide; glyburide; glimepiride; gliclazide; glipentide; gliquidone; glisolamide; tolazamide; and tolbutamide, and the like; (5) meglitinides such as repaglinide, metiglinide (GLUFAST) and nateglinide, and the like; (6) alpha glucoside hydrolase inhibitors such as acarbose; adiposine; camiglibose; emiglitate; miglitol; voglibose; pradimicin-Q;
salbostatin; CKD-711; MDL-25,637; MDL-73,945; and MOR 14, and the like; (7) alpha-amylase inhibitors such as tendamistat, trestatin, and Al-3688, and the like; (8) insulin secreatagogues such as linogliride nateglinide, mitiglinide (GLUFAST), IDl 101 A-4166, and the like; (9) fatty acid oxidation inhibitors, such as clomoxir, and etomoxir, and the like; (10) A2 antagonists, such as mϊdaglizole; isaglidole; deriglidole; idazoxan; earoxan; and fluparoxan, and the like; (11) insulin or insulin mimetics, such as biota, LP-100, novarapid, insulin detemir, insulin lispro, insulin glargine, insulin zinc suspension (lente and ultralente); Lys-Pro insulin, GLP-I (17-36), GLP-I (73-7) (insulintropin); GLP-I (7-36)-NH2) exenatide/Exendin-4, Exenatide LAR, Linaglutide, AVEOOlO, CJC 1131, BIM51077, CS 872, THO318, BAY-694326, GPOlO, ALBUGON (GLP-I fused to albumin), HGX-007 (Epac agonist), S-23521, and compounds disclosed in WO 04/022004, WO 04/37859, and the like; (12) non-thiazolidinediones such as JT- 501 , and farglitazar (GW-2570/GI-262579), and the like; (13) PPARα/y dual agonists such as AVE 0847, CLX-0940, GW-1536, GW1929, GW-2433, KRP-297, L-796449, LBM 642, LR-90, LY510919, MK-0767, ONO 5129, SB 219994, TAK-559, TAK-654, 677954 (GlaxoSmithkline), E-3O3O (Eisai), LY510929 (Lilly), AKl 09 (Asahi), DRF2655 (Dr. Reddy), DRF8351 (Dr.
Reddy), MC3002 (Maxocore), TY51501 (ToaEiyo), naveglitazar, muraglitizar, peliglitazar, tesaglitazar (GALIDA), reglitazar (JTT-501 ), chiglitazar, and those disclosed in WO 99/16758, WO 99/19313, WO 99/20614, WO 99/38850, WO 00/23415, WO 00/23417, WO 00/23445, WO 00/50414, WO 01/00579, WO 01/79150, WO 02/062799, WO 03/033481, WO 03/033450, WO 03/033453; and (14) other insulin sensitizing drugs; (15) VPAC2 receptor agonists; (16) GLK modulators, such as PSNl 05, RO 281675, RO 274375 and those disclosed in WO 03/015774, WO 03/000262, WO 03/055482, WO 04/046139, WO 04/045614, WO 04/063179, WO
04/063194, WO 04/050645, and the like; (17) retinoid modulators such as those disclosed in WO 03/000249; (18) GSK 3beta/GSK 3 inhibitors such as 4-[2-(2-bromophenyl)-4-(4-fluorophenyl- lH-imidazol-5-yl]pyridine; CT21022, CT20026, CT-98023, SB-216763, SB410111, SB-675236, CP-70949, XD4241 and those compounds disclosed in WO 03/037869, 03/03877, 03/037891, 03/024447, 05/000192, 05/019218 and the like; (19) glycogen phosphorylase (HGLPa) inhibitors, such as AVE 5688, PSN 357, GPi-879, those disclosed in WO 03/037864, WO 03/091213, WO 04/092158, WO 05/013975, WO 05/013981, US 2004/0220229, and JP 2004- 196702, and the like; (20) ATP consumption promoters such as those disclosed in WO
03/007990; (21) fixed combinations of PPARγ agonists and metformin such as AVANDAMET; (22) PPAR pan agonists such as GSK 677954; (23) GPR40 (G-protein coupled receptor 40) also called SNORF 55 such as BG 700, and those disclosed in WO 04/041266, 04/022551,
03/099793 ; (24) GPRl 19 (also called RUP3 ; SNORF 25) such as RUP3 , HGPRBMY26, PFI 007, SNORF 25; (25) adenosine receptor 2B antagonists such as ATL-618, AT1-802, E3080, and the like; (26) carnitine palmitoyl transferase inhibitors such as ST 1327, and ST 1326, and the like; (27) Fructose 1 ,6-bisρhospohatase inhibitors such as CS-917, MB7803, and the like; (28) glucagon antagonists such as AT77077, BAY 694326, GW 4123X, NN2501, and those disclosed in WO 03/064404, WO 05/00781 , US 2004/0209928, US 2004/029943, and the like; (30) glucose-6-phosρhase inhibitors; (31) phosphoenolpyruvate carboxykinase (PEPCK) inhibitors; (32) pyruvate dehydrogenase kinase (PDK) activators; (33) RXR agonists such as MC 1036, CS00018, JNJ 10166806, and those disclosed in WO 04/089916, US 6759546, and the like; (34) SGLT inhibitors such as AVE 2268, KGT 1251, T1095/RWJ 394718; (35) BLX-1002;
(b) lipi d lowering agents such as (1) bile acid sequestrants such as, cholestyramine, colesevelem, colestipol, dialkylaminoalkyl derivatives of a cross-linked dextran; Colestid®; LoCholest®; and Questran®, and the like; (2) HMG-CoA reductase inhibitors such as atorvastatin, itavastatin, pitavastatin, fluvastatin, lovastatin, pravastatin, rivastatin,
rosuvastatin, simvastatin, rosuvastatin (ZD-4522), and the like, particularly simvastatin; (3) HMG-CoA synthase inhibitors; (4) cholesterol absorption inhibitors such as FMVP4 (Forbes Medi-Tech), KT6-971 (Kotobuki Pharmaceutical), FM-VAl 2 (Forbes Medi-Tech), FM-VP-24 (Forbes Medi-Tech), stanol esters, beta-sitosterol, sterol glycosides such as tiqueside; and azetidinones such as ezetimibe, and those disclosed in WO 04/005247 and the like; (5) acyl coenzyme A -cholesterol acyl transferase (ACAT) inhibitors such as avasimibe, eflucimibe, pactimibe (KY505), SMP 797 (Sumitomo), SM32504 (Sumitomo), and those disclosed in WO 03/091216, and the like; (6) CETP inhibitors such as JTT 705 (Japan Tobacco), torcetrapib, CP 532,632, BAY63-2149 (Bayer), SC 591, SC 795, and the like; (7) squalene synthetase inhibitors; (8) antioxidants such as probucol, and the like; (9) PP ARa agonists such as beclofibrate, benzafibrate, ciprofibrate, clofibrate, etofϊbrate, fenofϊbrate, gemcabene, and gemfibrozil, GW 7647, BM 170744 (Kowa), LY518674 (Lilly), GW590735 (Glaxo Smithkline), KRP-101
(Kyorin), DRF10945 (Dr. Reddy), NS-220/R1593 (Nippon Shinyaku/Roche, ST1929 (Sigma Tau) MC3001/MC3004 (MaxoCore Pharmaceuticals, gemcabene calcium, other fibric acid derivatives, such as Atromid®, Lopid®, and Tricor®, and those disclosed in US 6,548,538, and the like; (10) FXR receptor modulators such as GW 4064 (GlaxoSmithkline), SR 103912, QRX401, LN-6691 (Lion Bioscience), and those disclosed in WO 02/064125, WO 04/045511, and the like; (11) LXR receptor modulators such as GW 3965 (GlaxoSmithkline), T9013137, and XTCOl 79628 (X-Ceptor Therapeutics/Sanyo), and those disclosed in WO 03/031408, WO 03/063796, WO 04/072041, and the like; (12) lipoprotein synthesis inhibitors such as niacin; (13) renin angiotensin system inhibitors; (14) PPAR δ partial agonists, such as those disclosed in WO 03/024395; (15) bile acid reabsorption inhibitors, such as BARI 1453, SC435, PHA384640, S8921, AZD7706, and the like; and bile acid sequesterants such as colesevelam (WELCHOL/ CHOLESTAGEL), (16) PPARγ agonists such as GW 501516 (Ligand, GSK), GW 590735, GW- 0742 (GlaxoSmithkline), T659 (Amgen/Tularik), LY934 (Lilly), NNC610050 (Novo Nordisk) and those disclosed in WO97/28149, WO 01/79197, WO 02/14291, WO 02/46154, WO
02/46176, WO 02/076957, WO 03/016291, WO 03/033493, WO 03/035603, WO 03/072100, WO 03/097607, WO 04/005253, WO 04/007439, and JP10237049, and the like; (17) triglyceride synthesis inhibitors; (18) microsomal triglyceride transport (MTTP) inhibitors, such as implitapide, LAB687, JTT130 (Japan Tobacco), CP346086, and those disclosed in WO
03/072532, and the like; (19) transcription modulators; (20) squalene epoxidase inhibitors; (21) low density lipoprotein (LDL) receptor inducers; (22) platelet aggregation inhibitors; (23) 5-LO or FLAP inhibitors; and (24) niacin receptor agonists including HM74A receptor agonists; (25) PPAR modulators such as those disclosed in WO 01/25181, WO 01/79150, WO 02/79162, WO 02/081428, WO 03/016265, WO 03/033453; (26) niacin-bound chromium, as disclosed in WO 03/039535; (27) substituted acid derivatives disclosed in WO 03/040114; (28) infused HDL such as LUV/ETC-588 (Pfizer), APO-Al Milano/ETC216 (Pfizer), ETC-642 (Pfizer), ISIS301012, D4F (Bruin Pharma), synthetic trimeric ApoAl, Bioral Apo Al targeted to foam cells, and the like; (29) IBAT inhibitors such as BARI143/HMR145A/ HMR1453 (Sanofi-Aventis,
PHA384640E (Pfizer), S8921 (Shionogi) AZD7806 (AstrZeneca), AKl 05 (Asah Kasei), and the like; (30) Lp-PLA2 inhibitors such as SB480848 (GlaxoSmithldine), 659032 (GlaxoSmithkline), 6771 16 (GlaxoSmithkline), and the like; (31) other agents which affect lipic composition including ETC1001/ESP31015 (Pfizer), ESP-55016 (Pfizer), AGIl 067 (AtheroGenics), AC3056 (Amylin), AZD4619 (AstrZeneca);
(c) anti-hypertensive agents such as (1) diuretics, such as thiazides, including chlorthalidone, chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide, and hydrochlorothiazide; loop diuretics, such as bumetanide, ethacrynic acid, furosemide, and torsemide; potassium sparing agents, such as amiloride, and triamterene; and aldosterone antagonists, such as spironolactone, epirenone, and the like; (2) beta-adrenergic blockers such as acebutolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, carteolol, carvedilol, celiprolol, esrnolol, indenolol, metaprolol, nadolol, nebivolol, penbutolol, pindolol, propanolol, sotalol, tertatolol, tilisolol, and timolol, and the like; (3) calcium channel blockers such as amlodipirte, aranidipine, azelnidipme, baraidipine, benidipine, bepridil, cinaldipine, clevidipine, diltiazem, efonidipine, felodipine, gallopamil, isradipine, lacidipine, lemildipine, lercanidipine, nicardipine, nifedipine, mlvadipine, nimodepine, msoldipine, nitrendipine, manidipine, pranidipine, and verapamil, and the like; (4) angiotensin converting enzyme (ACE) inhibitors such as benazepril; captopril; cilazapril; delapril; enalapril; fosinopril; imidapril; losinopril; moexipril; quinapril; quinaprilat; ramipril; perindopril; perindropril; quanipril; spirapril;
tenocapril; trandolapril, and zofenopril, and the like; (5) neutral endopeptidase inhibitors such as omapatrilat, cadoxatril and ecadotril, fosidotril, sampatrilat, AVE7688, ER4030, and the like; (6) endothelin antagonists such as tezosentan, A308165, and YM62899, and the like; (7) vasodilators such as hydralazine, clonidine, minoxidil, and nicotinyl alcohol, and the like; (8) angiotensin II receptor antagonists such as candesartan, eprosartan, irbesartan, losartan, pratosartan, tasosartan, telmisartan, valsartan, and EXP-3137, FI6828K, and RNH6270, and the like; (9) α/β adrenergic blockers as nipradilol, arotinolol and amosulalol, and the like; (10) alpha 1 blockers, such as terazosin, urapidil, prazosin, bunazosin, trimazosin, doxazosin, naftopidil, mdoramin, WHIP 164, and XENOlO, and the like; (11) alpha 2 agonists such as lofexidine, tiamenidine, moxonidine, rilmenidine and guanobenz, and the like; (12) aldosterone inhibitors, and the like; (13) angiopoietin-2-binding agents such as those disclosed in WO 03/030833; and
(d) anti-obesity agents, such as (1) 5HT (serotonin) transporter inhibitors, such as paroxetine, fluoxetine, fenfluramine, fluvoxamine, sertraline, and imipramine, and those disclosed in WO 03/00663, as well as serotonin/noradrenaline re uptake inhibitors such as sibutramine (MERIDIA/REDUCTIL) and dopamine uptake inhibitor/Norepenephrine uptake inhibitors such as radafaxine hydrochloride, 353162 (GlaxoSmithMine), and the like; (2) NE (norepinephrine) transporter inhibitors, such as GW 320659, despiramine, talsupram, and nomifensine; (3) CBl (carmabinoid-1 receptor) antagonist/inverse agonists, such as rimonabant (ACCOMPLIA Sanofi Synthelabo), SR-147778 (Sanofi Synthelabo), AVE1625 (Sanofi- Aventis), BAY 65-2520 (Bayer), SLV 319 (Solvay), SLV326 (Solvay), CP945598 (Pfizer), E- 6776 (Esteve), 01691 (Organix), ORG14481 (Organon), VER24343 (Vernalis), NESS0327 (Univ of Sassari/Univ of Cagliari), and those disclosed in US Patent Nos. 4,973,587, 5,013,837, 5,081,122, 5,112,820, 5,292,736, 5,532,237, 5,624,941, 6,028,084, and 6,509367; and WO 96/33159, WO97/29079, WO98/31227, WO 98/33765, WO98/37061, WO98/41519,
WO98/43635, WO98/43636, WO99/02499, WO00/10967, WO00/10968, WO 01/09120, WO 01/58869, WO 01/64632, WO 01/64633, WO 01/64634, WO 01/70700, WO 01/96330, WO 02/076949, WO 03/006007, WO 03/007887, WO 03/020217, WO 03/026647, WO 03/026648, WO 03/027069, WO 03/027076, WO 03/027114, WO 03/037332, WO 03/040107, WO
04/096763, WO 04/111039, WO 04/111033, WO 04/111034, WO 04/111038, WO 04/013120, WO 05/000301, WO 05/016286, WO 05/066126 and EP-658546 and the like; (4) ghrelin agonists/antagonists, such as BVT81-97 (BioVitrum), RC 1291 (Rejuvenon), SRD-04677 (Sumitomo), unacylated ghrelin (TheraTechnologies), and those disclosed in WO 01/87335, WO 02/08250, WO 05/012331, and the like; (5) H3 (histamine H3) antagonist/inverse agonists, such as thioperamide, 3-(lH-imidazol-4-yl)propyl N-(4-pentenyl)carbamate), clobenpropit, iodophenpropit, imoproxifan, GT2394 (Gliatech), and A331440, and those disclosed in WO 02/15905; and O- [3 -(lH~imidazol-4-yl)propanoI] carbamates (Kiec-Kononowicz, K. et al., Pharmazie, 55:349-55 (2000)), piperidine-containing histamine H3-receptor antagonists
(Lazewska, D. et al, Pharmazie, 56:927-32 (2001), benzophenone derivatives and related compounds (Sasse, A. et al., Arch. Pharm.(Weinheim) 334:45-52 (2001)), substituted N- phenylcarbamates (Reidemeister, S. et al., Pharmazie, 55:83-6 (2000)), and proxifan derivatives (Sasse, A. et al., J. Med. Chera.. 43:3335-43 (2000)) and histamine H3 receptor modulators such as those disclosed in WO 03/024928 and WO 03/024929; (6) melanin-concentrating hormone 1 receptor (MCHlR) antagonists, such as T-226296 (Takeda), T71 (Takeda/Amgen), AMGN- 608450, AMGN-503796 (Amgen), 856464 (GlaxoSmithkline), A224940 (Abbott), A798 (Abbott), ATC0175/AR224349 (Arena Pharmaceuticals), GW803430 (GlaxoSmithkine), NBI- IA (Neurocrine Biosciences), NGX-I (Neurogen), SNP-7941 (Synaptic), SNAP9847 (Synaptic), T-226293 (Schering Plough), TPI- 1361-17 (Saitama Medical School/University of California Irvine), and those disclosed WO 01/21169, WO 01/82925, WO 01/87834, WO 02/051809, WO 02/06245, WO 02/076929, WO 02/076947, WO 02/04433, WO 02/51809, WO 02/083134, WO 02/094799, WO 03/004027, WO 03/13574, WO 03/15769, WO 03/028641, WO 03/035624, WO 03/033476, WO 03/033480, WO 04/004611, WO 04/004726, WO 04/011438, WO 04/028459, WO 04/034702, WO 04/039764, WO 04/052848, WO 04/087680; and Japanese Patent
Application Nos. JP 13226269, JP 1437059, JP2004315511, and the like; (7) MCH2R (melanin concentrating hormone 2R) agonist/antagonists; (8) NPYl (neuropeptide Y Yl) antagonists, such as BMS205749, BIBP3226, J-115814, BIBO 3304, LY-357897, CP-671906, and GI-264879A; and those disclosed in U.S. Patent No. 6,001,836; and WO 96/14307, WO 01/23387, WO 99/51600, WO 01/85690, WO 01/85098, WO 01/85173, and WO 01/89528; (9) NPY5
(neuropeptide Y Y5) antagonists, such as 152,804, S2367 (Shionogi), E-6999 (Esteve), GW- 56918OA, GW-594884A (GlaxoSmithklme), GW-587081X, GW-548118X; FR 235,208;
FR226928, FR 240662, FR252384; 1229U91, GI-264879A, CGP71683A, C-75 (Fasgen) LY- 377897, LY366377, PD-160170, SR-120562A, SR-120819A,S2367 (Shionogi), JCF-104, and H409/22; and those compounds disclosed in U.S. Patent Nos. 6,140,354, 6,191,160, 6,258,837, 6,313,298, 6,326,375, 6,329,395, 6,335,345, 6,337,332, 6,329,395, and 6,340,683 ; and EP- 01010691, EP-01044970, and FR252384; and PCT Publication Nos. WO 97/19682, WO
97/20820, WO 97/20821, WO 97/20822, WO 97/20823, WO 98/27063, WO 00/107409, WO 00/185714, WO 00/185730, WO 00/64880, WO 00/68197, WO 00/69849, WO 01/09120, WO 01/14376, WO 01/85714, WO 01/85730, WO 01/07409, WO 01/02379, WO 01/02379, WO 01/23388, WO 01/23389, WO 01/44201, WO 01/62737, WO 01/62738, WO 01/09120, WO 02/20488, WO 02/22592, WO 02/48152, WO 02/49648, WO 02/051806, WO 02/094789, WO 03/009845, WO 03/014083, WO 03/022849, WO 03/028726, WO 05/014592, WO 05/01493; and Norman et al., I Med. Chem. 43:4288-4312 (2000); (10) leptin, such as recombinant human leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl human leptin (Amgen); (11) leptin derivatives, such as those disclosed in Patent Nos. 5,552,524; 5,552,523; 5,552,522; 5,521,283; and WO 96/23513; WO 96/23514; WO 96/23515; WO 96/23516; WO 96/23517; WO 96/23518; WO 96/23519; and WO 96/23520; (12) opioid antagonists, such as nalmefene (Revex ®), 3-methoxynaltrexone, naloxone, and naltrexone; and those disclosed in WO
00/21509; (13) orexin antagonists, such as SB-334867-A (GlaxoSmithkline); and those disclosed in WO 01/96302, 01/68609, 02/44172, 02/51232, 02/51838, 02/089800, 02/090355, 03/023561, 03/032991, 03/037847, 04/004733, 04/026866, 04/041791, 04/085403, and the like; (14) BRS3 (bombesin receptor subtype 3) agonists; (15) CCK-A (cholecystokinin-A) agonists, such as AR- R 15849, GI 181771, JMV-180, A-71378, A-71623, PD170292, PD 149164, SR146131, SR125180, butabindide, and those disclosed in US 5,739,106; (16) CNTF (ciliary neurotrophic factors), such as GΪ-181771 (Gϊaxo-SmithKline); SR146131 (Sanofϊ Synthelabo); butabindide; and PD170,292, PD 149164 (Pfizer); (17) CNTF derivatives, such as axokine (Regeneron); and those disclosed in WO 94/09134, WO 98/22128, and WO 99/43813; (18) GHS (growth hormone secretagogue receptor) agonists, such as NN703, hexarelin, MK-0677, SM- 130686, CP- 424,391, L-692,429 and L- 163,255, and those disclosed in U.S. Patent No. 6358951, U.S. Patent Application Nos. 2002/049196 and 2002/022637; and WO 01/56592, and WO 02/32888; (19) 5HT2c (serotonin receptor 2c) agonists, such as APD3546/AR10A (Arena Pharmaceuticals), ATH88651 (Athersys), ATH88740 (Athersys), BVT933 (Biovitrum/GSK), DPCA37215 (BMS), IK264; LY448100 (Lilly), PNU 22394; WAY 470 (Wyeth), WAY629 (Wyeth), WAY161503 (Biovitrum), R-1065, VR1065 (Vernalis/Roche) YM 348; and those disclosed in U.S. Patent No. 3,914,250; and PCT Publications 01/66548, 02/36596, 02/48124, 02/10169, 02/44152;
02/51844, 02/40456, 02/40457, 03/057698, 05/000849, and the like; (20) Mc3r (melanocortin 3 receptor) agonists; (21) Mc4r (melanocortin 4 receptor) agonists, such as CHIR86036 (Chiron), CHIR915 (Chiron); ME-10142 (Melacure), ME-10145 (Melacure), HS-131 (Melacure),
NBI72432 (Neurocrine Biosciences), NNC 70-619 (Novo Nordisk), TTP2435 (Transtech)and those disclosed in PCT Publications WO 99/64002, 00/74679, 01/991752, 01/0125192,
01/52880, 01/74844, 01/70708, 01/70337, 01/91752, 01/010842, 02/059095, 02/059107, 02/059108, 02/059117, 02/062766, 02/069095, 02/12166, 02/11715, 02/12178, 02/15909, 02/38544, 02/068387, 02/068388, 02/067869, 02/081430, 03/06604, 03/007949, 03/009847, 03/009850, 03/013509, 03/031410, 03/094918, 04/028453, 04/048345, 04/050610, 04/075823, 04/083208, 04/089951, 05/000339, and EP 1460069, and US 2005049269, and JP2005042839, and the like; (22) monoamine reuptake inhibitors, such as sibutratmine (Meridia ®/Reductil®) and salts thereof, and those compounds disclosed in U.S. Patent Nos. 4,746,680, 4,806,570, and 5,436,272, and U.S. Patent Publication No. 2002/0006964, and WO 01/27068, and WO
01/62341; (23) serotonin reuptake inhibitors, such as dexfenfluramine, fluoxetine, and those in U.S. Patent No. 6,365,633, and WO 01/27060, and WO 01/162341; (24) GLP-I (glucagon-like peptide 1) agonists; (25) Topiramate (Topimax®); (26) phytopharm compound 57 (CP 644,673); (27) ACC2 (acetyl-CoA carboxylase-2) inhibitors; (28) β3 (beta adrenergic receptor 3) agonists, such as rafebergron/AD9677/TAK677 (Dainippon/ Takeda), CL-316,243, SB 418790, BRL- 37344, L-796568, BMS-196085, BRL-35135A, CGP12177A, BTA-243, GRC1087 (Glenmark Pharmaceuticals) GW 427353 (solabegron hydrochloride), Trecadrine, Zeneca D7114, N-5984 (Nisshin Kyorin), LY-377604 (Lilly), KT07924 (Kissei), SR 59119A5 and those disclosed in US Patent Nos. 5,705,515, US 5,451,677; and WO94/18161, WO95/29159, WO97/46556,
WO98/04526 WO98/32753, WO 01/74782, WO 02/32897, WO 03/014113, WO 03/016276, WO 03/016307, WO 03/024948, WO 03/024953, WO 03/037881, WO 04/108674, and the like; (29) DGATl (diacylglycerol acyltransferase 1) inhibitors; (30) DGAT2 (diacylglycerol acyltransferase 2)inhibitors; (31) FAS (fatty acid synthase) inhibitors, such as Cerulenin and C75; (32) PDE (phosphodiesterase) inhibitors, such as theophylline, pentoxifylline, zaprinast, sildenafil, amrinone, milrinone, cilostamide, rolipram, and cilomilast, as well as those described in WO 03/037432, WO 03/037899; (33) thyroid hormone β agonists, such as KB-2611
(KaroBioBMS), and those disclosed in WO 02/15845; and Japanese Patent Application No. JP 2000256190; (34) UCP-I (uncoupling protein 1), 2, or 3 activators, such as phytanic acid, 4-[(E)- 2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-l-propenyl]benzoic acid (TTNPB), and retinoic acid; and those disclosed in WO 99/00123; (35) acyl-estrogens, such as oleoyl-estrone, disclosed in del Mar-Grasa, M. et al., Obesity Research, 9:202-9 (2001); (36) glucocorticoid receptor antagonists, such as CP472555 (Pfizer), KB 3305, and those disclosed in WO
04/000869, WO 04/075864, and the like; (37) 1 1 β HSD-I (11-beta hydroxy steroid
dehydrogenase type 1) inhibitors, such as BVT 3498 (AMG 331), BVT 2733, 3-(l-adamantyl)-4- ethyl-5-(ethylthio)-4H-l,2,4-triazole, 3-(l-adamantyl)-5-(3,4,5-trimethoxyphenyl)-4-methyl-4H- 1,2,4-triazoIe, S-adamantanyM^^J^^lOJ ^π^a-decahydro-l^-triazolo^- a][l l]annulene, and those compounds disclosed in WO 01/90091, 01/90090, 01/90092,
02/072084, 04/01 1410, 04/033427, 04/041264, 04/027047, 04/056744, 04/065351, 04/089415, 04/037251, and the like; (38) SCD-I (stearoyl-CoA desaturase-1) inhibitors; (39) dipeptidyl peptidase IV (DPP-4) inhibitors, such as isoleucine thiazolidide, valine pyrrolidide, sitagliptin, saxagliptin, NVP-DPP728, LAF237 (vildagliptin), P93/01, TSL 225, TMC-2A/2B/2C, FE 999011, P9310/K364, VIP 0177, SDZ 274-444, GSK 823093, E 3024, SYR 322, TS021, SSR 162369, GRC 8200, K579, NN7201, CR 14023, PHX 1004, PHX 1149, PT-630, SK-0403; and the compounds disclosed in WO 02/083128, WO 02/062764, WO 02/14271, WO 03/000180, WO 03/000181, WO 03/000250, WO 03/002530, WO 03/002531, WO 03/002553, WO 03/002593, WO 03/004498, WO 03/004496, WO 03/005766, WO 03/017936, WO 03/024942, WO 03/024965, WO 03/033524, WO 03/055881, WO 03/057144, WO 03/037327, WO
04/041795, WO 04/071454, WO 04/0214870, WO 04/041273, WO 04/041820, WO 04/050658, WO 04/046106, WO 04/067509, WO 04/048532, WO 04/099185, WO 04/108730, WO
05/009956, WO 04/09806, WO 05/023762, US 2005/043292, and EP 1 258 476; (40) lipase inhibitors, such as tetrahydrolipstatin (orlistat/XENICAL), ATL962 (Alizyme/Takeda),
GT389255 (Genzyme/Peptimmune)Triton WR1339, RHC80267, lipstatin, teasaponin, and diethylυmbelliferyl phosphate, FL-386, WAY- 121898, Bay-N-3176, valilactone, esteracin, ebelactone A, ebelactone B, and RHC 80267, and those disclosed in WO 01/77094, WO
04/111004, and U.S. Patent Nos. 4,598,089, 4,452,813, 5,512,565, 5,391,571, 5,602,151,
4,405,644, 4,189,438, and 4,242,453, and the like; (41) fatty acid transporter inhibitors; (42) dicarboxylate transporter inhibitors; (43) glucose transporter inhibitors; and (44) phosphate transporter inhibitors; (45) anorectic bicyclic compounds such as 1426 (Aventis) and 1954 (Aventis), and the compounds disclosed in WO 00/18749, WO 01/32638, WO 01/62746, WO 01/62747, and WO 03/015769; (46) peptide YY and PYY agonists such as PYY336
(Nastech/Merck), AC 162352 (IC Innovations/Curis/Amylin), TM3O335/TM3O338 (7TM
Pharma), PYY336 (Emisphere Tehcnologies), PEGylated peptide YY3-36, those disclosed in WO 03/026591, 04/089279, and the like; (47) lipid metabolism modulators such as maslinic acid, erythrodiol, ursolic acid uvaol, betulinic acid, betulin, and the like and compounds disclosed in WO 03/011267; (48) transcription factor modulators such as those disclosed in WO 03/026576; (49) McSr (melanocortin 5 receptor) modulators, such as those disclosed in WO 97/19952, WO 00/15826, WO 00/15790, US 20030092041, and the like; (50) Brain derived neutotropic factor (BDNF), (51) McIr (melanocortin 1 receptor modulators such as LK- 184 (Proctor & Gamble), and the like; (52) 5HT6 antagonists such as BVT74316 (BioVitrum), BVT5182c (BioVitrum), E-6795 (Esteve), E-6814 (Esteve), SB399885 (GlaxoSmithkline),
SB271046 (GlaxoSmithkline), RO-046790 (Roche), and the like; (53) fatty acid transport protein 4 (FATP4); (54) acetyl-CoA carboxylase (ACC) inhibitors such as CP640186, CP610431, CP640188 (Pfizer); (55) C-terminal growth hormone fragments such as AOD9604 (Monash Univ/Metabolic Pharmaceuticals), and the like; (56) oxyntomodulin; (57) neuropeptide FF receptor antagonists such as those disclosed in WO 04/083218, and the like; (58) amylin agonists such as Symlin/pramlintide/AC137 (Amylin); (59) Hoodia and trichocaulon extracts; (60) BVT74713 and other gut lipid appetite suppressants; (61) dopamine agonists such as bupropion (WELLBUTRIN/GlaxoSmithkline); (62) zonisamide (ZONEGRAN/Dainippon/Elan), and the like.
Examples of specific compounds that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention further include specific CBl antagonists/inverse agonists include those described in WO03/077847, including: N-[3-(4-chlorophenyl)-2(5)-phenyl- 1 (5)-methylpropyl]-2-(4- trifluoromethyl-2-pyrimidyloxy)-2-methylpropaiiamide, JV- [3 -(4-chlorophenyl)-2-(3- cyanophenyl)-l-methylpropyl]-2-(5-trifluoromethyl-2-pyridyloxy)-2-methylpropanamideJ JV-[3- (4-chlorophenyl)-2-(5 -chloro- 3 -pyridyl)- 1 -methylpropyl] -2-(5-trifluoromethyl-2-pyridyloxy)-2- methylpropanamide, and pharmaceutically acceptable salts thereof; as well as those in
WO05/00Θ809, which includes the following: 3-{ l-[bis(4-chloroρhenyl)methyl]azetidin-3- ylidene}-3~(3 ,5-difluorophenyl)-2,2-dimethylpropanenitrileJ 1 -{ 1 -[ 1 ~(4- chlorophenyl)pentyl]azetidin-3-yl} - 1 -(3,5-difluorophenyl)-2-methylpropan-2-ol. 3-((S)-(4- chlorophenyl) (3-[(1S)-I -(3 ,5-difluorophenyl)-2-hydroxy-2-methylρroρyl] azetidin- 1 - yl}methyl)benzonitrile? 3-((S)-(4-chlorophenyl){3-[(lS)-l-(355-difluorophenyl)-2-fluoro-2- methylpropyl] azetidin- 1 -yl} methyl)benzonitrile, 3-((4-chlorophenyl) { 3 - [ 1 -(3 ,5-difiuorophenyl)- 2,2-dimethylpropyl] azetidin- 1 -yl } methyl)benzonitriles 3 -(( 1 S)- 1 - { 1 - [(-S)-(3 -cyanophenyl)(4- cyanophenyl)methyI]azetidin-3-yl}-2-fluoro-2-methylpropyl)-5-fluorobenzonitrile, 3-[(S)-(4- chlorophenyl)(3-{(l S)-2-fluoro-l-[3-fluoro-5-(4H-l,2?4-triazol-4-yl)phenyl]-2- methylpropyl} azetidin- l-yl)methyl]benzonitrile, and 5-((4-chlorophenyI){3-[(l S)-I -(3,5- difluorophenyl)-2-iluoro-2-methylpropyl]azetidin- 1 -yl } methyl)thiophene-3 -carbonitrile, and pharamecueitcally acceptable salts thereof; as well as: 3-[(S)-(4-chlorophenyiχ3-{(15)-2-fluoro- 1 - [3 -fluoro-5-(5-oxo-4,5-dihydro- 1 ,3 ,4-oxadiazol-2-yl)phenyl] -2-methylpropyl } azetidin- 1 - yl)methyl] benzonitrile, 3 -[(5)-(4-chlorophenyl)(3 - { ( 1 S)-2-fluoro- 1 - [3 -fluoro-5-( 1 ,3 ,4-oxadiazol- 2-yl)phenyl]-2-methylpropyl}azetidin-l-yl)me%l]benzoiiitrile? 3-[(^-(3-{(15)-l-[3-(5-amino- 153s4-oxadiazol-2-yl)-5-fluorophenyl]-2-fluoro-2-methylpropyl} azetidin- 1 -yl)(4- chlorophenyl)methyl]ben2onitrile, 3 - [(5)-(4-cyanophenyl)(3 - { ( 15)-2-fluoro- 1 - [3 -fluoro-5 -(5 -oxo- 4,5 -dihydro- 1 ?3 ,4-oxadiazol-2-yl)phenyl] -2-methylpropyl } azetidin- 1 -yl)methyl]benzonitrile, 3 - [(S)-(3- { ( 1 S)- 1 - [3 -(5 -amino- 1 , 3 ,4-oxadiazol-2-yl>5-fluorophenyl] -2-fluoro-2-methylpropyl } azetidin-l-yl)(4-cyanophenyl)methyl]benzonitrile; 3-[(JS)-(4-cyanophenyl)(3-{(lsS)-2-fiuoro-l-[3- fluoro-5-(l ,354-oxadiazol-2-yl)phenyl]-2-methylpropyl}azetidin-l -yl)methyl]benzonitrUe, 3-[(5)- (4-chlorophenyl)(3- {(15)-2-fluoro-l -[3 -fluoro-5 -(1 ,2,4-oxadiazol-3-yl)phenyl]-2- methylpropyl } azetidin- 1 -yl)methyl]benzomtrile3 3 -[( 1 S)- 1 -( 1 - { (S)-(4-cyanophenyl) [3 -( 1 ,2,4- oxadiazol-3-yl)phenyl]-methyl}azetidin-3-yl)-2-fluoro-2-methylpropyl]-5-fluorobenzomtrile, 5- (3- { 1 -[I -(diphenylmethyl)azetidin-3-yl]-2-fluoro-2-methylpropyl} -5 -fluorophenyl)- lH-tetrazole, 5-(3-{ l-[l-(diphenyImethyl)azetJdin-3-yI]-2-fluoro-2-methylpropyl}-5-fluorophenyl)-l-methyl- 1 H-tetrazole, 5-(3-{ l-[l-(d iphenylmethyl)azetidin-3 -yl] -2-fluoro-2-methy lpropyl } -5- fluorophenyl)-2-methyl-2H-tetrazole; 3 - [(4-chlorophenyl)(3 - { 2-fluoro- 1 - [3 -fϊuoro-5-(2-methyl- 2H4etrazol-5-yl)phenyI]-2-methylpropyl}azetidui-l-yl)methyl]benzonitrile, 3-[(4- chlorophenyl)(3- { 2-fluoro- 1 - [3 -fluoro- 5 -( 1 -methyl- 1 #-tetrazol-5-yl)ρheny 1] -2- methylpropyl} azetidin- 1 ~yl)methyl] benzonitrile, 3-[(4-cyanophenyl)(3-{2-fluoro-l-[3-fluoro-5- ( 1 -methyl- 1 /f-tetrazol-5 -yl)phenyl] -2-methylpropyl } azetidin- 1 -yl)methyl] benzonitrile, 3 - [(4- cyanophenyl)(3-{2-fluoro-l-[3-fluoro-5-(2-melhyl-2H-tetrazol-5-yl)phenyl]-2- methylpropyl } azetidin- 1 -yl)methyl] benzonitrile, 5- { 3 -[(5)- { 3- [( 1 S)- 1 -(3 -bromo- 5 -fluorophenyl)- 2-fluoro-2-methylpropyl] azetidin- 1 -yl } (4-chlorophenyl)methyl] phenyl } - 1 ,3 ,4-oxadiazol-2 (3H)- one, 3-[(1S)- 1-(1 - {(S)-(4-chlorophenyl)[3-(5-oxo-4,5-dihydro- 1 ,3,4-oxadiazol-2- yl)phenyl]methyl} azetidin-3 -yl)-2-fiuoro-2-methylpropyl] -5 -fluorobenzonitrile, 3-[(1S)-I-(I- {(S)-(4-cyanophenyl)[3-(5-oxo-4,5-dihydro-l ,354-oxadiazol-2-yl)phenyl]methyl } azetidin-3 -yl)- 2-fluoro-2-methylpropyl] -5-fluorobenzonitrile, 3 -[( 1 S)- 1 -( 1 - { (S)-(4-cyanophenyl)[3 -( 1 ,3 ,4- oxadiazol-2-yl)phenyl] methyl } azetidin-3 -yl)-2-fJuoro-2-methyipropyl] - 5-fluorobenzonitrile, 3 - [( 1 S)- 1 -( 1 - { (S)-(4-chlorophenyl) [3-( 1 ,3 ,4-oxadiazol-2-yl)phenyl] methyl} azetidin-3 -yl)-2-fluoro- 2-methylρroρyl]-5-fluorobenzonitrile5 3-((lS)-l-{ l-[(S)-[3-(5-ammo-l,3,4-oxadiazol-2- yl)phenyl] (4-chloroρhenyl)melhyl]azetidin-3 -yl } -2-fluoro-2-methylpropyl)-5 -fluorobenzonitπle, 3-((1S)-I - { 1 -[(S)-[3-(5-amino-l ,3,4~oxadiazol-2-yl)ρhenyl](4-cyanophenyl)methyl]azetidin-3- yl}-2-fluoro-2-methylpropyl)-5-lluorobenzonitrile5 3-[(1S)-I -(I -{(S)-(4-cyanophenyl)[3-(l ,2,4- oxadiazol-3 -yl)phenyl] methyl } azetidin- 3 -yl)-2-fluoro-2-methylρropyl] -5-fluorobenzonitriles 3 - [( 1 S)- 1 -( 1 - { (S)-(4-chlorophenyl) [3-( 1 ,2,4-oxadiazol-3 -yl)ρhenyl] methyl } azetidin-3 -yl)~2-fluoro- 2-methylpropyl]-5-fluorobenzonitrile, 5 -[3-((S)-(4-chloroρhenyl) { 3 - [( 1 S)- 1 -(3 ,5-difluorophenyl)- 2-fluoro-2-methylpropyl] azetidin- l-yl}methyl)phenyl]~l, 354-oxadiazol-2(3i/)-one;> 5-[3-((S)-(4- chlorophenyl) {3-[(lS)-l-(3,5 -difluorophenyl)-2-fluoro-2-methylpropyl] azetidin- 1 - yl}methyl)phenyl]-l,3,4-oxadiazol-2(3H)-one! 4-{(S)-{3-[(lS)-l-(355-difluorophenyl)-2-fluoro- 2~methylpropyl]azetidin-l -yl} [3-(5-oxo-4,5-dihydro-l,3J4-oxadiazol-2-yl)phenylJmethyl}- benzonitrile, ACOMPLIA (rimonabant, N-(l-piperidinyl)-5-(4-chlorophenyl)-l-(2j4- dichlorophenyl)-4-methylpyrazole-3-carboxamide, SR.141716A), 3-(4-chlorophenyl-N'-(4- chlorophenyl)sulfonyl-N-methyl-4-phenyl-4,5-dihydro-lH-ρyrazole-l-carboxamide (SLV-319), taranabant, N-[(l S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-l -methylρropyl]-2-methyl-2-[[5- (trifluoromethyl)-2-pyridinyl]oxy]propanamide, and pharmaceutically acceptable salts thereof- Specific NPY5 antagonists that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: 3-oxo-N-(5-phenyl-2-pyrazinyl)-spiro[isobenzofuran-l (3H),4'-piperidine]- 1'- carboxamide, 3 -oxo-N-(7-trifiuoromethylpyrido [3 ,2-b]pyridin-2-yl)sρiro- [isobenzofuran- l(3H),4'~piperidine]~l '-carboxamide, N-[5-(3-fluorophenyl)-2-pyrimidinyl]-3-oxospiro- [isobenzofuran- 1 (3H),4'-piperidine]~ 1 '-carboxamide, trans-3 '-oxo-N-(5-phenyl-2- pyrimidinyl)spiro [cyclohexane- 1 , 1 ' (3 ' H)-isobenzofuran] -4-carboxamϊde , trans-3 ' -oxo-N- [ 1 -(3 - quinolyl)-4-imidazolyl] spiro [cyclohexane- 1 , 1' (3 ' H)-isobenzofuran] -4-carboxamide, trans-3-oxo- N-(5-phenyl-2-pyrazinyl)spiro [4-azaiso-benzofuran- 1 (3H), 1 ' -cyclohexane] -4' -carboxamide, trans-N- [5-(3-fluoroρhenyl)-2-pyrimidinyl] -3-oxospiro [5 -azaisobenzofuran- 1 (3H), 1 ' - cyclohexane] -4 ' -carboxamide, trans-N-[5-(2-fluoroρhenyl)-2-pyrimidinyl] -3 -oxospiro [5- azaisobenzofuran- 1 (3 H) , 1 ' -cyclohexane] -4 ' -carboxamide, trans-N- [ 1 - (3 , 5 -difluorophenyl)-4- imidazolyl]-3-oxospiro [7~azaisobenzofuran- 1 (3 H), 1 ' -cyclohexane] -4 ' -carboxamide„ trans-3 -oxo- N-(I -phenyl-4-pyrazolyl)spiro[4-azaisobenzofυran-l (3H)51 '-cyclohexane] -4 '-carboxamide, trans-N- [ 1 -(2-fluorophenyl)-3 -pyrazolyl] - 3 -oxospiro [6-azaisobenzofuran- 1 (3 H) , 1 ' -cyclohexane] - 4'-carboxamide, trans-3 -oxo-N-(l-phenyl-3-pyrazolyl)spIro[6-azaisobenzofbran-l (3H)5F- cyclohexane] -4' -carboxamide, trans-3-oxo-N-(2-phenyl- 1 ,2,3-triazol-4-yl)spiro[6- azaisobenzofuxan- 1(3 H), I' -cyclohexane] -4 '-carboxamide? and pharmaceutically acceptable salts and esters thereof.
Specific ACC- 1/2 inhibitors that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: r-[(4J8-dimethoxyquinolin-2-yl)carbonyl]-6-(l/i-tetrazol-5-yl)spiro[chroman-2J41- ρiperidin]-4-one; (5- { 1 '-[(4,8-dimethoxyquinolin-2-yl)carbonyl]-4-oxospiro[chroman-2,41- piperidin]-6-yl}-2H-tetrazol-2-yl)methyl pivalate; 5-{ 1 '-[(8-cycIopropyl-4-methoxyquinolin-2- yl)carbonyl]-4-oxospiro[chroman-2,4'-piperidin]-6-yl}nicotinic acid; 1 '-(8-methoxy-4- moφholin-4-yl-2-naphthoyl)-6-(l/-/1-tetrazol-5-yl)spiro[chroman-254I-piperidin3-4-one; and 1 '- [(4-ethoxy-8-ethylquinolin-2-yl)carbonyl3-6-(lH-tetrazol-5-yl)spiro[chroman-2,4'-piperidin]-4- one; and pharmaceutically acceptable salts and esters thereof. MK-3887, L-001738791.
Specific MCHlR antagonist compounds that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: 1 -{4-[(l -ethylazetidin-3-yl)oxy]phenyl} -4-[(4-fluorobenzyI)oxy]pyridin- 2( 1 H)-one, 4- [(4-fluorobenzyl)oxy] - 1 - {4- [( 1 -isopropylazetidin-3-yl)oxy]phenyl} pyridin-2( 1 H)- one, l-[4-(azetidin-3-yloxy)phenylJ-4-[(5-chloropyridin-2-yl)methoxy]pyridin-2(lH)-one, 4-[(5- chloroρyridin-2-yl)methoxy] - 1 - {4-[( 1 -ethylazetidin-3-yl)oxy]phenyl } pyridin-2( 1 H)-one, 4- [(5- chloroρyridin-2-yl)methoxy] - 1 - {4-[( 1 -propyIazetidin-3-yl)oxy]phenyl } pyridin-2( 1 HJ-one, and A- [(5 -chloropyr idin-2-yl)methoxy] - 1 -(4- { [(25)- 1 -ethylazetidϊn-2-yl] methoxy } phenyl)pyridin- 2(lH)-one, or a pharmaceutically acceptable salt thereof.
A specific DP-IV inhibitor that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention is 7- [(3R)-3-amino-4-(2J4,5-trifluorophenyl)butanoyl]-3-(trifiuoromethyl)-5,6,7,8-tetrahydro4f2>4- triazolo[4,3-a]ρyrazine, or a pharmaceutically acceptable salt thereof.
Specific Η3 (histamine H3) antagonists/inverse agonists that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: those described in WO05/077905, including:3-{4- [( 1 -cyclobutyl-4-piperidinyl)oxy] phenyl } -2-ethylpyrido [2, 3 -d] -pyrimidin-4(3H)-one, 3- { 4-[( 1 - cyclobutyl-4-piperidinyl)oxy] phenyl }-2-methylpyrido [4,3 -d]pyrimidin-4(3H)-one, 2-ethyl-3-(4- {3-[(3S)-3-methylpiperidin-l-yl]propoxy}phenyl)pyrido[2,3-d]pyrimidin-4(3H)-one 2-methyl-3- (4- {3-[(3 S)-3-methylpiperidin- 1 -yl]propoxy}phenyl)pyrido[4,3-d]pyrimidin-4(3H)-one, 3-{4- [( 1 -cyclobutyl-4-piperidinyl)oxy]phenyl } -2, 5 -dimethyl-4(3 H)-quinazolinone, 3 - {4- [( 1 - cyclobutyl-4-piperidinyl)oxy]phenyl } -2~methyl-5-trifIuorornethyl-4(3 H)-quinazolinone, 3 - {4- [(I ~cyclobutyl-4-piperidinyl)oxy]phenyl} -5-methoxy-2-methyl-4(3H)-quinazoIinone? 3- {4-{(l - cyclobutylpiperidin-4-yl)oxy]phenyl } -5-fluoro-2-methyl-4(3H)-quinazolmone, 3- {4-[( 1 - cyclobutylpiperidin-4-yl)oxy]phenyl}-7-fluoro-2-methyl-4(3H)-quinazolinone, 3-{4-[(l- cyclobutylpiperidin-4-yl )oxy] phenyl } -6-methoxy-2-methyl-4(3 H)-quinazolinone5 3-{4-[(l- cyclobutylpiperidin-4-yl)oxy]ρhenyl}-6-fluoro-2-methyl-4(3H)-quinazolinone, 3-{4-[(l- cyclobutylpiperidin-4-yl)oxy]ρhenyl}-8-fluoro-2-methyl-4(3H)-quinazolinone;, 3- {4-[(l - cyclopentyl-4-piperidinyl)oxy]phenyl } -2-methylpyrido [4,3 -d]pyrimi din-4(3 H)-one, 3 - {4- [( 1 - cydobutylpiperidin-4-yl)oxy]phenyl } -6-fluoro-2-methy lpyrido [3 ,4-d] pyrimidin-4(3 H)-one, 3 - { 4- [(l-cyclobutyl-4-piperidinyl)oxy]phenyl}-2-ethylpyrido[4,3-d]pyrimidin-4(3H)-one, 6-methoxy~ 2-raethy 1-3- { 4- [3 -( 1 -piperidinyl)ρropoxy]phenyl } pyrido [3 ?4-d]pyrimidin-4(3 H)-one, 6-methoxy- 2-methyl-3-{4-[3-(l-pyrrolidinyl)propoxy]pheByl}pyrido[3f4~d]pyrimidin-4(3H)-one, 2,5- dimethyl-3-{4-[3-(l-pyrrolidinyl)ρropoxy]ρhenyl}-4(3H)-quinazolinone, 2-methyl-3-{4-[3-(l- pyiτoIidinyI)propoxy]phenyl}-5-trifluoromethyl-4(3H)-quinazolinone, 5-fluoro-2-methyl-3-{4- [3-(l -piperidinyl)propoxy]phenyl}-4(3H)~quinazolinone, 6-methoxy-2-methyl-3- {4-[3-(l - piperidinyl)propoxy]phenyl}-4(3H)-quinazoHnone5 5-methoxy-2-methyl-3-(4-{3-[(3S)-3- methylpiperidin- 1 -yl] propoxy } phenyl)-4(3 H)-quinazolinone, 7-methoxy-2-methyl-3 -(4- {3 - [(3 S)- 3 -methylpiperidin- 1 -yl]propoxy } phenyl)-4(3 H)-quinazolinone, 2-methyl-3 -(4- { 3 - [(3 S)-3 - methylpiperidin-l-yl]propoxy}phenyl)ρyrido[2J3-d]pyrimidin-4(3H)-one> 5-fluoro-2-methyl-3- (4- { 3 - [(2R)-2-methylpyrrolidin- 1 -yl] propoxy } phenyl)-4(3H)-quinazolinone, 2-methyl-3-(4- { 3 - [(2R)-2-methylpyrrolidin- 1 -yl]propoxy } ρhenyl)pyrϊdo [4.3-d]pyrimidin-4(3H)-one, 6-methoxy-2- methyl-3-(4-{3-[(2R)-2-methylpyrrolidin- 1 -yl]propoxy}phenyl)-4(3H)-quinazolinones 6- methoxy-2-methyl-3-(4~{3-[(2S)-2-methylpyrrolidin-l-yl]propoxy}phenyl)-4(3H)-quinazolinone! and pharmaceutically acceptable salts thereof.
Specific CCKlR agonists that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: 3 -(4- { [ 1 -(3 -ethoxyphenyl)-2-(4-methylphenyl)- 1 H -imidazol-4-yl] carbonyl } - 1 - piperazinyl)- 1 -naphthoic acid; 3 -(4- { [ 1 -(3-ethoxyphenyl)-2-(2-fluoro-4-methylρhenyl)- 1 H - imidazol-4-yl]carbonyl } - 1 -piperazinyl)- 1 -naphthoic acid; 3-(4- { [ 1 -(3 ~ethoxyρhenyl)-2-(4- fluorophenyl)- 1 H -imidazol-4-yl]carbonyl } - 1 -piperazinyl)- 1 -naphthoic acid; 3 -(4- { [ 1 -(3 - ethoxyphenyl)-2-(2,4-difluorophenyl)- 1 H -imidazol-4-yl] carbonyl } - 1 -piperazinyl)- 1 -naphthoic acid; and 3 -(4- { [ 1 -(2 , 3 -dihydro- 1 ,4-benzodioxin-6-yl)-2-(4-£luorophenyl)- 1 //-imidazol-4- yl]carbonyl}-l -piperazinyl)- 1 -naphthoic acid; and pharmaceutically acceptable salts thereof. MK-8406
Specific MC4R agonists that can be used in combination with a composition of the present invention or in a treatment that includes a composition of the present invention include: l) (5JS)-l'~{[(3JR,4^)-l-fer?-butyl-3-(2,3,4-trifluorophenyl)piperidin-4-yl]carbonyl}-3- chloro-2-methyI-5-[l-methyl-l-(l-methyl-lH-l,254-triazol-5-yl)ethyl]-5H-spiro[faro[3,4- &]pyridine-7,4'-piperidine]; 2) (5i?)-l'-{[(3i?>4J?)-l-fert-butyl-3-(253,4-trifluorophenyl)-piperidin- 4-yl]carbonyl}-3-chloro-2-methyl-5-[l-methyl-l-(l-methyl-lH-l,2s4-triazol-5-yl)ethyl]-5H- spiro[furo[3,4-6]pyridine-7s4'-piperidine]; 3) 2-(lI-{[(3-?>4/.)-l-ferr-butyl-4-(2,4- difluorophenyl)pyπOlidin-3-yl]carbonyl}-3-chloro-2-methyl-5H-spiro[furo[3,4-ό]pyridine-7,4'- piperidin]-5-yl)-2-methylpropanenitrile; 4) l'-{[(35'J4JR)-l-^rt-butyl-4-(2?4- difluorophenyl)pyrrolidin-3-yl] carbonyl } -3-chloro-2-methyl-5- [ 1 -methyl- 1 -( 1 -methyl- IH-I f2,4- triazol-5-yl)ethyl]-5H-spiro[furo[3s4-6]pyridine-754'-piperidine]; 5) N-[(3i?!4Jff)-3-({3-chloro-2- methyl-5-[ 1-methyl-l -(I -methyl- IH- 15254-triazoI-5-yl)ethyl]-l 'H55H-spiro[furo-[3 ,4-έ]pyridine- 7,4'-piperidin] - 1 '-yl } carbonyl)-4-(2,4-difluorophenyl)-cyclopentyl] -jV-methyltetraliydro-2H- pyran-4-amine; 6) 2-[3-chloro-l<-({(liϊ,2i?)-2-(2J4-diflιιorophenyl)-4-[methyl(tetraliydro-2H- pyran-4-yl)amino] -cyclopentyl } -carbonyl)-2-methyl-5H-spiro [furo [3 ,4-&]pyridine-7,4!- piperidin]~5-yl]-2-methyl-propane-nitrile; and pharmaceutically acceptable salts thereof.
Methods of administrating the pharmacological compositions comprising the one or more neuromedin U receptor agonists to an individual include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (for example, oral mucosa, rectal and intestinal mucosa, and the like), ocular, and the like and can be administered together with other biologically-active agents. Administration can be systemic or local, hi addition, it may be advantageous to administer the composition into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter attached to a reservoir (for example, an Ommaya reservoir). Pulmonary administration may also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. It may also be desirable to administer the one or more neuromedin U receptor agonists locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant.
Various delivery systems are known and can be used to administer the
neuromedin U receptor agonists including, but not limited to, encapsulation in liposomes, microparticles, microcapsules; minicells; polymers; capsules; tablets; and the like. In one embodiment, the neuromedin U receptor agonist may be delivered in a vesicle, in particular a liposome. In a liposome, the neuromedin U receptor agonist is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,837,028 and U.S. Patent No. 4,737,323. In yet another embodiment, the neuromedin U receptor agonist can be delivered in a controlled release system including, but not limited to: a delivery pump (See, for example, Saudek, et ah, New Engl. J. Med. 321 : 574 (1989) and a semi-permeable polymeric material (See, for example, Howard, etai, J. Neurosurg. 71: 105 (1989)). Additionally, the controlled release system can be placed in proximity of the therapeutic target (for example, the brain), thus requiring only a fraction of the systemic dose. See, for example, Goodson, In: Medical Applications of Controlled Release, 1984. (CRC Press, Bocca Raton, FIa.).
The amount of the compositions comprising the neuromedin U receptor agonist which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques by those of average skill within the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the attending physician will decide the amount of the composition with which to treat each individual patient. Initially, the attending physician will administer low doses of the composition and observe the patient's response. Larger doses of the composition may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further, hi general, the daily dose range lies within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases. However, suitable dosage ranges for intravenous administration of the compositions comprising the neuromedin U receptor agonist are generally about 5-500 micrograms (μg) of active compound per kilogram (Kg) body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient. Ultimately the attending physician will decide on the appropriate duration of therapy using compositions comprising the neuromedin U receptor agonist of the present invention. Dosage will also vary according to the age, weight and response of the individual patient.
Further provided is a pharmaceutical pack or kit, comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions and neuromedin U receptor agonists. Optionally associated with such containers) may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
The following examples are intended to promote a further understanding of the present invention.
EXAMPLE 1
This example shows the synthesis of NMU receptor Agonists.
The neuromedin U receptor agonists (See Table 1) were synthesized by solid phase using Fmoc/tBu chemistry on a peptide synthesizer ABI433A (Applied Biosystems). For each peptide 0.75 g of a resin aminomethylated polystirene LL (100-200 mesh, 0.41 mmol/g) (Novabiochem) resin derivatized with a modified Rink linker p- [(R5S)-Ot- [9H-Fluoren-9-yl- methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid (Rink, Tetrahedron Lett.
28:3787-3789 (1987); Bernatowicz et al., Tetrahedron Lett. 30:4645-4667 (1989)) was used. The acylation reactions were performed for 60 minutes with four-fold excess of activated amino acid over the resin free amino groups. The amino acids were activated with equimoϊar amounts of HBTU (2-(lH-benzotriazole-l-yl)-l,lf3?3-tetramethyluronium hexafluorophosphate) and a 2- fold molar excess of DIEA (N,N-diisopropylethylamine) in DMF.
Alternatively, the peptides were synthesized by solid phase using Fmoc/t-Bu chemistry on a peptide multisynthesizer Simphony (Protein Technologies Inc.) or APEX396 (AAPTEC). For each peptide, 0.5 g of a resin aminomethylated polystirene LL (100-200 mesh, 0.41 mmol/g) (Novabiochem) resin derivatized with a modified Rink linker p-[(R,S)-α-[9H- Fluoren-9-yl-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid (Rink, Tetrahedron Lett. 28:3787-3789 (1987); Bernatowicz et al., Tetrahedron Lett. 30:4645-4667 (1989)) was used. All the amino acids were dissolved at a 0.5 M concentration in a solution of 0.5 M HOBt (Hydroxybenzotriazole) in DMF. The acylation reactions were performed for 60 minutes with 5- fold excess of activated amino acid over the resin free amino groups. The amino acids were activated with equimolar amounts of HBTU (2-(lH-benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluorophosphate), solution 0.5 M in DMF, and a 2-fold molar excess of DIEA (N,N-diisoproρylethylamine) solution 2 M in NMP.
The side chain protecting groups were: tert-butyl for Asp, GIu, (D)GIu, GIa, hGlu, CbF, Ser and Tyr; trityl for Asn, Cys and GIn; 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl for Arg and Harg; the alpha-carboxyl protecting group of gamma-Glu was tert-butyl The N-terminal acetylation reaction was performed at the end of the peptide assembly by reaction with a 10-fold excess of acetic anhydride in DMF. The N-terminal palmltoylation reaction (for NMU34, 38, 39 and 40) was performed at the end of the peptide assembly by reaction with a four-fold excess of activated palmitic acid over the resin free amino groups. The palmitic acici was activated with equimolar amounts of DIPC (1,3-Dϊisopropylcarbodiimide) and HOBt (Hydroxybertzotriazole) in DMF.
At the end of the syntheses, the dry peptide-resins were individually treated with 20 raL of the cleavage mixture, 88% TFA, 5% phenol, 2% triisopropylsilane and 5% water (Sole and Barany, J. Org. Chem. 57: 5399-5403 (1992)) for 2.5 hours at room temperature. Each resin was filtered and the solution was added to cold methyl-t-butyl ether in order to precipitate the peptide. After centrifugation, the peptide pellets were washed with fresh cold methyl-t-butyl ether to remove the organic scavengers. The process was repeated twice. Final pellets were dried, resuspended in H20, 20% acetonitrile, and lyophilized.
The crude peptides were purified by reverse-phase HPLC using Waters RCM Delta-PakTM C4 or C18 cartridges (40 x 200 mm, 15 μm) or ReproSil-Pur C4 (50 x 150 mm, 10 μm) ( Dr. Maisch GmbH) or Phenomenex Proteo C12 (50 x 150 mm, 10 μm) and using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, flow rate 80 mL/min, or Waters X- Bridge C^ (19 x 150 mm, 10 μm), flow rate 30 mL/min. Analytical HPLC was performed on a Phenomenex Jupiter C4 column (150 x 4.6 mm, 5 μm) or ReproSil-Pur 300 C4 column (150 x 4.6 mm, 5 μm) ( Dr. Maisch GmbH) or Beckman Ultrasphere Cl g column (250 x 4.6 mm, 5 μm), flow rate one mL/min, using H2O, 0.1% TFA (A) and CH3CN, 0.1% TFA (B) as solvents. Alternatively, the peptides were characterized on an Alliance Waters Chromatograph, with a ACE C-4 (300 A), 3 μm column, 150 x 4.6 mm, (CPS analitica p/n ACE-213-1546), at 450C. The purified peptides were characterized by electrospray mass spectrometry on a Micromass LCZ platform.
The synthesis of peptide NMUlOl was performed by dissolving the thiol containing NMU peptide precursor in sodium phosphate 0.2M , urea 8M, EDTA 4mM, pH 6.5. A three molar excess of N-ethylmaleimide was added. After one hour incubation, the peptide was purified by HPLC.
PEGylation of Neuromedin U (NMlJ) analogs
PEGylation reactions were run under conditions permitting amide bond (NMU58, 108, 1 19, 134, 137 and 139) or thioether bond formation (NMU51, 52, 53, 54, 80, 81, 33, 100). The PEGylated NMU peptides were then isolated using cation exchange chromatography (FXC) followed by size exclusion chromatography (SEC) or reverse-phase (RP) HPLC. Cation exchange chromatography (IXC) was carried out on MacroCap SP (GE Healthcare) column (26 x 120 mm) with a linear gradient of NaCl (0-0.6M) in 3.5 column volumes in formic acid 0.05%, flow rate loading one mL/min, gradient elution 6 mL/min. Size exclusion chromatography
(SEC) was carried out on TSK-HW50 (Tosoh) column (21 x 700 mm) in acetic acid 0.1% (w/v), 30% acetonitrile, flow rate one mL/min. RP-HPLC was performed using a semi-preparative Waters RCM Delta-Pale™ C4 cartridge (40 x 100 mm, 15 μm) and using as eluents (A) 0.2% acetic acid in water and (B) 0.2% acetic acid in acetonitrile, and the following gradient: 5%-5% B (in five minutes) - 50% B (in 10 minutes) - 80% B (in two minutes), flow rate 80 mL/min. PEGylated NMU analogs were characterized using RP-HPLC and MALDI-Tof Mass
Spectrometry.
NMU58, NMUl 08, NMUl 19, NMU 134, NMUl 37 and NMUl 39 peptides were synthesized from the NMU peptide precursors to produce derivatives with PEG covalently attached via an amide bond. Synthesis of N MU 58
10 mg of peptide precursor (3.2 μmoles) were dissolved at 6.7 mg/mL concentration in 0.2 M HEPES5 pH 7.3. Then 413 mg of SUNBRIGHT GL2-400GS2 (NOF Corporation) (9.6 μmoles) dissolved at 100 mg/mL concentration in water (1:3 mole/mole ratio of peptide to PEG) were added to this solution. After 14 hours incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange
chromatography (IXC). The IXC purified PEGylated-peptide was further purified by size exclusion chromatography (SEC) and characterized by RP-HPLC and MALDI-Tof.
Synthesis of NMUl 08
10.1 mg of peptide precursor (5.4 μmoles) were dissolved at 10 mg/mL concentration in 8M urea, 0.2 M HEPES, pH 7.3. Then 464 mg of SUNBRIGHT GL2-400GS2 (NOF Corporation) (10.8 μmoles) dissolved at 100 mg/mL concentration in water (1:2 mole/mole ratio of peptide to PEG) were added to this solution. After 2 hours incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange chromatography (IXC). The IXC purified PEGylated-peptide was further purified by RP-HPLC and characterized by RP-HPLC and MALDI-Tof.
Synthesis of NMUl 19
6.46 mg of peptide precursor (2.7 μmoles) were dissolved at 10 mg/mL concentration in 8M urea, 0.2 M HEPES, pH 7.3. Then 230 mg of SUNBRIGHT GL2-400GS2 (NOF Corporation) (5.4 μmoles) dissolved at 100 mg/mL concentration in water (1:2 mole/mole ratio of peptide to PEG) were added to this solution. After 4 hours incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange
chromatography (IXC). The IXC purified PEGylated-peptide was further purified by RP-HPLC and characterized by RP-HPLC and MALDI-Tof.
Synthesis of NMUl 34 20 mg of peptide precursor (2.9 μmoles) were dissolved at 10 mg/mL concentration in 8M urea, 0.2 M HEPES5 pH 7.3. Then 810 mg of SUNBRIGHT ME-400HS (NOF Corporation) (18.9 μmoles) dissolved at 100 mg/mL concentration in water (1:3.5 mole/mole ratio of peptide to PEG) were added to this solution. After 4 hours incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange chromatography (IXC). The IXC purified PEGylated-peptide was further purified by size exclusion chromatography (SEC) and characterized by RP-HPLC and MALDI-Tof.
Synthesis ofNMU137
10 mg of peptide precursor (3.3 μmoles) were dissolved at 10 mg/mL concentration in 8M urea, 0.2 M HEPES, pH 7.3. Then 283 mg of SUNBRJGHT ME-400HS (NOF Corporation) (6.6 μmoles) dissolved at 100 mg/mL concentration in water (1:2 mole/mole ratio of peptide to PEG) were added to this solution. After 14 hours incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange
chromatography (IXC). The IXC purified PEGylated-peptide was further purified by size exclusion chromatography (SEC) and characterized by RP-HPLC and MALDI-Tof.
Synthesis ofNMU139
11.1 mg of peptide precursor (3.6 μmoles) were dissolved at 10 mg/mL concentration in 0.2 M HEPES, pH 7.3. Then 472 mg of SUNBRIGHT ME-400HS (NOF
Corporation) (11 μmoles) dissolved at 100 mg/mL concentration in water (1 :3 mole/mole ratio of peptide to PEG) were added to this solution. After 14 hours incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange chromatography (IXC).
The IXC purified PEGylated-peptide was further purified by size exclusion chromatography (SEC) and characterized by RP-HPLC and MALDI-Tof.
NMU51, NMU52, NMU53, NMU54, NMU80, NMU81, NMU33 and NMUlOO peptides were synthesized from the thiol-containing NMU peptide precursors to produce derivatives with PEG covalently attached via a thioether bond. Synthesis ofNMUSl, NMU52, NMU53 and NMU54
10 mg of peptide precursor (3.2 μmoles) were dissolved at 10 mg/mL concentration in 0.15 M sodium phosphate, pH 6.5, urea 8M5 4 mM EDTA. Then 137 mg of SUNBRIGHT GL2-400MA (NOF Corp.) (3.2 μmoles) dissolved at 100 mg/mL concentration in water (1:1 mole/mole ratio of peptide to PEG) were added to this solution. After one hour incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange chromatography (IXC). The IXC purified PEGylated-peptide was further purified by RP-HPLC and characterized by RP-HPLC and MALDI-Tof. Synthesis ofNMU80
10 mg of peptide precursor (3.2 μmoles) were dissolved at 10 mg/mL concentration in 0.15 M sodium phosphate, pH 6.5, urea 8M, 4 rnM EDTA. Then 120 mg of 4OkDa methoxy poly(ethylene glycol)maleimido-propionamide (Chirotech, Product Code 008- 016) (3.2 μmoles) dissolved at 100 mg/mL concentration in water (1 :1 mole/mole ratio of peptide to PEG) were added to this solution. After one hour incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange chromatography (FXC). The IXC purified PEGylated-peptide was further purified by RP-HPLC and characterized by RP- HPLC and MALDI-Tof.
Synthesis ofNMUSl
10 mg of peptide precursor (3.2 μmoles) were dissolved at 20 mg/mL concentration in 0.2 M TRIS, pH 8, urea 8M, 4 roM EDTA. Then 200 mg of SUNBRIGHT ME- 400IA (NOF Corporation) (4.8 μmoles) dissolved at 100 mg/mL concentration in water (1 :1*5 mole/mole ratio of peptide to PEG) were added to this solution. After one hour incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange chromatography (IXC). The IXC purified PEGylated-peptide was further purified by RP-HPLC and characterized by RP-HPLC and MALDI-Tof.
Synthesis ofNMU33
40 mg of peptide precursor (12.8 μmoles) were dissolved in 2.0 mL of 0.1 M sodium phosphate, urea 8M, 4 mM EDTA5 pH 7.0 Then 7 mg of SUNBRIGHT ME-050MA (NOF Corp.) (14 μmoles) dissolved at 140 mg/mL concentration in water (1 :1.1 mole/mole ratio of peptide to PEG) were added to this solution. After one hour incubation, the PEGylated peptide solution was acidified to 1% formic acid and purified by cation exchange
chromatography (IXC). The IXC purified PEGylated-peptide was further purified by size exclusion chromatography (SEC) and characterized by RP-HPLC and MALDI-Tof. Synthesis of NMUJ00
10 mg of peptide precursor (2.6 μmoles) were dissolved in 3.5 mL of 0.15 M sodium phosphate, pH 6.5, urea 8M, 4 mM EDTA. Then 111 mg of SUNBRIGHT GL2-400MA (NOF Corp.) (2.6 μmoles) dissolved at 100 mg/mL concentration in water (1 :1 mole/mole ratio of peptide to PEG) were added to this solution. After one hour incubation, the PEGylated peptide solution was acidified to 1 % formic acid and purified by cation exchange
chromatography (IXC). The IXC purified PEGylated-peptide was further purified by RP-HPLC and characterized by RP-HPLC and MALDI-Tof. Cholesteroylation of Neuromedin U (NMU) analogs
Derivatizations with cholesterol were run under conditions permitting thioether bond formation. The cholesteroylated neuromedin U receptor agonists were then purified by RP- HPLC and characterized by electrospray mass spectrometry.
Synthesis ofNMU36
40 mg of peptide precursor (12 μmoles) were dissolved at 10 mg/mL concentration in DMSO. Then 15 mg of cholest-5-en-3-yl l-bromo-2~oxo-6,9,12,15-tetraoxa-3- azaoctadecan- 18-o ate (bromoacetyl-oxa^-cholesterol) (18 μmoles), dissolved at 20 mg/mL concentration in THF (1:1.5 mole/mole ratio of peptide to bromoacetyl-Oxa4-cholesterol), and 47.5 μL of DIEA (N,N-diisopropylethylamine) (1% by volume) were added to this solution. After one hour incubation, the cholesteroylated peptide was purified by reverse-phase HPLC on a Waters RCM Delta-Pak™ C4 cartridge (40 x 100 mm, 15 μm), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and the following gradient: 55%-55% B (in five minutes) - 75% B (in 20 minutes) - 80% B (in two minutes), flow rate 80 mL/min. The purified product was characterized by RP-HPLC and electrospray mass spectrometry.
Synthesis ofNMU37
40 mg of peptide precursor (12 μmoles) were dissolved at 10 mg/mL concentration in DMSO. Then 15 mg of cholest~5-en-3-yl N-[43-(2,5-dioxo-2,5-dihyάro-lH- pyrrol-1 -yl)-41 -oxo-4,7, 10,13,16.19,22,25,28,31 ,34,37-dodecaoxa-40-azatritetracontan- 1 - oyl]glycinate (maleimide-oxa^-cholesterol) (13 μmoles), dissolved at 20 mg/mL concentration in THF (1:1.1 mole/mole ratio of peptide to maleimide-oxa^-cholesterol), and 47.5 μL of DIEA (N,N-diisoproρylethylamine) (1% by volume) were added to this solution. After one hour incubation, the cholesteroylated peptide was purified by reverse-phase HPLC on a Waters RCM Delta-Pak™ C4 cartridge (40 x 100 mm, 15 μm), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and the following gradient: 45%-45% B (in five minutes) - 65% B (in 20 minutes) - 80% B (in two minutes), flow rate 80 mL/min. The purified product was characterized by RP-HPLC and electrospray mass spectrometry.
Synthesis ofNMU44
30 mg of peptide precursor (23 μmoles) were dissolved at 10 mg/mL concentration in DMSO. Then 15 mg of cholest-5-en-3-yl N-[43-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)-41-oxo-4,7510,13, 16,19,22,25,28,3 l,34,37-dodecaoxa-40-azatritetracontan-l- oyljglycinate (maleimide-oxa^-cholesterol) (25 μmoles), dissolved at 20 mg/mL concentration in THF (1 :1.1 mole/mole ratio of peptide to maleimide-oxaπ-cholesterol), and 40 μL of DIEA (N,N-diisopropylethylamine) (1% by volume) were added to this solution. After one hour incubation, the cholesteroylated peptide was purified by reverse-phase HPLC on Waters RCM Delta-Pak™ C4 cartridges (25 x 200 mm, 15 μm), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and the following gradient: 70%~70% B (in five minutes) - 90% B (in 20 minutes) - 90% B (in two minutes), flow rate 30 mL/min. The purified product was characterized by RP-HPLC and electrospray mass spectrometry.
EXAMPLE 2
This example shows the in vitro activity of NMU analogs on NMU receptors. The NMU receptors signal primarily through Gαq/j { proteins; therefore FLIPR, a calcium mobilization assay, was used to measure functional activity using ceil lines expressing the human and mouse NMU receptors.
FLIPR assay. Stable cell lines expressing human and or rodent NMURl or human NMUR2 receptors were plated at a density of 12,000 cells per well overnight on poly- lysine coated 384- well black- walled plates. The following day, the media was removed from the plates and the cells were subsequently loaded with Fluo-3 (Molecular Probes), a calcium sensitive dye, diluted in FLIPR buffer (IX Hank's buffered saline containing 20 mM HEPES5 0.1% BSA, 2.5 mM probenecid (Sigma) and 1.6 mM TR40). All reagents are from Invitrogen unless otherwise noted. Peptide solutions were resuspended in saline at a stock concentration of 2 mM and diluted in FLIPR buffer on the day of the experiment to a 4 μM working stock solution. After a 90 minute incubation at room-temperature, cell plates were loaded onto a FLIPR (Molecular Devices) to monitor cellular fluorescence (excitation = 488 nM; emission = 540 nM) before and after compound/peptide addition. Eight to twelve point dose responses were tested on NMUR-expressing cell lines using FLIPR with lμM peptide as the highest dose. The response after peptide addition was taken as the maximum fluorescence units minus the fluorescence immediately prior to stimulation for each well. EC50 values were calculated using GraphPad Prism (San Diego, CA) software.
In vitro responses of human NMU and NMU peptide analogs in the FLIPR assay for human NMURl and NMUR2 are presented (Table 3 and Table 4). EC50S are reported in nM values. Table 5 shows the EC50S for mouse NMU receptors. Percent activity refers to the maximum response at 1 μM compared to the hNMU response at the same concentration.
Modification of the N terminus of NMU with PEGylation, cholesteroylation, or palmitoylation results in a rightward shift in the in vitro potency. Modification of P3 residue in the NMU8 sequence showed trends to a NMURl -selective peptide since there was little or no activity at the NMUR2 receptor (NMU8-P3- 11 through NMU8-P3-47).
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000053_0002
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000055_0002
EXAMPLE 3
This examples shows in vivo activity of the NMU analogs using feeding studies.
NMURl knockout (Nmurl-/-) mice were generated using standard homologous recombination techniques. Nmurl mice were subsequently transferred to Taconic Farms where they were either maintained on a 75% C57BL/6 x 25% 129S6/SvEv mixed genetic background or backcrossed ten generations to C57BL/6. NMUR2 knockout (Nmur2-/-) mice were licensed from Deltagen Inc., San Mateo, CA and subsequently transferred to Taconic Farms where they were either maintained on a 75% C57BL/6 x 25% 129/OlaHsd mixed genetic background or backcrossed for ten generations to C57BL/6. Mice were individually housed in Tecniplast cages in a conventional SPF facility. Mice were initially maintained on a regular chow diet and then early in their life were switched to a high fat diet (D 12492: 60 % kcal from fat; Research Diets, Inc., New Brunswick, NJ) with ad libitum access to water in a 12-hour light/12-hour dark cycle.
Ad libitum fed male diet-induced obese mice were weighed and dosed either i.p. or s.c. about 30 minutes prior to the onset of the dark phase of the light cycle and provided with a preweighed aliquot of high fat diet D 12492 which was then weighed 2 hours and 18 hours (day 1), 42 hours (day 2), 66 hours (day 3), and 90 hours (day 4) after the onset of the initial dark phase. Mice were weighed at the 18, 42, 66, and 90 hour time points. Data showed the outcome of the feeding study (all values are reported as mean ± SEM and data was analyzed using a two- tailed unpaired Student's t test; p values < 0.05 were reported as significant and are denoted with an asterisk).
Figures IA and IB show that acute peripheral administration of palmitoylated NMU analogs, significantly reduced food intake in diet-induced obese mice for two days post dose. Figure 2 illustrates the finding that the anorectic effects of NMU38, a palmitoylated NMU analog, are mediated by the contribution of both the NMURl and NMUR2 receptors. Acute administration of NMU38 was highly efficacious in wild-type animals but the anorectic effect was diminished in both Nmurl- and Nmur2-deficient animals on day 1 post dose. The effects on food intake were completely gone on day 2 post dose in the Nmurl -deficient mice. The data indicate that NMU38 evokes reductions in food intake on day 2 primarily through the NMURl receptor. Figure 3 illustrate the finding that cholesteroylated NMU analogs can reduce food intake for up to two days following a single administration. Figure 2 illustrates the finding that the anorectic effects of NMU37, a cholesleroylated NMU analog, are mediated by the contribution of both the NMURl and NMUR2 receptors. Acute administration of NMU38 was highly efficacious in wild-type animals but the anorectic effect was diminished in both Nmurl - and Nmur2-deficient animals on day 1 post dose. The effects on food intake were completely gone on day 2 post dose in both the Nmurl -and Nmur2-deficient mice. Figure 5 illustrates the finding that the PEGylated NMU analogs, NMU80 and NMU81 , can reduce food intake for up to three days following a single subcutaneous dose. NMU80 reduced food intake for two days while NMU81 evoked significant reductions in food intake for three days post dose.
While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

Claims

WHAT IS CLAIMED:
1. A composition comprising the formula (I) Zl-peptide-Z2 wherein the peptide has the amino acid sequence Xl~χ2-χ3_χ4-χ5.χ6.χ7_χ8- X9-xl0.xl Lxl2-Xl3.xl4.xl5.xl6-Xl7-xl8-xl9-x20-x21-x22.x23.x24.x25 (SEQ ID NO:1), wherein amino acids 1 to 17 can be any amino acid or absent; wherein amino acid χl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid χl9 is AIa, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid χ20 jg absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid χ21 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; χ22 1S Arg, Ly s, Harg, Ala, or Leu; amino acid X23 is pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid χ25 is Asn, any D- or L-amino acid, NIe or D-NIe, D-AIa or Ala;
Zl is optionally a protecting group that, if present, is joined to the N-termi.nus amino group; and
Z2 is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group, and pharmaceutically acceptable salts thereof.
2. The composition of claim 1, wherein the peptide has the amino acid sequence Xl-χ2-χ3.χ4.χ5.χ6-χ7_χ8.χ9.χlθ-χl l-χl2-χl3-χl4-χl5.χl6,χl7.χl8.phe- Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO:2), wherein amino acids 1 to 17 can be any amino acid or absent.
3. The composition of claim 1 , wherein the composition comprises a compound selected from the compounds represented by SEQ ID NOs: 7 to 56.
4. The composition of claim 1, wherein the composition comprises a compound selected from the compounds represented by SEQ ID NOs: 57 to 132.
5. The composition of claim 1 , wherein the composition comprises NMU80 orNMUδl.
6. A method for treating a metabolic disorder in an individual comprising a neuromedin U receptor agonist or pharmaceutically acceptable salt thereof that has the formula Zl-peptϊde-Z2 wherein the peptide has the amino acid sequence Xl-χ2.χ3.χ4.χ5.χ6-χ7_χ8- X9.χlθ-χl l-xl2.xB.xl4.xl5.xl6-Xl7.xl8-Xl9-x20.x2Lx22.x23.x24.x25 (SEQ ID NO: I), wherein amino acids 1 to 17 can be any amino acid or absent; wherein amino acid Xl8 is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, Ala, D-Lys, an aromatic amino acid, a des-amino acid or an acyl group; amino acid X* 9 [s Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 j$ absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-Leu, D-AIa or Ala, or any D- or L-amino acid; amino acid χ21 is Phe, NMe~Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; χ22 is Arg, Lys, Harg, Ala, or Leu; amino acid X23 \s pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid χ25 is Asn, any D- or L-amino acid, Me or D-NIe5 D-AIa or Ala;
Z^ is optionally a protecting group that, if present, is joined to the N-terminus amino group; and
Z2 is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group, and pharmaceutically acceptable salts thereof.
7. The method of claim 6, wherein the peptide has the amino acid sequence Xl-X2-χ3-χ4-χ5-χ6-χ7-χ8.χ9-χl0-χl Lχl2.χl3-χl4.χl 5.χl6-χl7.χl8-Phe-Leu-Phe-
Arg-Pro-Arg-Asn (SEQ ID NO:2), wherein amino acids 1 to 17 can be any amino acid or absent.
8. The method of Claim 6, wherein the metabolic disorder is obesity.
9. The method of claim 6, wherein the neuromedin U receptor agonist is selected from the agonists represented by SEQ ID NOs: 7 to 56.
10. The method of claim 6, wherein the neuromedin U receptor agonist is selected from the agonists represented by SEQ ID NOs:57 to 132.
11. The method of claim 6, wherein the neuromedin agonist is NMU80 or NMU81.
12. The use of the composition of any one of Claims 1-5 in the manufacture of a medicament for treatment of a metabolic disorder.
13. The use of the composition of any one of Claims 1 -5 in the manufacture of a medicament for treatment of obesity.
14. The use of the composition of any one of Claims 1-5 in the manufacture of a medicament for treatment of type II diabetes.
15. A pharmaceutical composition comprising the neuromedin U receptor agonist of any one of Claims 1-5 and a pharmaceutically acceptable carrier.
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