WO2010138343A1 - Neuromedin u receptor agonists - Google Patents

Neuromedin u receptor agonists Download PDF

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Publication number
WO2010138343A1
WO2010138343A1 PCT/US2010/035260 US2010035260W WO2010138343A1 WO 2010138343 A1 WO2010138343 A1 WO 2010138343A1 US 2010035260 W US2010035260 W US 2010035260W WO 2010138343 A1 WO2010138343 A1 WO 2010138343A1
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WIPO (PCT)
Prior art keywords
amino acid
ala
leu
phe
peptide
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PCT/US2010/035260
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French (fr)
Inventor
Antonello Pessi
Elisabetta Bianchi
Paolo Ingallinella
Armin Lahm
Philippe Neuner
Fabio Talamo
Gaetano Barbato
Donald J. Marsh
Andrea Peier
Original Assignee
Merck Sharp & Dohme Corp.
Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa
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Publication of WO2010138343A1 publication Critical patent/WO2010138343A1/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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site

Definitions

  • the present invention relates to neuromedin U (NMU) receptor agonists wherein the NMU peptide or analog thereof is covalently conjugated to a carrier protein.
  • the present invention relates to NMU receptor agonists wherein the carrier protein is serum albumin (HSA), lactoferrin, immunoglobulin, or immunoglobulin Fc fragment.
  • the present invention relates to NMU receptor agonists wherein the NMU peptide or analog thereof is conjugated to the carrier protein in a thioether acetamide linkage or a beta-thiosulfonyl linkage.
  • the NMU receptor agonists can. be used for the treatment of metabolic diseases such as obesity and diabetes.
  • 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.
  • mice overexpressing NMU are lean and hypophagic (Kowalski et al, J. Endocrinol.185: 151-164 (2005)).
  • the internal energy status of an animal affects expression and release of NMU as well (Wren et al, op. cit.).
  • 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 administration of NMU either centrally or peripherally reduce food intake in mice in a dose-dependent fashion.
  • NMU centrally administered NMU
  • Nmur2 ⁇ " NMUR2-deficient mice
  • Nmurl ⁇ NMURl- deficient mice
  • peripherally administered NMU are absent in Nmurl ⁇ mice and present in Nmur2 ⁇ " 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.
  • NMU receptor agonists administered peripherally have a short half- life. Therefore, there is a need for NMU receptor agonists that have an extended half-life in plasma serum.
  • 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.
  • the carrier protein enhances the half-life of the NMU peptide in the plasma serum compared to the half-life of a non-conjugated NMU peptide under similar conditions.
  • the linkage between NMU peptide and the carrier protein is a stable linkage, which further enhances the stabilizing effect of the carrier protein on the NMU peptide by increasing the length of time in which NMU peptide is conjugated to the carrier protein.
  • the stable linkages are formed by reacting a non-maleimido or non-succinimidyl reactive group that is capable of reacting with a functional group on the carrier protein covalently attached to a spacer moiety at or near the N- terminus of the NMU peptide.
  • reactive groups include but are not limited to iodoacetamide, bromoacetamide, and vinyl sulfonate reactive groups.
  • the NMU receptor agonists herein have a linkage between the peptide and the carrier protein that is stable for greater than 20 to 48 hours or more.
  • the carrier protein can be any protein, in particular embodiments, the earner protein is selected from the group consisting of serum albumin, immunoglobulins, lactoferrin, and Fc fragments.
  • the carrier protein is non- immunogenic in the host.
  • 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 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.
  • 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)
  • the peptide has the amino acid sequence Xl- ⁇ 2- ⁇ 3- ⁇ 4- ⁇ 5- ⁇ 6- ⁇ 7- ⁇ 8_ ⁇ 9- ⁇ l0 . ⁇ l l_ ⁇ l2_ ⁇ l3_ ⁇ l4. ⁇ l5_ ⁇ l6. ⁇ l7. ⁇ l 8. ⁇ l9. ⁇ 20. ⁇ 21- ⁇ 22. ⁇ 23. ⁇ 24 .
  • amino acids 1 to 17 can be any amino acid or absent; 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 ⁇ l9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20
  • the peptide has the amino acid sequence ⁇ l- ⁇ 2. ⁇ 3. ⁇ 4_ ⁇ 5_ ⁇ 6- ⁇ 7. ⁇ 8- ⁇ 9. ⁇ l 0- ⁇ l 1. ⁇ l2. ⁇ 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-Val- Asp-Glu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-Gln-Ser-Arg-Gly-Xl8-Xl9. ⁇ 20_ ⁇ 21. ⁇ 22_ ⁇ 23.
  • 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 1 9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid
  • amino acid ⁇ 20 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, AIa or Trp
  • ⁇ 22 is Arg, Lys, Harg, AIa, or Leu
  • amino acid ⁇ 23 Js Pro Ser, Sar, Ala or Leu
  • amino acid X 1 9 is Ala,
  • the peptide comprises the amino acid sequence Xl—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, 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 3 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 X5 is Arg, Lys
  • Spacer Zl has a proximal end and a distal end: the distal end is covalently attached to the NMU peptide and the proximal end is covalently attached to a reactive group capable of reacting with a functionality on the carrier protein to form a stable covalent linkage Z2.
  • the stable linkage has a half-life of more than 20 hours in 10% plasma serum at 37°C.
  • the linkage has a half-life of more than 36 hours in 10% plasma serum at 37°C.
  • the linkage has a half-life of more than 48 hours in 10% plasma serum at 37°C.
  • Z2 does not include maleimido (3-thiosuccinimidyl ether) or succinimidyl linkages.
  • ZX is a spacer comprising one to four units of Ttds (13- amino-4,7 f 10-trioxa-tridecayl succinamic acid), which in further aspects comprises two units of Ttds (13-amino-4,7,10-trioxa-tridecayl succinamic acid).
  • the spacer includes on the proximal end a reactive group selected from the group consisting of iodoacetamide, bromoacetamide, and vinyl sulfonate. The iodoacetamide or bromoacetamide reactive group reacts with thio functionalities on the carrier protein to form stable tbioether acetamide linkages.
  • the linkage Z ⁇ is a thioether acetamide linkage or beta-thiosulfonyl linkage.
  • the peptide has the amino acid sequence of human NMU peptide as shown in SEQ ID NO: 10 and which can optionally have a protecting group at the C- terminus.
  • an neuromedin U receptor agonist is provided as shown in Figure IB that has the formula
  • spacer Zl comprises two units of Ttds (13-amino-4,7,10-trioxa- tridecayl succinamic acid) in which the distal end of the spacer is covalently linked to the N- terminus of the NMU peptide and the proximal end of the space is covalently linked to the thiol group of cysteine 34 of human serum albumin in a thioether acetamide linkage (Z ⁇ ).
  • the agonist further includes an amide protecting group (Z3) at the C-temiinus of the peptide.
  • 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.
  • 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 ⁇ !8 is absent, Tyr or D-Tyr, Leu, Phe, VaI 5 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, sarcosine (Sar), D-Leu, NMe-Leu
  • Carrier protein Pr can be any protein but in particular embodiments, the Pr is selected from the group consisting of serum albumin, immunoglobulins, lactoferrin, Fab fragment, scFv, and Fc fragment.
  • 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-X2- ⁇ 3-. ⁇ 4- ⁇ 5- ⁇ 6- ⁇ 7- ⁇ 8- ⁇ 9- ⁇ l O. ⁇ l I. ⁇ l 2. ⁇ l3, ⁇ l4_ ⁇ l 5_ ⁇ l6_ ⁇ l7- ⁇ 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-Val- Asp-Glu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-Gln-Ser-Arg-Gly-Xl8_ ⁇ l9- ⁇ 20- ⁇ 21- ⁇ 22- ⁇ 23.
  • amino acid ⁇ l8 is absent, Tyr or D-Tyr, Leu, Phe 5 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, an aliphatic amino acid, an aromatic amino acid, Ala or Trp
  • ⁇ 22 is Arg, Lys, Harg, Ala, or Leu
  • amino acid ⁇ 23 is Pro, Ser, Sar, Ala or Leu
  • amino acid ⁇ l8 is absent, Tyr
  • the peptide comprises the amino acid sequence Xl— ⁇ 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 X2 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 X4 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid ⁇ 5 is Arg, Lyse-N-linked
  • Spacer Zl has a proximal end and a distal end: the distal end is covalently attached to the NMU peptide and the proximal end is covalently attached to a reactive group capable of reacting with a functionality on the carrier protein to form a stable covalent linkage Z2.
  • the stable linkage has a half-life of more than 20 hours in 10% plasma serum at 37°C.
  • the linkage has a half-life of more than 36 hours in 10% plasma serum at 37 0 C.
  • the linkage has a half-life of more than 48 hours in 10% plasma serum at 37 0 C.
  • Z ⁇ does not include maleimido (3-thiosuccinimidyl ether) or succinimidyl linkages.
  • Zl is a spacer comprising one to four units of Ttds (13- amino-4,7,10-trioxa-tridecayl succinamic acid), which in further aspects comprises two units of Ttds (13-amino-4,7,10-trioxa-tridecay ⁇ succinamic acid).
  • the spacer includes on the proximal end a reactive group selected from the group consisting of iodoacetamide, bromoacetamide, and vinyl sulfonate. The iodoacetamide or bromoacetamide reactive group reacts with thio functionalities on the carrier protein to form stable thioether acetamide linkages.
  • the linkage Z2 is a thioether acetamide linkage or beta-thiosulfonyl linkage.
  • the peptide has the amino acid sequence of human NMU peptide as shown in SEQ ID NO: 10 and which can optionally have a protecting group at the C-terminus.
  • the neuromedin U receptor agonist has the formula
  • peptides that can be conjugated to a carrier protein to form neuromedin U receptor agonists that have the formula
  • 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 ⁇ !9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid ⁇ 20 is absent, Leu, GIy, sarcosine (Sar), D -Leu,
  • the peptide has the amino acid sequence Xl- ⁇ 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-
  • amino acid ⁇ l8 is absent, Tyr or D-Tyr s Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, AIa 5 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 ⁇ 20 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-
  • 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 AIa 5 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 ⁇ 5 is Arg, Lys
  • Spacer Zl has a proximal end and a distal end: the distal end is covalently attached to the NMU peptide and the proximal end is covalently attached to one or more reactive groups R n capable of reacting with a functionality on the carrier protein to form a stable covalent linkage.
  • the reactive groups form a stable linkage that has a half-life of more than 20 hours in 10% plasma serum at 37°C.
  • the linkage has a half-life is more than 36 hours in 10% plasma serum at 37 0 C.
  • the linkage has a half-life of more than 48 hours in 10% plasma serum at 37°C.
  • R n does not include maleimido or succinimidyl reactive groups.
  • Zl is a spacer comprising one to four units of Ttds (13- araino-4,7,10-trioxa-tridecayl succinamic acid), which in further aspects comprises two units of Ttds (13-amino-4,7,10-trioxa-tridecayl succinamic acid).
  • the spacer includes on the proximal end at least one reactive group R n selected from the group consisting of iodoacetamide, bromoacetamide, and vinyl sulfonate. The iodoacetamide or bromoacetamide reactive group reacts with thio functionalities on the carrier protein to form stable thioether acetamide linkages.
  • the vinyl sulfonate reactive group reacts with thio functionalities on the carrier protein to form stable beta-thiosulfonyl linkages.
  • the linkage formed by the iodoacetamide, bromoacetamide, and vinyl sulfonate reactive groups are thioether acetamide or beta-thiosulfonyl linkages, respectively.
  • R n is an
  • ETAC reactive group capable of forming a three carbon bridge between two cysteine residues in a carrier protein involved in a disulfide linkage.
  • the ETAC can be a bis-sulfone or a mono- sulfone.
  • the peptide has the amino acid sequence of human NMU peptide as shown in SEQ ID NO: 10 and which can optionally have a protecting group at the C- terminus.
  • the neuromedin U receptor agonist has the formula
  • I is an iodo group.
  • 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: 10 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.
  • the carrier protein when the carrier protein is serum albumin or lactoferrin, the serum albumin or lactoferrin will usually be of human origin or have an amino acid sequence substantially the same as the amino acid sequence of human serum albumin (HSA) or lactoferrin.
  • the human serum albumin or lactoferrin can be recombinantly produced. Further included are human serum albumin or lactoferrin fusion proteins.
  • the carrier protein is an immunoglobulin or antibody molecule
  • the immunoglobulin can be an IgG molecule and includes IgGi, ⁇ gQ2 » IgCb, ⁇ d IgG4 and subspecies thereof.
  • the immunoglobulin is selected from the group consisting of IgA 5 IgM, IgE 5 camel heavy chain, and llama heavy chain.
  • the IgG can be a catalytic antibody, for example as described in U.S. Patent Application No s. 7205136; 4888281 ; 5037750 to Schochetman et al., U.S. Patent Application Nos, 5733757; 5985626; and 6368839 to Barbas, III et al.
  • the terms "antibody,” “immunoglobulin/' “immunoglobulins” and “immunoglobulin molecule” are used interchangeably,
  • Fc fragment refers to the 'fragment crystallized' C-terminal region of the antibody containing the Co2 and CH3 domains.
  • Fab fragment refers to the 'fragment antigen binding' region of the antibody containing the VJJ, CH 1 * VL and CL domains. Fc fragments include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation and those produced recombinantly.
  • scFv refers to a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine or glycine.
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (1975) Nature, 256:495, or may be made by recombinant DNA methods (See, for example, U.S. Pat. No. 4,816,567 to Cabilly et al).
  • Immunoglobulins, ScFv, Fabs, and Fc fragments further include immunoglobulins chimeric, human, and humanized antibodies and Fc fragments; immunoglobulin and Fc fusion proteins; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (See, for example, Intracellular Antibodies: Research and Disease Applications, (Marasco, ed., Springer- Verlag New York, Inc., 1998).
  • Figure IA shows the synthesis of HSA-NMU43 conjugate in which NMU43 is conjugated to HSA by means of a maleimido reactive group.
  • NMU43 having a (Ttds)2 spacer with a maleimide reactive group is conjugated to Cys34 of HSA in a 3-thiosuccinimidyl ether linkage.
  • Figure IB shows the synthesis of HSA-NMU79 conjugate in which NMU79 is conjugated to HSA by means of a iodoacetamide reactive group.
  • NMU79 having a (Ttds)2 spacer with an iodoacetamide reactive group is conjugated to Cys34 of HSA in an thioether acetamide linkage.
  • FIG. 1A shows that acute administration of HSA-NMU43 significantly reduces food intake in diet-induced obese mice.
  • Figure 2B shows that acute administration of HSA-NMU43 significantly reduces body weight in diet-induced obese mice.
  • Figure 3 A shows that acute administration of HSA-NMU43 significantly reduces food intake and body weight in Nmurl- and Nmur2-deficient mice.
  • Figure 3B shows that acute administration of HSA-NMU43 significantly reduces food intake and body weight in Nmurl- and Nmur2-deficient mice after two days.
  • Figure 3 C shows that acute administration of HSA-NMU43 significantly reduces food intake and body weight in Nmurl- and Nmur2-deficient mice after 3 days.
  • Figure 4A shows that acute administration of HSA-NMU79 dose-dependently reduces food intake in wild-type mice. Significant effects on food intake were observed for three days after a single dose.
  • Figure 4B shows that acute administration of HSA-NMU79 dose-dependently reduces body weight in wild-type mice. Significant effects on food intake were observed for three days after a single dose.
  • Figure 5A shows that acute administration of HSA-NMU79 dose-dependently enhances glucose excursion during an OGTT. Significant effects on glucose were observed at 0.3 and 1 mpk of NMU79.
  • Figure SB shows the area under the curve (AUC) for the time course of the OGTT in Figure 5A.
  • Figure 6A-D show LC-MS analyses of HSA-NMU conjugates with different spacers.
  • Figure 7 shows a time-course analysis of HSA-NMU43 conjugate in 10% mouse plasma (mass spectrum reconstructions).
  • Figure 8 shows a time-course analysis of HSA-NMU79 conjugate in 10% mouse plasma (mass spectrum reconstructions).
  • FIG. 9 shows a schematic view of an ETAC reaction using ETAC reagent (4- [2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid) in the context of an IgGj isotype. Insertion of ETAC-bridges into the IgGi molecule is shown and the function group on the ETAC-bridges available for conjugation are shown by the X.
  • ETAC reagent 4- [2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid
  • Figure 10 shows a schematic view of preparing NMU-(Ttds)2-ETAC precursor molecule for conjugating to a carrier protein in a disulfide bridge between two cysteine residues involved in a disulfide linkage.
  • Compound 3 is a bis-sulfone precursor and compound 4 is a mono-sulfone precursor.
  • Figure 11 shows the synthesis of Methyl Ketone-ETAC (MKE) 5 from ETAC 4.
  • MKE Methyl Ketone-ETAC
  • the present invention provides neuromedin U receptor agonists in which the neuromedin U (NMU) peptide or analog thereof is conjugated to a carrier protein in a covalent linkage that is more stable than a maleimido (3-thiosuccinimidyl ether) or succinimidyl linkage under physiological conditions and compositions comprising the same.
  • Suitable carrier proteins are preferably non-immunogenic and include but are not limited to human serum albumin (HSA), immunoglobulins, lactoferrin, and immunoglobulin Fc fragments.
  • Conjugating a peptide to a carrier protein can enhance the half-life of the peptide in serum by either steric hindrance in which the peptide by virtue of its conjugation to the carrier protein is rendered less accessible to endogenous proteases or by compartmentalizing the peptide in a way that avoids or delays clearance of the peptide from serum or both.
  • conjugating a small peptide to carrier proteins that increases the molecular weight of the peptide to greater than 40 kDa will substantially reduce renal clearance, which can substantially increase the half- life of the peptide in blood or plasma serum.
  • Conjugation linkages with enhanced stability as described herein extends the time the peptide is conjugated to the carrier protein.
  • 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 glycosylation structures, and can further include fusions to heterologous proteins, polypeptides, and peptides.
  • 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 drugs 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-HIV 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 (V ⁇ ) 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 are conjugated with the linkage to the carrier protein.
  • linkage to the carrier protein is by means of a maleimido or succinimidyl reactive group that targets a functional group on the carrier protein.
  • NMU peptides conjugated to human serum albumin (HSA) as shown herein has an efficacy profile that is similar to the profile of NMU PEGylated at the JV-terminus as disclosed in Published PCT Application No. WO2007109135. Therefore, one or more of the neuromedin U receptor agonists according to the present invention can be administered to an individual to treat a metabolic disorder afflicting the individual.
  • a metabolic disorder 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 according to the present invention 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 a 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 used to treat multiple disorders in an individual.
  • 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.
  • neuromedin U receptor agonists of the present invention comprise the general 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, 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 Ala, Trp, Tyr, Phe, GIu 5 Nva, NIe or an aromatic amino acid; amino acid ⁇ 20 is absent, Leu, GIy, sarcosine (Sar), D-Leu, NMe-
  • Carrier protein Pr can be any protein but in particular embodiments, the Pr is selected from the group consisting of serum albumin (HSA), immunoglobulins, lactoferrin, and Fc fragments.
  • HSA serum albumin
  • immunoglobulins immunoglobulins
  • lactoferrin lactoferrin
  • Fc fragments Fc fragments
  • the peptide has the amino acid sequence ⁇ l- ⁇ 2. ⁇ 3. ⁇ 4. ⁇ 5_ ⁇ 6- ⁇ 7_ ⁇ 8. ⁇ 9. ⁇ l O. ⁇ l 1. ⁇ l 2. ⁇ l 3. ⁇ l4 j ⁇ l 5. ⁇ l 6_ ⁇ l 7_ ⁇ l 8-Phe-Leu-Phe-Arg-Pro-Arg-Asn
  • amino acids 1 to 17 can be any amino acid or absent.
  • the peptide comprises the amino acid sequence Phe- Arg- VaI-
  • 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 Xl9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid
  • amino acid ⁇ 20 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,
  • the peptide comprises the amino acid sequence ⁇ l ⁇ 2- X3. ⁇ 4. ⁇ 5- ⁇ 6- ⁇ 7. ⁇ 8 (SEQ ID N0:4) wherein amino acid Xl is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu 5 Asp, AIa, 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 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 neuromedin U receptor agonist is conjugated to the cysteine at position 34 of human serum albumin (HSA).
  • HSA human serum albumin
  • Spacers (Z 1) are chemical moieties that covalently link or connect reactive groups to the modified NMU peptide.
  • the spacer has a distal end and a proximal end.
  • the distal end of the spacer is attached to the jV-terminus of the peptide by means of an amide linkage.
  • the proximal end of the spacer has a functional or reactive group that is available for conjugating the peptide-spacer to an amino acid in the carrier protein.
  • spacers may comprise one or more alkyl groups such as methyl, ethyl, propyl, butyl, etc.
  • alkoxy groups alkenyl groups, alkynyl groups, or amino groups substituted by alkyl groups, cycloalkyl groups, polycyclic groups, aryl groups, polyaryl groups, substituted aryl groups, heterocyclic groups, and substituted heterocyclic groups.
  • the linker is a derivative of a compound selected from the group consisting of acyloxymethylketones; aziridines; diazomethyl ketones; epoxides; iodo-, bromo-or chloroacetamides; ⁇ -haloesters; ⁇ -haloketones; sulfoniums; chloroethylsulfides; O-alkylisoureas; alkyl halides; vinylsulfones; acrylamides; acrylates; vinylpyridines; organometallic compounds; aryldisul fides; thiosulfonates; aldehydes; nitriles; ⁇ - diketones; ⁇ -ketoamides; ⁇ -ketoesters; diaminoketones; sernicarbazones; and dihydrazides.
  • Spacers may also comprise poly ethoxy aminoacids such as AEA ( (2-amino) ethoxy acetic acid) or a AEEA ([2-(2-amino) ethoxy)] ethoxy acetic acid).
  • AEA (2-amino) ethoxy acetic acid
  • AEEA [2-(2-amino) ethoxy)] ethoxy acetic acid
  • Useful spacers include polyethylene glycol polymers including but not limited to polymers comprising one, two, three, four, five, or more units or molecules of 13-amino-4,7,10- trioxa-tridecayl succinamic acid (Ttds or PEG- 13) units in tandem or ethoxy polymers comprising one to twenty-four or more ethoxy units.
  • Ttds or PEG- 13 13-amino-4,7,10- trioxa-tridecayl succinamic acid
  • the examples illustrate embodiments in which the NMU-HSA conjugate comprises a Ttds dimer or trimer polymer or an ethoxy polymer that includes 24 ethoxy units.
  • the chemical structure of the spacer is not critical since it serves primarily to provide a suitable distance between the reactive group and the peptide; however, in certain embodiments, the spacer may itself provide improved properties to the compositions of the present invention.
  • the spacer may itself provide improved properties to the compositions of the present invention. For example, as shown by use of the Ttds dimer and trimer spacers in Example 5, a sufficiently long and flexible spacer between the functional reactive group and the N-terminus of NMU appears to facilitate conjugation to Cys34 of HSA and also to minimize attachment of multiple copies of NMU peptide to the HSA at sites other than the cysteine residue at position 34.
  • a spacer that is about 22 to 150 A in length, which in particular embodiments is about 44 A to about 66 to 88 A length is particularly useful for conjugating NMU or analogs thereof to HSA.
  • One Ttds unit has a length of about 22 A.
  • HSA-NMU conjugates that have a maleimido (3-thiosuccinimidyl ether ) linkage have an apparent half-life in 10% plasma serum at 37°C of about 20 hours whereas HSA-NMU conjugates that have the more stable acetamide linkage are stable for the duration of the experiment, i.e., have a half-life of greater than 48 hours in 10% plasma serum at 37 0 C.
  • An example of another reactive group that forms more stable linkages than maleimido reactive groups are vinyl sulfonate groups.
  • the advantage of using a reactive group that forms a covalent bond that is stable for long periods of time in serum as in the conjugates herein is that it enables the neuromedin U receptor agonists herein to be administered once weekly or even bi-weekly.
  • the neuromedin U receptor agonists will be administered intravenously. Therefore, reactive groups that form stable covalent linkages to be able to administer the neuromedin U receptor agonists on a weekly or bi-weekly basis is a desirable.
  • the linkage in the conjugates herein should have a half-life in a solution containing about 10% plasma serum at 37 0 C that is greater than 20 hours as determined by LC-MS.
  • the linkage should have a half-life greater than 36 hours, and in further still embodiments, the linker should have a half-life that is greater than 48 hours.
  • 72- comprises two stable linkages to the carrier protein such as those formed by an equilibrium transfer alkylating cross-link (ETAC) reagent or variant thereof.
  • ETAC reagents have been described in Liberatore et al., Bioconjugate Chem, 1: 36-50 (1990), Shaunak et al, Nature Chem. Biol. 2: 312-313 (2006), Balan et al., Bioconjugate Chem. 18: 61-76 (2007), Brocchini et al, Adv. Drug Del. Rev.
  • ETAC reagents comprise three reactive groups: two of the reactive groups are available for conjugating to the thiol groups of two cysteine residues involved in a disulfide linkage or bond on the carrier protein to form a three carbon covalent bridge between the two cysteine residues, which maintains the disulfide linkage and thus the tertiary structure of the protein.
  • the third reactive group is conjugated to the proximal end of the spacer Z ⁇ .
  • the third reactive group is conjugated to the proximal end of the spacer Z* prior to conjugating to the carrier protein and in other embodiments, the third reactive group is conjugated to the proximal end of the spacer Zl subsequent to conjugating to the carrier protein.
  • Figure 9 provides an example of an ETAC reagent (4-[2,2-bis[(p ⁇ tolylsulfonyl)- methyl]acetyl]benzoic acid) and Figure 11 provides an example of an ETAC reagent modified to have a methyl ketone group (compound 5).
  • ETAC reagents are particularly useful for conjugating peptides to proteins such as antibodies and Fc fragments which rely upon disulfide bonds for stabilization.
  • the IgG molecule is partially reduced. This provides one or more reduced cysteine residues for conjugating the NMU peptide and spacer to the IgG molecule using the a reactive group that reacts with thiol groups.
  • the resulting conjugates are less susceptible to papain cleavage, the standard method to generate Fc fragments conjugated to the NMU peptide from full antibodies. If instead, the IgG molecule is first cleaved by papain and the Fc fragment thus generated is then partially reduced and conjugated to NMU stability of the Fc fragment is usually compromised because the Fc fragment cannot form the proper disulfide bonds needed to maintain its structural integrity.
  • the ETAC reagents and variants thereof overcome both of the above disadvantages because the ETAC reagent forms a bridge between two disulfide groups on the partially reduced IgG or Fc fragment thus the stabilizing role of the disulfide bridge at the conjugation location is reestablished.
  • linkage Z2 is a non-maleimido (3-thiosuccinimidyl ether) or succinimidyl linkage in which a functionality on the carrier protein (Pr) is covalently joined to spacer Z ⁇ by means of a reactive group on spacer T ⁇ and which linkage is more stable than a maleimido (3- thiosuccinimidyl ether ) linkage toward hydrolytic cleavage in vivo.
  • Functionalities are groups on the carrier protein to which the reactive group on the linker can react to form a covalent bond.
  • Functionalities include hydroxyl groups for bonding to ester reactive entities; thiol groups for bonding to nialamides, maleimido, iodoacetyl, bromoacetyl, vinyl sulfone, imidates, and thioester groups; amino groups for bonding to carboxy, phosphoryl, or acyl groups on reactive entities; and carboxyl groups for bonding to amino groups.
  • HSA human serum albumin
  • Functionalities can further include unnatural amino acids for which a there is a reactive group that can form a linkage with the unnatural amino acid (See Published U.S. Application No. 20080255045 and U.S. Patent Nos.
  • a carrier protein can be engineered to include one or more non-natural amino acids capable of reacting with a reactive group anywhere within the carrier molecule.
  • compounds can be provided in which the NMU peptide conjugated at any location within the carrier protein or to one, two, three, or more locations within the carrier molecule.
  • a precursor molecule is prepared that comprises the NMU peptide or analog thereof co valently linked to the distal end of spacer Zl that has a non-maleimido or succinimidyl reactive group at the proximal end.
  • These peptides that can be conjugated to a carrier protein to form neuromedin U receptor agonists have the formula
  • amino acids 1 to 17 can be any amino acid or absent; wherein amino acid X 1 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 Xl9 1S Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid ⁇ 20 is absent, Leu, GIy, sarcosine (S ar), D-Leu,
  • the peptide has the amino acid sequence ⁇ l-X2- ⁇ 3-. ⁇ 4- ⁇ 5- ⁇ 6- ⁇ 7. ⁇ 8- ⁇ 9. ⁇ l0 ⁇ l l. ⁇ l2. ⁇ l3 m ⁇ 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-
  • 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 AIa, 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-
  • the peptide comprises the amino acid sequence Xl-X2- ⁇ 3- ⁇ 4- ⁇ 5.. ⁇ 6- ⁇ 7- ⁇ 8 (SEQ ID N0:4) wherein amino acid ⁇ l is absent, Tyr or D-Tyr, 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 ⁇ 2 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 X4 is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; amino acid X5 is Arg, Lyse-N-N
  • Spacer Zl has a proximal end and a distal end; the distal end is covalently attached to the NMU peptide and the proximal end is covalently attached to one or more reactive groups R n capable of reacting with a functionality on the carrier protein to form a stable covalent linkage.
  • the reactive groups form a stable linkage that has a half-life of more than 20 hours in 10% plasma serum at 37 0 C.
  • the linkage has a half-life is more than 36 hours in 10% plasma serum at 37 0 C.
  • the linkage has a half-life of more than 48 hours in 10% plasma serum at 37 0 C.
  • R ⁇ does not include maleimido or succinimidyl reactive groups.
  • Zl is a spacer comprising one to four units of Ttds (13- amino-4,7,10-trioxa-tridecayl succinamic acid), which in further aspects comprises two units of Ttds (13-amino-4,7,10-trioxa-tridecayl succinamic acid).
  • the spacer includes on the proximal end at least one reactive group R n selected from the group consisting of iodoacetamide, bromoacetamide, vinyl sulfonate, and ETAC.
  • the iodoacetamide or bromoacetamide reactive group reacts with thio functionalities on the carrier protein to form stable thioether acetamide linkages.
  • the vinyl sulfonate reactive group reacts with thio functionalities on the carrier protein to form stable beta-thiosulfonyl linkages.
  • the ETAC reacts with thio functionalities and is capable of forming a three carbon bridge between thiol groups on the carrier protein involved in a disulfide bond.
  • the precursor molecule is a compound comprising the formula SRGYFLFRPRN-CONH 2
  • I is an iodo group.
  • This compound is reactive with the cysteine residues, for example, the cysteine residue at position 34 in human serum albumin (HSA).
  • HSA human serum albumin
  • use of the above compound enabled HSA-NMU conjugates to be made wherein greater than 90% or greater than 95% of the conjugates comprise one molecule of NMU per molecule of HSA as determined by mass spectrometry (See Table 4 in Example 5).
  • the above compound enabled HSA-NMU conjugates to be made wherein proportion of conjugates having two molecules of NMU per molecule of HSA is less than 10% or 5% as determined by mass spectroscopy and having three or more molecules of NMU per molecule of HSA is not detectable by mass spectrometry.
  • Compounds that have spacers of similar length but of different composition are expected to provide similar results.
  • the Rn-Zl -peptide precursor molecule is conjugated to a recombinant antibody or Fc fragment in which the TV-terminus or the C-terminus of the antibody or Fc fragment includes a cysteine residue, which provides a functionality for reacting with the iodoacetamide or bromoacetamide reactive group of the R ⁇ -Zl-peptide precursor molecule to conjugate the R n -Zl -peptide precursor to the antibody or Fc fragment.
  • the antibody or Fc fragment is conjugated to the 2 ⁇ -peptide precursor molecule via an ETAC reactive group capable for forming a three carbon bridge between two cysteine residues in the antibody or Fc fragment involved in a disulfide bond.
  • Figure 10 provides a scheme for the construction of an R n -Z ⁇ -peptide precursor molecule comprising an ETAC reactive group.
  • 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 C 1-6 ester, or converted to an amide of formula NRR2 wherein R and R2 are each independently H or C 1-6 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.
  • Hydroxyl groups of the neuromedin U receptor agonist amino acid side chains may be converted to C I Brush6 alkoxy or to a C I Conduct6 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 Cl faced6 alkyl, Cl ⁇ 6 allkoxy, 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.
  • the present invention includes diastereomers as well as their racermc and resolved enantiomerically pure forms.
  • the neuromedin U receptor agonists can contain D -amino acids, L-amino acids, unnatural amino acids, amino acid analogs, rare amino acids, or a combination thereof.
  • the amino acids are in the L-form with particular amino acids in D-form.
  • 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/m2).
  • 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.
  • the increased risks associated with obesity occur at a lower Body Mass Index
  • BMI in Asians.
  • "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, hyperuricacideraia, 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, emmeniopathy, 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.
  • 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.
  • 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 useful 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.
  • 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.
  • 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, tromethamine, and the like.
  • basic ion exchange resins such
  • 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
  • references to the neuromedin U receptor agonists of the general formula (I) are meant to also include the pharmaceutically acceptable salts.
  • 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.
  • pharmaceutical 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 amelioration of such conditions.
  • 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.
  • an individual active ingredient administered alone
  • 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.
  • 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 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 (CBl) receptor antagonists; glucagon like peptide 1 (GLP- 1) receptor agonists; glucagon receptor agonists and antagonists
  • 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); rosiglitazone (AVANDIA); troglitazone; rivoglitazone, BRL49653; CLX-0921; 5-BTZD, GW- 0207, LG-100641, R483, and LY-300512, and the like and compounds disclosed in WO97/10813, 97/27857, 97/281 15, 97/28137, 97/27847, 03/000685, and 03/027112 and
  • glitazones e.g. ciglitazone; darglitazone; englitazone; isaglitazone (MCC-555); pioglitazone (ACTOS); rosiglitazone (AVANDIA); tro
  • SPPARMS selective PPAR gamma modulators
  • Tl 31 Amgen
  • FK614 Flujisawa
  • netoglitazone and metaglidasen
  • biguanides such as buformin; metformin; and phenformin, and the like
  • PPP-IB protein tyrosine phosphatase- IB inhibitors
  • 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 03/000
  • SGLT inhibitors such as AVE 2268, KGT 1251, T1095/RWJ 394718; (35) BLX-1002; (b) lipid 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, pravastatin, fluvastatin, lovastatin, pravastatin, rivastatin, rosuvastatin, simvastatin, rosuvastatin (ZD-4522), and the like, particularly simvastatin; (3) HMG-CoA synthase
  • NS-220/R1593 Nippon Shinyaku/Roche, STl 929 (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;
  • FXR receptor modulators such as GW 4064 (GlaxoSmithkline), SR 103912,
  • LXR receptor modulators such as GW 3965 (GlaxoSmithkline), T9013137, and XTCO 179628 (X-Ceptor Therapeutics/Sanyo), and those disclosed in WO 03/031408, WO 03/063796, WO 04/072041, and the like
  • lipoprotein synthesis inhibitors such as niacin
  • renin angiotensin system inhibitors e.g., renin angiotensin system inhibitors
  • PPAR ⁇ partial agonists such as those disclosed in WO 03/024395
  • bile acid reabsorption inhibitors such as BARI 1453, SC435, PHA384640, S8921, AZD7706, and the like
  • bile acid sequesterants such as colesevelam (WELCHOL/ CHOLESTAGEL),
  • PPAR ⁇ agonists such as
  • 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, esmolol, indenolol, metaprolol, nadolol, nebivolo
  • 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)propanol]carbamates (Kiec-Kononowicz, K.
  • MCHlR melanin-concentrating hormone 1 receptor
  • T-226296 Takeda
  • T71 Takeda/Amgen
  • AMGN- 608450 AMGN-503796
  • Amgen 856464
  • A798 Abbott
  • ATCOl 75/AR224349 Arena Pharmaceuticals
  • GW803430 GaxoSmithkine
  • NBI- IA Neurorocrine Biosciences
  • NGX-I Neurogen
  • SNP-7941 Synaptic
  • SNAP9847 Synaptic
  • T-226293 Schering Plough
  • TPI- 136 M 7 Saitama Medical School/University of California Irvine
  • NPY5 neuropeptide Y Y5-5 antagonists, such as 152,804, S2367 (Shionogi), E-6999 (Esteve), GW- 569180A, GW-594884A (GlaxoSmithkline), 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
  • 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 03/0228
  • leptin such as recombinant human leptin (PEG-OB, Hoffman La Roche) and recombinant methionyl human leptin (Amgen);
  • 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 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,
  • 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, VRl 065 (Vernalis/Roche) YM 348; and those disclosed in U.S.
  • GLP-I glucagon-like peptide 1 agonists
  • Topiramate Topimax®
  • phytopharm compound 57 CP 644,673
  • ACC2 acetyl-CoA carboxylase-2
  • ⁇ 3 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)
  • 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 5 6,7 5 8-tetrahydro-5,5,8 J 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.
  • glucocorticoid receptor antagonists such as CP472555 (Pfizer), KB 3305, and those disclosed in WO 04/000869, WO 04/075864, and the like; (37) 11 ⁇ HSD-I (1 1-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-triazole, 3-adamantanyl-4,5,6 ⁇ 7,8,9,10,l l,12,3a-decahydro-l,2,4-triazolo[4,3- a][l ljannulene
  • glucocorticoid receptor antagonists such as CP472555
  • GT389255 (Genzyme/Peptimmune)Triton WRl 339, RHC80267, lipstatin, teasaponin, and diethylumbelliferyl 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 04/1 11004, and U.S. Patent Nos.
  • 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: JV-[3-(4-chlorophenyl)-2(5>-phenyH(5)-methylpropyl]-2-(4- trifluoromethyl»2 ⁇ pyrimidyloxy)-2-memylpropanamide, iV-f3-(4-chlorophenyl)-2-(3- cyanophenyl)-l-methylpropyl]-2-(5-trifluoromethyl-2-pyridyloxy)-2-methylpropanamide, JV-[3- (4-chlorophenyl)-2-(5-chloro-3-pyridyl)-l-methylpropyl]-2-(5-trifluoromethyl-2-pyridyloxy)-2- methylpropanamide, and pharmaceutically acceptable salts
  • 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 5 8-dimethoxyquinolin-2-yl)carbonyl]-6-(lH-tetrazol-5-yl)s ⁇ iro[chroman-2,4'- piperidin]-4-one; (5- ⁇ r-[(4,8-dimethoxyquinolin-2-yl)carbonyl]-4-oxospiro[chroman-2 5 4'- piperidin]-6-yl ⁇ 2i ⁇ -tetrazol-2-yl)methyl pivalate; 5- ⁇ l'-[(8-cyclopropyl-4-methoxyquinolin-2- yl)carbonyl]-4-oxospiro[chroman ⁇ 2,4'-piperidin]-6-yl ⁇ nicotinic acid; 1 '-(8-methoxy-4- morpholin ⁇ 4-yl ⁇
  • 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: l ⁇ 4-[(l-ethylazetidin-3-yl)oxy]phenyl ⁇ -4-[(4-fluorobenzyl)oxy]pyridin- 2(IH)-OUS, 4- [(4-fluorobenzyl)oxy] - 1 - ⁇ 4- [( 1 -isopropylazetidin-3 -yl)oxy] phenyl ⁇ pyri din-2( 1 H)- one, 1 -[4-(azetidin-3-yloxy)phenyl]-4-[(5-chloropyridin-2-yl)methoxy]pyridin-2(lH)-one :i 4-[(5- chloropyridin-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-(2,4,5-trifluoro ⁇ henyl)butanoyl]-3-(trifluoromethyl)-5,6 > 7,8-tetrahydro-l ,2,4- triazolo[4,3-a]pyrazine, or a pharmaceutically acceptable salt thereof.
  • H3 (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- [(l-cyclobutyl-4-piperidinyl)oxy]phenyl ⁇ -2-ethylpyrido[2,3-d]-pyrimidin-4(3H)-one, 3- ⁇ 4-[(l- cyclobutyl-4-piperidinyl)oxy] phenyl ⁇ -2-methylpyrido[4 ,3 -d]pyrimidin-4(3 H)-one ?
  • 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- ⁇ [l-(3 -ethoxyphenyl)-2-(4-methyiphenyl)- 1 H -imidazol-4-yl] carbonyl ⁇ - 1 - piperazinyl)- 1 -naphthoic acid; 3-(4- ⁇ [ 1 -(3 -ethoxyphenyl)-2-(2-fluoro-4-methylphenyl)- 1 H - imidazol-4-yl] carbonyl ⁇ - 1 -piperazinyl)- 1 -naphthoic acid; 3 -(4- ⁇ [ 1 -(3 -ethoxyphenyl)-2-(4- fluorophenyl)- 1 H -imidazol-4-yl] carbonyl ⁇ - 1 -piperazin
  • 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 : 1 ) (5S)- 1 '- ⁇ [(3 ⁇ , 4R)- 1 -terf-butyl-3 -(2,3 ,4-trifluorophenyl)piperidin ⁇ 4-yl] carbonyl ⁇ -3 - chloro-2-methyl-5- [ 1 -methyl- 1 -( 1 -methyl- 1 H- 1 ,2,4-triazol-5-yl)ethyl]-5/f ⁇ spiro[furo [3 ,4-
  • 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.
  • 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
  • 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 al., New Engl. J. Med.
  • 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 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.
  • 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.
  • 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.
  • 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 container(s) 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.
  • HSA-NMU conjugates Preparation of several human Serum Albumin (HSA)-NMU conjugates was as follows. Conjugation to Human Serum Albumin (HSA) takes advantage of the presence of the unpaired Cys34 jn the protein primary structure. The site of conjugation on the NMU peptide of the present invention is chosen taking into account the structure of NMU and its interactions with the NMU receptors. Hence, the conjugation was site-specific through the addition of either a maleimido or a haloacetyl functionality and spacer at the JV-terminus of the peptide sequence.
  • HSA Human Serum Albumin
  • NMU Neuromedin U
  • NMU peptide precursors (SEQ ID NOs: 5 to 9) were synthesized by solid phase using Fmoc/tBu chemistry on a peptide synthesizer ABI433A (Applied Biosystems).
  • ABI433A Applied Biosystems
  • the peptides were synthesized by solid phase using Fmoc/t-Bu chemistry on a peptide multisynthesizer Simphony (Protein Technologies Inc.) or APEX396 (AAPPTEC).
  • Simphony Protein Technologies Inc.
  • APEX396 APEX396
  • a resin aminomethylated polystirene LL 100-200 mesh, 0.41 mmol/g
  • 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-diisopropyIethyIamine) solution 2 M in NMP.
  • the side chain protecting groups were: tert-butyl for Asp, GIu, Ser and Tyr; trityl for Asn, and GIn; 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl for Arg.
  • the NMU peptide sequence was modified linking to the N-terminus of the NMU two Ttds spacers (13-amino-4 5 7,10-trioxa-tridecayl succinamic acid, NeoMPS Part# FAl 8801) and a maleimidobutiryl reactive group for covalently joining to Cys34 of the HSA.
  • the NMU peptide sequence was modified by linking to the N-terminus of the NMU two Ttds spacers and an iodoacetyl reactive group for covalently joining to Cys34 of the HSA.
  • the NMU peptide sequences spanning residues 12-25 and 18-25, respectively, were modified by linking to the N-terminus of the NMU two Ttds spacers and an iodoacetyl reactive group for covalently joining to Cys34 of the HSA.
  • the NMU peptide sequence spanning residues 18-25 was modified by linking to the N-terminus of NMU an Oxa24 spacer and an iodoacetyl reactive group for covalently joining to Cys34 o f the HSA.
  • the N-terminal derivatization with the maleimido group was performed at the end of the peptide assembly by reaction with a four-fold excess of activated 4- maleimidobutyric acid (Fluka, Cat No 63174) over the resin free amino groups for one hour.
  • the 4-maleimidobutyric acid was activated with equimolar amounts of DIPC (1,3- Diisopropylcarbodiimide) and HOBt (Hydroxybenzotriazole) in DMF.
  • the N-terminal derivatization with the iodoacetyl group was performed at the end of the peptide assembly by reaction with a three-fold excess of iodoacetic anhydride (Aldrich, Cat No 284262) over the resin free amino groups for 15 minutes.
  • the acylation reaction for linking the Oxa24 spacer to the N-terminus was performed for two hours using a two-fold excess of Fmoc-N-amido-dPEG24-acidTM (Quanta Biodes ⁇ gn Ltd., Product No 10313) over the resin amino groups.
  • the Fmoc-N-amido-dPEG24-acidTM was dissolved in DCM
  • the dry peptide-resins were individually treated with 20 mL of the cleavage mixture, 88% TFA, 5% phenol, 2% triisopropylsilane and 5% water (Sole, N. A. and G. Barany, 1992, J. Org. Chem. 57:5399-5403) 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 H2O, 20% acetonitrile, and lyophilized. The crude peptides were purified by reverse-phase HPLC using semi-preparative
  • ReproSil-Pur C4 column 25 x 200 mm, 15 ⁇ m) or Waters X-Bridge C18 (19 x 150 mm, 10 ⁇ m) and using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, flow rate 30 mL/min.
  • Analytical HPLC was performed on a Waters UPLC Acquity BEH 130A Cl 8 column (2.1 x 100 mm, 1.7 ⁇ ra), flow rate 0.4 mL/min, 45 0 C.
  • the purified peptides were characterized by electrospray mass spectrometry on a Micromass LCZ platform.
  • HSA-NMU conjugates were then isolated through size exclusion chromatography (SEC) and characterized by ESI Q-ToF Hybrid System (Q-Star XL, Applied Biosystems).
  • a first step the Cys34 in the HSA was reduced.
  • an aliquot of 200 ⁇ L (50 mg) of human serum albumin (Cortex, Cat. No. CP0925U; 25% solution) was diluted with 2.3 niL PBS pH 7.4 (20 mg HSA/mL; 300 ⁇ M) and the resulting mixture was reacted with three molar equivalent of dithiothreitol (25 ⁇ L; 0.1 M solution of DTT, Aldrich 45,777-9) for one hour at room temperature.
  • the crude reaction mixture was loaded on a PD-10 pre-packed desalting column (GE Healthcare, cat.17-0851-01) and eluted with 3.5 mL of PBS pH 6.5.
  • concentration of HSA was determined by UV absorbance at 280 nm ( ⁇ ⁇ 33 500 M ⁇ l cirri).
  • the percentage of the fully reduced Cys34 sulfhydryl group in the sample was determined by mass spectroscopy on a Q-Star XL Applied Biosystems spectrometer and was estimated as 90-92% (MW 66440) together with a major contamination of 8-10% of HSA Cys34 of higher oxidation species (MW 66472).
  • NMU peptide precursor Conjugation of the NMU peptide precursor to the HSA was by means of a maleimido reactive group to make a comparator molecule. 2.1 mL of the solution of Cys- reduced HSA (14.3 mg/niL; 215 ⁇ M in PBS pH 6.5) was reacted with 1.2 molar equivalent of the NMU peptide precursor (SEQ ID NO: 5) (2.1 mg of peptide in 0.6 mL in PBS pH 6.5) for 18 hours at room temperature.
  • HSA-NMU peptide conjugate was purified by size exclusion chromatography (SEC) using a High-Load 26/60, Superdex 75 prep, grade (Amersham Biosciences) in PBS pH 7.4, flow rate 2.5 mL/min.
  • SEC size exclusion chromatography
  • the purified HSA-NMU conjugate was characterized by ESI Q-ToF Mass spectrometry: found MW 70291 Da; calculated MW 70290 Da.
  • the conjugate net content (17 mg) was determined by UV absorbance at 280 nm (total HSA content) and by mass spectrometry (HSA-NMU conjugate relative content).
  • Figure IA shows the structure of the NMU43 peptide precursor and the HSA-NMU43 conjugate.
  • HSA-NMU79 conjugate was as follows. Purification of rHSA was by IEX chromatography. In order to isolate a free thiol Cys34 albumin sample, an aliquot (30 mg) of recombinant human serum albumin (rHSA, Recombumin ® Novozymes) was purified by ion exchange (IEX) chromatography on a Resource Q anion exchange column 6 mL (Amersham Biosciences, Cat Nr. 640262) and using as buffer eluents (A) 25 mM phosphate pH 7.0 and (B) 125 mM phosphate pH 7.0, flow rate 2.5 mL/min.
  • IEX ion exchange
  • NMU peptide precursor Conjugation of the NMU peptide precursor to the HSA was by means of an iodoacetamide reactive group.
  • 3.5 mL of a solution of IEX purified rHSA (6.25 mg/mL; 95 ⁇ M in 50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5 buffer) was reacted with 1.3 molar equivalent of the NMU peptide precursor (SEQ ID NO: 6) (1.75 mg; 455 nmol resuspended in 100 ⁇ L H2 ⁇ ) for 24 hours at room temperature.
  • ESI Q-ToF mass spectrometry analysis showed
  • the resulting crude rHSA-NMU peptide conjugate was purified by size exclusion chromatography (SEC) using a High-Load 26/60, Superdex 75 prep, grade (Amersham Biosciences) in PBS pH 7.4, flow rate 2.5 mL/min.
  • the purified rHSA-NMU conjugate was characterized by ESI Q-ToF Mass spectrometry: found MW 68779 Da; calculated MW 68784 Da.
  • the conjugate net content (10.3 mg) was determined by UV absorbance at 280 nm.
  • Preparation of HSA-NMUl 04 conjugate was as follows.
  • ESI Q-ToF mass spectrometry analysis showed > 98% completed reaction.
  • the resulting crude rHSA-NMU peptide conjugate was purified by size exclusion chromatography (SEC) using a High-Load 26/60, Superdex 75 prep, grade (Amersham Biosciences) in PBS pH 7.4, flow rate 2.5 niL/min.
  • the purified rHSA-NMU conjugate was characterized by ESI Q-ToF Mass spectrometry: found MW 68192 Da; calculated MW 68198 Da.
  • the conjugate net content (4.25 mg) was determined by UV absorbance at 280 nm.
  • HSA Human Serum Albumin
  • 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-Iysine 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, 0.1% BSA, 2.5 mM probenecid (Sigma) and 1.6 mM TR40). All reagents are from Invitrogen unless otherwise noted.
  • 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% 129S ⁇ /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 Lp, 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 I) 5 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;/? values ⁇ 0.05 were reported as significant and are denoted with an asterisk).
  • FIGs 2A and 2B show that acute peripheral administration of HSA-NMU43 significantly reduced food intake in diet-induced obese mice for two days post dose. At the highest dose tested, food intake was reduced by 80% relative to vehicle treated animals. On day 2 post dose of HSA-NMU, food intake was reduced by 40%. Significant reductions in body weight were also observed.
  • Figures 3 A, 3B, and 3C illustrate the finding that the anorectic effects of HSA- NMU43 are mediated by the contribution of both the NMURl and NMUR2 receptors. Acute administration of HSA-NMU43 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 Nmur2-deficient mice. The data indicate that HS A-NMU43 evokes reductions in food intake on day 2 primarily through the NMUR2 receptor.
  • NMURl knockout mice were generated using standard homologous recombination techniques as described in the previous example.
  • Ad libitum fed male diet- induced obese mice were weighed and dosed either Lp. 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 I) 5 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).
  • FIG. 4 A and 4B shows that NMU79 can reduce food intake for up to three days following a single administration.
  • NMU79 dose-dependently reduced food intake with a minimal efficacious dose of lmpk for overnight food intake.
  • the effects on glucose excursion of HSA-conjugated NMU analogs were evaluated in an oral glucose tolerance test (OGTT).
  • a glucose challenge (given orally at 2 g/kg) was then administered. Blood samples were taken after 15, 30, 60, 90, and 120 minutes and glucose levels were determined with the glucose oxidase method.
  • Figure SA shows that the glucose levels at multiple time points after the challenge were reduced at 0.3 and lmpk NMU79.
  • Figure 5B shows the area under the curve (AUC) for the time course of the study. No effect on glucose excursion was observed at 0.1 mpk NMU79.
  • EXAMPLE 5 In the this example, different length spacers with a haloacetyl functional group for conjugating NMU to HSA were evaluated.
  • the conjugation conditions were as reported for the preparation of HSA-NMU79 conjugate in Example 1.
  • the spacers tested were (Ttds)2, (Ttds)3, and Txa-(Ttds)2-
  • the presence of spacer comprising two or three Ttds units ((Ttds)2 or (Ttds)3) yielded a homogeneous conjugate bearing mostly only one copy of NMU peptide precursor per molecule of HSA, as targeted (most likely at the free Cys 34 of HSA) ( Figure 6 A and 6B).
  • haloacetyl group at the N-terminus of NMU peptide sequence might not be able to insert into the HSA Cys 34 's pocket unless a sufficiently long and flexible spacer exists between the functional reactive group and the N-terminus of NMU.
  • a spacer comprising two, three, or more Ttds units allows conjugation of NMU to HSA to be achieved with good yields, yielding a highly pure conjugate in which there is essentially one NMU molecule per molecule of HSA, or wherein at least about 95% of the HSA molecules have one molecule of NMU attached thereto.
  • This example provides a prophetic example of conjugating NMU79 to an Fc fragment of an antibody that avoids denaturing the internal disulfide bonds in the Fc fragment.
  • a recombinant Fc fragment is provided that has an iV-terminal cysteine residue (CysFc).
  • Conjugation of the NMU79 peptide precursor to the CysFc is by means of an iodoacetamide or bromoacetamide reactive group.
  • the resulting CysFcTM NMU79 peptide conjugate can be purified by size exclusion chromatography (SEC) or Protein A affinity chromatography.
  • SEC size exclusion chromatography
  • the purified CysFc-NMU79 conjugate can be characterized by ESI Q- ToF Mass spectrometry.
  • This example provides another prophetic example for conjugating NMU precursors having iodoacetamide or bromoacetamide reactive groups to the cysteine groups of Fc fragments prepared from antibodies.
  • NMU precursors having iodoacetamide or bromoacetamide reactive groups can be conjugated to the now available reduced cysteine residues.
  • the resulting peptide-conjugated IgG is much less susceptible to Papain cleavage, the standard protocol to generate Fc fragments from full antibodies. If the antibody is first cleaved by papain and the Fc fragment thus generated is then partially reduced and the reduced cysteine residues conjugated to NMU, stability of the Fc fragment is usually compromised.
  • a conjugation protocol based on Equilibrium transfer alkylating cross-link can be used.
  • EAC Equilibrium transfer alkylating cross-link
  • Liberatore et ai Bioconjugate Chem. 1: 36- 50 (1990) describes this alternative conjugation strategy that retains the covalent disulfide linkages of antibodies or Fc-fragment by creating disulfide bridges (See Figure 9 for a schematic of the synthesis of ETAC).
  • ETAC reagents are three-functionalized reagents, two of which react with free Cys thiols generating a covalent bridge leaving one function available for conjugation. In the reaction, temporarily reduced intra- or inter-molecular covalent bonds are re-established thus maintaining their stabilizing role in the diraeric Fc fragment (or a complete IgG).
  • ETAC (4-[2,2-bis[(p-to ⁇ ylsulfonyl)- methyl] acetyl]benzoic acid; 1.) is reacted with iV-hydroxysuccinimide (NHS) as described in Shaunak et al:, Nature Chem. Biol. 2: 312-313 (2006). Briefly, as an example, under an argon atmosphere, a stirred suspension of ⁇ . (2 g, 4 mmol), NHS (0.483 g, 4.2 mmol) and anhydrous dichloromethane (5 ml, Aldrich) is cooled using an ice bath.
  • NHS iV-hydroxysuccinimide
  • Neat 1 ,3-diisopropylcarbodiimide (657 ⁇ L, 4.2 mmol, DIPC) is then added dropwise. After 1.5 hours, a further 60 ⁇ L of DIPC is added, and after 3 hours, the reaction mixture is passed through a non-absorbent cotton wool filter. The homogeneous filtrate is diluted with dichloromethane (about 30 mL), washed with water (2 x 15 mL) and dried with magnesium sulfate. Filtration under gravity and removal of volatiles under vacuum provides an ETAC reagent with an active NHS ester (ETAC-NHS ester, a). Compound 2 is reacted with H2N-(Ttds)2 ⁇ NMU peptide to produce BTAC-NH-
  • NMU peptide (3) is redissolved in acetone (15 mL) with gentle warming. The flask containing the stirred solution is then placed in an ice bath to precipitate the desired product 3 which is isolated using a # 3 sintered glass funnel and washed with chilled acetone (about 30 mL).
  • the ETAC-NH-(Ttds)2 ⁇ NMU peptide (3) can be then be conjugated to the Fc fragment as described in Liberatore et at (op. cit.) and shown in Figure 9.
  • the bis-sulfone ETAC-NH-(Ttds)2-NMU peptide (3) is converted to a mono-sulfone (4) by incubating overnight in phosphate buffered saline (PBS) at pH 7.8 (See Shaunak et ah, (op. cit).
  • PBS phosphate buffered saline
  • Compound 4 can then be conjugated to the Fc fragment following procedures described in Shaunak et al. (ibid.), Balan et al. (Bioconjugate Chem. 18: 61-76 (2007)), Brocchini et al. (Adv. Drug Del. Rev. 60: 3-12 (2008)), and U.S. Pub. Application No. 20060210526.
  • a modified ETAC compound, Methyl-Ketone Methyl-Ketone
  • MKE has been prepared. This compound is linked to the antibody as a bridge between two cysteine residues forming a disulfide linkage where it provides a stable ketone-functionality to which a peptide can be conjugated at a later stage. On one side this approach stabilizes the antibody or antibody-fragment by re-establishing covalent bonds. However, it also offers the additional advantage that large amounts of "conjugation-ready" MKE modified antibodies (or fragments thereof) can be prepared, stored away and subsequently conjugated when needed.
  • MKE Methyl Ketone-ETAC

Abstract

Neuromedin U receptor agonists comprising neuromedin U or derivative or analog thereof conjugated to a carrier protein for use in the treatment of metabolic disorders such as obesity and diabetes are disclosed.

Description

TITLE OF THE INVENTION NEUROMEDIN U RECEPTOR AGONISTS
BACKGROUND OF THE INVENTION (1) Field of the Invention
The present invention relates to neuromedin U (NMU) receptor agonists wherein the NMU peptide or analog thereof is covalently conjugated to a carrier protein. In particular embodiments, the present invention relates to NMU receptor agonists wherein the carrier protein is serum albumin (HSA), lactoferrin, immunoglobulin, or immunoglobulin Fc fragment. In further still embodiments, the present invention relates to NMU receptor agonists wherein the NMU peptide or analog thereof is conjugated to the carrier protein in a thioether acetamide linkage or a beta-thiosulfonyl linkage. The NMU receptor agonists can. be used for the treatment of metabolic diseases such as obesity and diabetes.
(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 et al, J. Endocrinol.185: 151-164 (2005)). The internal energy status of an animal affects expression and release of NMU as well (Wren et al, op. cit.).
Two high affinity NMU 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 administration 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^ mice and present in Nmur2~ " 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" transgenic mice, body weight, body composition and food intake are largely unaffected by chronic peripheral administration of rat NMU-23.
In general, NMU receptor agonists administered peripherally have a short half- life. Therefore, there is a need for NMU receptor agonists that have an extended half-life in plasma serum.
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. The carrier protein enhances the half-life of the NMU peptide in the plasma serum compared to the half-life of a non-conjugated NMU peptide under similar conditions. The linkage between NMU peptide and the carrier protein is a stable linkage, which further enhances the stabilizing effect of the carrier protein on the NMU peptide by increasing the length of time in which NMU peptide is conjugated to the carrier protein. The stable linkages are formed by reacting a non-maleimido or non-succinimidyl reactive group that is capable of reacting with a functional group on the carrier protein covalently attached to a spacer moiety at or near the N- terminus of the NMU peptide. Examples of reactive groups include but are not limited to iodoacetamide, bromoacetamide, and vinyl sulfonate reactive groups. The NMU receptor agonists herein have a linkage between the peptide and the carrier protein that is stable for greater than 20 to 48 hours or more. While the carrier protein can be any protein, in particular embodiments, the earner protein is selected from the group consisting of serum albumin, immunoglobulins, lactoferrin, and Fc fragments. In general, preferably the carrier protein is non- immunogenic in the host. 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)
pr-Z2-Zl-peptide-Z3
wherein the peptide has the amino acid sequence Xl-χ2-χ3-χ4-χ5-χ6-χ7-χ8_ χ9-χl0.χl l_χl2_χl3_χl4.χl5_χl6.χl7.χl 8.χl9.χ20.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 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 χl9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 |g absent, Leu, GIy, sarcosine (S ar), 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 X24 js 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; Zl is a spacer; Z2 is one or more non-maleimido (3-thiosuccinimidyl ether) or non-succϊmmidyl linkages; and Z3 is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group. Carrier protein Pr can be any protein preferably non-immunogenic but in particular embodiments, the Pr is selected from the group consisting of serum albumin, immunoglobulins, lactoferrin, and Fc fragments.
In particular aspects, the peptide has the amino acid sequence χl-χ2.χ3.χ4_χ5_ χ6-χ7.χ8-χ9.χl 0-χl 1.χl2.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-Val- Asp-Glu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-Gln-Ser-Arg-Gly-Xl8-Xl9.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 X19 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, AIa or Trp; χ22 is Arg, Lys, Harg, AIa, or Leu; amino acid χ23 Js Pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid X25 js 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 Xl—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-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, GIu3 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 X5 is Arg, Lys, Harg, Ala, or Leu; amino acid χ6 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.
Spacer Zl has a proximal end and a distal end: the distal end is covalently attached to the NMU peptide and the proximal end is covalently attached to a reactive group capable of reacting with a functionality on the carrier protein to form a stable covalent linkage Z2. In general, the stable linkage has a half-life of more than 20 hours in 10% plasma serum at 37°C. In particular embodiments, the linkage has a half-life of more than 36 hours in 10% plasma serum at 37°C. In further embodiments, the linkage has a half-life of more than 48 hours in 10% plasma serum at 37°C. Z2 does not include maleimido (3-thiosuccinimidyl ether) or succinimidyl linkages.
In particular aspects, ZX is a spacer comprising one to four units of Ttds (13- amino-4,7f10-trioxa-tridecayl succinamic acid), which in further aspects comprises two units of Ttds (13-amino-4,7,10-trioxa-tridecayl succinamic acid). In further aspects, the spacer includes on the proximal end a reactive group selected from the group consisting of iodoacetamide, bromoacetamide, and vinyl sulfonate. The iodoacetamide or bromoacetamide reactive group reacts with thio functionalities on the carrier protein to form stable tbioether acetamide linkages. The vinyl sulfonate reactive group reacts with thio functionalities on the carrier protein to form stable beta-thiosulfonyl linkages. Thus, in particular embodiments, the linkage Z^ is a thioether acetamide linkage or beta-thiosulfonyl linkage.
In further still aspects, the peptide has the amino acid sequence of human NMU peptide as shown in SEQ ID NO: 10 and which can optionally have a protecting group at the C- terminus. In particular aspects and by way of example, an neuromedin U receptor agonist is provided as shown in Figure IB that has the formula
Figure imgf000006_0001
. As shown in this aspect, spacer Zl comprises two units of Ttds (13-amino-4,7,10-trioxa- tridecayl succinamic acid) in which the distal end of the spacer is covalently linked to the N- terminus of the NMU peptide and the proximal end of the space is covalently linked to the thiol group of cysteine 34 of human serum albumin in a thioether acetamide linkage (Z^). The agonist further includes an amide protecting group (Z3) at the C-temiinus of the peptide.
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)
Pr~Z2-Zl-peptide-Z3
wherein the peptide has the amino acid sequence Xl-χ2-χ3-χ4-χ5-χ6-χ7-χ8- X9.χlθ.χl LX12-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 χ!8 is absent, Tyr or D-Tyr, Leu, Phe, VaI5 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 X21 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; Pr is a carrier protein; Zl is a spacer; Z2 is one or more non-maleimido (3-thiosuccinimidyl ether) or non-succinimidyl linkages; and 7? is NH2 or an optionally present protecting group that, if present, is joined to the
C-terminal carboxy group. Carrier protein Pr can be any protein but in particular embodiments, the Pr is selected from the group consisting of serum albumin, immunoglobulins, lactoferrin, Fab fragment, scFv, and Fc fragment.
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-X2-χ3-.χ4-χ5- χ6-χ7-χ8-χ9-χl O.χl I.χl 2.χl3,χl4_χl 5_χl6_χl7-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-Val- Asp-Glu-Glu-Phe-Gln-Ser-Pro-Phe-Ala-Ser-Gln-Ser-Arg-Gly-Xl8_χl9-χ20-χ21-χ22-χ23. χ24.χ25 (SEQ ID NO:3) wherein amino acid χl8 is absent, Tyr or D-Tyr, Leu, Phe5 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; 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 js Asn, any D- or L-amino acid, NIe or D-NIe5 D-AIa or Ala.
In a further still aspect, the peptide comprises the amino acid sequence Xl— 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 X2 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 X4 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 χό is Pro, Ser, Sar, AIa 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-Me5 D-AIa or Ala. Spacer Zl has a proximal end and a distal end: the distal end is covalently attached to the NMU peptide and the proximal end is covalently attached to a reactive group capable of reacting with a functionality on the carrier protein to form a stable covalent linkage Z2. In general, the stable linkage has a half-life of more than 20 hours in 10% plasma serum at 37°C. In particular embodiments, the linkage has a half-life of more than 36 hours in 10% plasma serum at 370C. In further embodiments, the linkage has a half-life of more than 48 hours in 10% plasma serum at 370C. Z^ does not include maleimido (3-thiosuccinimidyl ether) or succinimidyl linkages.
In particular aspects, Zl is a spacer comprising one to four units of Ttds (13- amino-4,7,10-trioxa-tridecayl succinamic acid), which in further aspects comprises two units of Ttds (13-amino-4,7,10-trioxa-tridecayϊ succinamic acid). In further aspects, the spacer includes on the proximal end a reactive group selected from the group consisting of iodoacetamide, bromoacetamide, and vinyl sulfonate. The iodoacetamide or bromoacetamide reactive group reacts with thio functionalities on the carrier protein to form stable thioether acetamide linkages. The vinyl sulfonate reactive group reacts with thio functionalities on the carrier protein to form stable beta-thiosulfonyl linkages. Thus, in particular embodiments, the linkage Z2 is a thioether acetamide linkage or beta-thiosulfonyl linkage.
In further still aspects of the method, the peptide has the amino acid sequence of human NMU peptide as shown in SEQ ID NO: 10 and which can optionally have a protecting group at the C-terminus. In particular aspects of the method, the neuromedin U receptor agonist has the formula
Figure imgf000008_0001
Further provided are peptides that can be conjugated to a carrier protein to form neuromedin U receptor agonists that have the formula
Rn-Zl-ρeptide-Z3
wherein the peptide has the amino acid sequence Xl-χ2-χ3-χ4_χ5-χ6-χ7-χ8-χ9-χl0.χl 1- χl 2-χl3-χl4-χl5-χl6-χl7-χl8-χl 9-χ20~χ2 Lχ22-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 χ!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 X2I is Phe, NMe-Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Tip; X22 is 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 χ25 is Asn, any D- or L- amino acid, NIe or D-NIe, D-AIa or Ala; Z^ is a spacer; Rn is one or more non-maleimido or non-succimidyl reactive groups; and 7? is NH2 or an optionally present protecting group that, if present, is joined to the C-termlnal carboxy group.
In particular aspects, the peptide has the amino acid sequence Xl-χ2-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-Val-
Asp-Glu-GlU"Phe-Gln-Ser-Pro-Phe-Ala-Ser-Gln-Ser-Arg-Gly-Xl8-Xl9.χ20.χ21-χ22.χ23. χ24.χ25 (SEQ ID NO:3) wherein amino acid χl8 is absent, Tyr or D-Tyrs Leu, Phe, VaI, GIn, NIe, GIu or D-GIu, Asp, AIa5 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 χ20 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; χ22 is 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 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~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 AIa5 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 χ5 is Arg, Lys, Harg, AIa, or Leu; amino acid χ6 is Pro, Ser, Sar, Ala or Leu; amino acid X? 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.
Spacer Zl has a proximal end and a distal end: the distal end is covalently attached to the NMU peptide and the proximal end is covalently attached to one or more reactive groups Rn capable of reacting with a functionality on the carrier protein to form a stable covalent linkage. In general, the reactive groups form a stable linkage that has a half-life of more than 20 hours in 10% plasma serum at 37°C. In particular embodiments, the linkage has a half-life is more than 36 hours in 10% plasma serum at 370C. In further embodiments, the linkage has a half-life of more than 48 hours in 10% plasma serum at 37°C. Rn does not include maleimido or succinimidyl reactive groups. In particular aspects, Zl is a spacer comprising one to four units of Ttds (13- araino-4,7,10-trioxa-tridecayl succinamic acid), which in further aspects comprises two units of Ttds (13-amino-4,7,10-trioxa-tridecayl succinamic acid). In particular aspects, the spacer includes on the proximal end at least one reactive group Rn selected from the group consisting of iodoacetamide, bromoacetamide, and vinyl sulfonate. The iodoacetamide or bromoacetamide reactive group reacts with thio functionalities on the carrier protein to form stable thioether acetamide linkages. The vinyl sulfonate reactive group reacts with thio functionalities on the carrier protein to form stable beta-thiosulfonyl linkages. Thus, in particular embodiments, the linkage formed by the iodoacetamide, bromoacetamide, and vinyl sulfonate reactive groups are thioether acetamide or beta-thiosulfonyl linkages, respectively. In further aspects, Rn is an
ETAC reactive group capable of forming a three carbon bridge between two cysteine residues in a carrier protein involved in a disulfide linkage. The ETAC can be a bis-sulfone or a mono- sulfone.
In further still aspects, the peptide has the amino acid sequence of human NMU peptide as shown in SEQ ID NO: 10 and which can optionally have a protecting group at the C- terminus. In particular aspects of the method, the neuromedin U receptor agonist has the formula
1 ^^^°^^0^^o^^^^lN^^~.o^^o^^o^^^ J^^ FRVDEEFQSPFASQSRGYFLFRPRN-CONH2 1 T o I {SEQ ID NO 10)
wherein I is an iodo group.
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: 10 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. In general, in any one of the above embodiments when the carrier protein is serum albumin or lactoferrin, the serum albumin or lactoferrin will usually be of human origin or have an amino acid sequence substantially the same as the amino acid sequence of human serum albumin (HSA) or lactoferrin. The human serum albumin or lactoferrin can be recombinantly produced. Further included are human serum albumin or lactoferrin fusion proteins. In the any one of the above embodiments or aspects in which the carrier protein is an immunoglobulin or antibody molecule, the immunoglobulin can be an IgG molecule and includes IgGi, ϊgQ2» IgCb, ^d IgG4 and subspecies thereof. In particular aspects of the above, the immunoglobulin is selected from the group consisting of IgA5 IgM, IgE5 camel heavy chain, and llama heavy chain. In further aspects, the IgG can be a catalytic antibody, for example as described in U.S. Patent Application No s. 7205136; 4888281 ; 5037750 to Schochetman et al., U.S. Patent Application Nos, 5733757; 5985626; and 6368839 to Barbas, III et al. As used herein, the terms "antibody," "immunoglobulin/' "immunoglobulins" and "immunoglobulin molecule" are used interchangeably,
The term "Fc" fragment refers to the 'fragment crystallized' C-terminal region of the antibody containing the Co2 and CH3 domains. The term "Fab" fragment refers to the 'fragment antigen binding' region of the antibody containing the VJJ, CH 1* VL and CL domains. Fc fragments include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation and those produced recombinantly. The term "scFv" refers to a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine or glycine.
The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (1975) Nature, 256:495, or may be made by recombinant DNA methods (See, for example, U.S. Pat. No. 4,816,567 to Cabilly et al).
Immunoglobulins, ScFv, Fabs, and Fc fragments further include immunoglobulins chimeric, human, and humanized antibodies and Fc fragments; immunoglobulin and Fc fusion proteins; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (See, for example, Intracellular Antibodies: Research and Disease Applications, (Marasco, ed., Springer- Verlag New York, Inc., 1998).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure IA shows the synthesis of HSA-NMU43 conjugate in which NMU43 is conjugated to HSA by means of a maleimido reactive group. NMU43 having a (Ttds)2 spacer with a maleimide reactive group is conjugated to Cys34 of HSA in a 3-thiosuccinimidyl ether linkage.
Figure IB shows the synthesis of HSA-NMU79 conjugate in which NMU79 is conjugated to HSA by means of a iodoacetamide reactive group. NMU79 having a (Ttds)2 spacer with an iodoacetamide reactive group is conjugated to Cys34 of HSA in an thioether acetamide linkage.
Figure 2A shows that acute administration of HSA-NMU43 significantly reduces food intake in diet-induced obese mice. Figure 2B shows that acute administration of HSA-NMU43 significantly reduces body weight in diet-induced obese mice.
Figure 3 A shows that acute administration of HSA-NMU43 significantly reduces food intake and body weight in Nmurl- and Nmur2-deficient mice. Figure 3B shows that acute administration of HSA-NMU43 significantly reduces food intake and body weight in Nmurl- and Nmur2-deficient mice after two days.
Figure 3 C shows that acute administration of HSA-NMU43 significantly reduces food intake and body weight in Nmurl- and Nmur2-deficient mice after 3 days.
Figure 4A shows that acute administration of HSA-NMU79 dose-dependently reduces food intake in wild-type mice. Significant effects on food intake were observed for three days after a single dose.
Figure 4B shows that acute administration of HSA-NMU79 dose-dependently reduces body weight in wild-type mice. Significant effects on food intake were observed for three days after a single dose. Figure 5A shows that acute administration of HSA-NMU79 dose-dependently enhances glucose excursion during an OGTT. Significant effects on glucose were observed at 0.3 and 1 mpk of NMU79.
Figure SB shows the area under the curve (AUC) for the time course of the OGTT in Figure 5A. Figure 6A-D show LC-MS analyses of HSA-NMU conjugates with different spacers. Figure 6A: spacer is (Ttds)2; Figure 6B: spacer is (Ttds)3; Figure 6C: spacer is Txa- (Ttds)2; Figure 6D: no spacer.
Figure 7 shows a time-course analysis of HSA-NMU43 conjugate in 10% mouse plasma (mass spectrum reconstructions). Figure 8 shows a time-course analysis of HSA-NMU79 conjugate in 10% mouse plasma (mass spectrum reconstructions).
Figure 9 shows a schematic view of an ETAC reaction using ETAC reagent (4- [2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid) in the context of an IgGj isotype. Insertion of ETAC-bridges into the IgGi molecule is shown and the function group on the ETAC-bridges available for conjugation are shown by the X.
Figure 10 shows a schematic view of preparing NMU-(Ttds)2-ETAC precursor molecule for conjugating to a carrier protein in a disulfide bridge between two cysteine residues involved in a disulfide linkage. Compound 3 is a bis-sulfone precursor and compound 4 is a mono-sulfone precursor. Figure 11 shows the synthesis of Methyl Ketone-ETAC (MKE) 5 from ETAC 4.
DETAILED DESCRIPTION OF THE INVENTION The present invention provides neuromedin U receptor agonists in which the neuromedin U (NMU) peptide or analog thereof is conjugated to a carrier protein in a covalent linkage that is more stable than a maleimido (3-thiosuccinimidyl ether) or succinimidyl linkage under physiological conditions and compositions comprising the same. Suitable carrier proteins are preferably non-immunogenic and include but are not limited to human serum albumin (HSA), immunoglobulins, lactoferrin, and immunoglobulin Fc fragments. Conjugating a peptide to a carrier protein can enhance the half-life of the peptide in serum by either steric hindrance in which the peptide by virtue of its conjugation to the carrier protein is rendered less accessible to endogenous proteases or by compartmentalizing the peptide in a way that avoids or delays clearance of the peptide from serum or both. In general, conjugating a small peptide to carrier proteins that increases the molecular weight of the peptide to greater than 40 kDa will substantially reduce renal clearance, which can substantially increase the half- life of the peptide in blood or plasma serum. Conjugation linkages with enhanced stability as described herein extends the time the peptide is conjugated to the carrier protein. This in turn facilitates and prolongs the effects of the carrier protein on the half-life of the peptide in blood or plasma serum. Conjugation of peptides to carriers 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 et al., 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: 18834888 (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 glycosylation 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 drugs 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-HIV 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 (Vø) 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. The conjugation further excludes embodiments where the linkage to the carrier protein is by means of a maleimido or succinimidyl reactive group that targets a functional group on the carrier protein.
As shown in the examples, NMU peptides conjugated to human serum albumin (HSA) as shown herein has an efficacy profile that is similar to the profile of NMU PEGylated at the JV-terminus as disclosed in Published PCT Application No. WO2007109135. Therefore, one or more of the neuromedin U receptor agonists according to the present invention can be administered 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 according to the present invention 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 a 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. In particular embodiments, the neuromedin U receptor agonists can be used to treat multiple disorders in an individual. As will be apparent to one of ordinary skill in the art in view of the disclosure herein, 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.
In general, the neuromedin U receptor agonists of the present invention comprise the general formula (I)
Pr-Z2-Zl-peptide-Z3
wherein the peptide has the amino acid sequence Xl-χ2.χ3.χ4.χ5.χ6-χ7.χ8- X9.Xl0.xl l.xl2.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 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, GIu5 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 AIa; Pr is a carrier protein; Zl is a spacer; Z2 is one or more non-maleimido (3-thiosuccinimidyl ether) or non-succinimidyl linkages; and 1? is NH2 or an optionally present protecting group that, if present, is joined to the
C-terminal carboxy group. Carrier protein Pr can be any protein but in particular embodiments, the Pr is selected from the group consisting of serum albumin (HSA), immunoglobulins, lactoferrin, and Fc fragments.
In particular aspects, the peptide has the amino acid sequence χl-χ2.χ3.χ4.χ5_ χ6-χ7_χ8.χ9.χl O.χl 1.χl 2.χl 3.χl4jχl 5.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-Gin-Ser-Arg-Gly-Xl 8-Xl 9.χ20.χ21 _χ22.χ23. χ24.χ25 (SEQ ID NO: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 Xl9 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 X21 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 X25 is Asn5 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- X3.χ4.χ5-χ6-χ7.χ8 (SEQ ID N0:4) wherein amino acid Xl is absent, Tyr or D-Tyr, Leu, Phe, VaI, GIn, NIe, GIu or D-GIu5 Asp, AIa, 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 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 χ6 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. In particular embodiments, the peptide comprises the amino acid sequence of human NMU, which is FRVDEEFQSPFASQSRGYFLFRPR (Phe-Arg-Val-Asp- Glu-Glu-Phe-Gln~Ser-Pro-Phe-Ala-Ser~Gln-Ser-Arg-GlyTyr-Phe-Leu-Phe-Arg-Pro-Arg: SEQ ID NO: 10). In an aspect exemplified in the examples, the neuromedin U receptor agonist is conjugated to the cysteine at position 34 of human serum albumin (HSA). In particular aspects, the neuromedin U receptor agonist has the formula
Figure imgf000016_0001
Spacers (Z 1) are chemical moieties that covalently link or connect reactive groups to the modified NMU peptide. The spacer has a distal end and a proximal end. The distal end of the spacer is attached to the jV-terminus of the peptide by means of an amide linkage. The proximal end of the spacer has a functional or reactive group that is available for conjugating the peptide-spacer to an amino acid in the carrier protein. In general, spacers may comprise one or more alkyl groups such as methyl, ethyl, propyl, butyl, etc. groups, alkoxy groups, alkenyl groups, alkynyl groups, or amino groups substituted by alkyl groups, cycloalkyl groups, polycyclic groups, aryl groups, polyaryl groups, substituted aryl groups, heterocyclic groups, and substituted heterocyclic groups. In particular aspects, the linker is a derivative of a compound selected from the group consisting of acyloxymethylketones; aziridines; diazomethyl ketones; epoxides; iodo-, bromo-or chloroacetamides; α-haloesters; α-haloketones; sulfoniums; chloroethylsulfides; O-alkylisoureas; alkyl halides; vinylsulfones; acrylamides; acrylates; vinylpyridines; organometallic compounds; aryldisul fides; thiosulfonates; aldehydes; nitriles; α- diketones; α-ketoamides; α-ketoesters; diaminoketones; sernicarbazones; and dihydrazides. Spacers may also comprise poly ethoxy aminoacids such as AEA ( (2-amino) ethoxy acetic acid) or a AEEA ([2-(2-amino) ethoxy)] ethoxy acetic acid).
Useful spacers include polyethylene glycol polymers including but not limited to polymers comprising one, two, three, four, five, or more units or molecules of 13-amino-4,7,10- trioxa-tridecayl succinamic acid (Ttds or PEG- 13) units in tandem or ethoxy polymers comprising one to twenty-four or more ethoxy units. The examples illustrate embodiments in which the NMU-HSA conjugate comprises a Ttds dimer or trimer polymer or an ethoxy polymer that includes 24 ethoxy units. In general, the chemical structure of the spacer is not critical since it serves primarily to provide a suitable distance between the reactive group and the peptide; however, in certain embodiments, the spacer may itself provide improved properties to the compositions of the present invention. For example, as shown by use of the Ttds dimer and trimer spacers in Example 5, a sufficiently long and flexible spacer between the functional reactive group and the N-terminus of NMU appears to facilitate conjugation to Cys34 of HSA and also to minimize attachment of multiple copies of NMU peptide to the HSA at sites other than the cysteine residue at position 34. Thus, a spacer that is about 22 to 150 A in length, which in particular embodiments is about 44 A to about 66 to 88 A length is particularly useful for conjugating NMU or analogs thereof to HSA. One Ttds unit has a length of about 22 A.
The inventors have found that iodoacetamide reactive groups form more stable linkages in serum than linkages formed with maleimido reactive groups. As shown in Example 6, HSA-NMU conjugates that have a maleimido (3-thiosuccinimidyl ether ) linkage have an apparent half-life in 10% plasma serum at 37°C of about 20 hours whereas HSA-NMU conjugates that have the more stable acetamide linkage are stable for the duration of the experiment, i.e., have a half-life of greater than 48 hours in 10% plasma serum at 370C. An example of another reactive group that forms more stable linkages than maleimido reactive groups are vinyl sulfonate groups. The advantage of using a reactive group that forms a covalent bond that is stable for long periods of time in serum as in the conjugates herein is that it enables the neuromedin U receptor agonists herein to be administered once weekly or even bi-weekly. Currently, the neuromedin U receptor agonists will be administered intravenously. Therefore, reactive groups that form stable covalent linkages to be able to administer the neuromedin U receptor agonists on a weekly or bi-weekly basis is a desirable. In general, the linkage in the conjugates herein should have a half-life in a solution containing about 10% plasma serum at 370C that is greater than 20 hours as determined by LC-MS. In further embodiments, the linkage should have a half-life greater than 36 hours, and in further still embodiments, the linker should have a half-life that is greater than 48 hours. In particular aspects, 72- comprises two stable linkages to the carrier protein such as those formed by an equilibrium transfer alkylating cross-link (ETAC) reagent or variant thereof. ETAC reagents have been described in Liberatore et al., Bioconjugate Chem, 1: 36-50 (1990), Shaunak et al, Nature Chem. Biol. 2: 312-313 (2006), Balan et al., Bioconjugate Chem. 18: 61-76 (2007), Brocchini et al, Adv. Drug Del. Rev. 60: 3-12 (2008), and U.S. Published Application No. 20060210526. In general, ETAC reagents comprise three reactive groups: two of the reactive groups are available for conjugating to the thiol groups of two cysteine residues involved in a disulfide linkage or bond on the carrier protein to form a three carbon covalent bridge between the two cysteine residues, which maintains the disulfide linkage and thus the tertiary structure of the protein. The third reactive group is conjugated to the proximal end of the spacer Zλ. In particular embodiments, the third reactive group is conjugated to the proximal end of the spacer Z* prior to conjugating to the carrier protein and in other embodiments, the third reactive group is conjugated to the proximal end of the spacer Zl subsequent to conjugating to the carrier protein.
Figure 9 provides an example of an ETAC reagent (4-[2,2-bis[(p~tolylsulfonyl)- methyl]acetyl]benzoic acid) and Figure 11 provides an example of an ETAC reagent modified to have a methyl ketone group (compound 5). These reagents are particularly useful for conjugating peptides to proteins such as antibodies and Fc fragments which rely upon disulfide bonds for stabilization.. For example, to provide a cysteine residue for conjugating NMU to IgG, the IgG molecule is partially reduced. This provides one or more reduced cysteine residues for conjugating the NMU peptide and spacer to the IgG molecule using the a reactive group that reacts with thiol groups. While IgG-NMU conjugates can be made, the resulting conjugates are less susceptible to papain cleavage, the standard method to generate Fc fragments conjugated to the NMU peptide from full antibodies. If instead, the IgG molecule is first cleaved by papain and the Fc fragment thus generated is then partially reduced and conjugated to NMU stability of the Fc fragment is usually compromised because the Fc fragment cannot form the proper disulfide bonds needed to maintain its structural integrity. The ETAC reagents and variants thereof overcome both of the above disadvantages because the ETAC reagent forms a bridge between two disulfide groups on the partially reduced IgG or Fc fragment thus the stabilizing role of the disulfide bridge at the conjugation location is reestablished.
Thus, linkage Z2 is a non-maleimido (3-thiosuccinimidyl ether) or succinimidyl linkage in which a functionality on the carrier protein (Pr) is covalently joined to spacer Z^ by means of a reactive group on spacer Tλ and which linkage is more stable than a maleimido (3- thiosuccinimidyl ether ) linkage toward hydrolytic cleavage in vivo. Functionalities are groups on the carrier protein to which the reactive group on the linker can react to form a covalent bond. Functionalities include hydroxyl groups for bonding to ester reactive entities; thiol groups for bonding to nialamides, maleimido, iodoacetyl, bromoacetyl, vinyl sulfone, imidates, and thioester groups; amino groups for bonding to carboxy, phosphoryl, or acyl groups on reactive entities; and carboxyl groups for bonding to amino groups. In the case of human serum albumin (HSA), the functionality that is usually targeted for conjugation is the cysteine residue at position 34. Functionalities can further include unnatural amino acids for which a there is a reactive group that can form a linkage with the unnatural amino acid (See Published U.S. Application No. 20080255045 and U.S. Patent Nos. 7,045,337 and 7,083,970). Therefore, a carrier protein can be engineered to include one or more non-natural amino acids capable of reacting with a reactive group anywhere within the carrier molecule. Thus, compounds can be provided in which the NMU peptide conjugated at any location within the carrier protein or to one, two, three, or more locations within the carrier molecule.
In general, a precursor molecule is prepared that comprises the NMU peptide or analog thereof co valently linked to the distal end of spacer Zl that has a non-maleimido or succinimidyl reactive group at the proximal end. These peptides that can be conjugated to a carrier protein to form neuromedin U receptor agonists have the formula
Rn-Zl-peptide-Z3
wherein the peptide has the amino acid sequence Xl-X2-χ3-χ4-χ5-χ6.χ7-χ8-χ9-χl0_χl 1. Xl2.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 X18 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 1S Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid χ20 is absent, Leu, GIy, sarcosine (S ar), 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 1S Arg, Lys, Harg, AIa5 or Leu; amino acid χ23 is Pro, Ser, Sar, Ala or Leu; amino acid χ24 is Arg, Harg or Lys; and amino acid χ25 js Asn, any D- or L- amino acid, NIe or D-NIe, D-AIa or Ala; Zl is a spacer; Rn is one or more non-maleimido or non-succinimidyl reactive groups; and Z3 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-X2-χ3-.χ4-χ5- χ6-χ7.χ8-χ9.χl0^χl l.χl2.χl3mχl4,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-G!u-Phe-Gln-Ser-Pro-Phe~Ala-Ser-Gln-Ser-Arg-Gly-Xl8-Xl9-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 AIa, 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, AIa or Trp; χ22 is Arg, Lys, Harg, Ala, or Leu; amino acid χ23 is Pro, Ser, Sar, Ala or Leu; amino acid X24 is Arg, Harg or Ly s; 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 Xl-X2- χ3-χ4-χ5..χ6-χ7-χ8 (SEQ ID N0:4) wherein amino acid χl is absent, Tyr or D-Tyr, 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 χ2 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 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 χ6 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-NIe5 D-AIa or Ala.
Spacer Zl has a proximal end and a distal end; the distal end is covalently attached to the NMU peptide and the proximal end is covalently attached to one or more reactive groups Rn capable of reacting with a functionality on the carrier protein to form a stable covalent linkage. In general, the reactive groups form a stable linkage that has a half-life of more than 20 hours in 10% plasma serum at 370C. In particular embodiments, the linkage has a half-life is more than 36 hours in 10% plasma serum at 370C. In further embodiments, the linkage has a half-life of more than 48 hours in 10% plasma serum at 370C. RΩ does not include maleimido or succinimidyl reactive groups. In particular aspects, Zl is a spacer comprising one to four units of Ttds (13- amino-4,7,10-trioxa-tridecayl succinamic acid), which in further aspects comprises two units of Ttds (13-amino-4,7,10-trioxa-tridecayl succinamic acid). In particular aspects, the spacer includes on the proximal end at least one reactive group Rn selected from the group consisting of iodoacetamide, bromoacetamide, vinyl sulfonate, and ETAC. The iodoacetamide or bromoacetamide reactive group reacts with thio functionalities on the carrier protein to form stable thioether acetamide linkages. The vinyl sulfonate reactive group reacts with thio functionalities on the carrier protein to form stable beta-thiosulfonyl linkages. The ETAC reacts with thio functionalities and is capable of forming a three carbon bridge between thiol groups on the carrier protein involved in a disulfide bond. In a specific embodiment, the precursor molecule is a compound comprising the formula SRGYFLFRPRN-CONH2
Figure imgf000020_0001
is provided wherein I is an iodo group. This compound is reactive with the cysteine residues, for example, the cysteine residue at position 34 in human serum albumin (HSA). As shown in Example 5, use of the above compound enabled HSA-NMU conjugates to be made wherein greater than 90% or greater than 95% of the conjugates comprise one molecule of NMU per molecule of HSA as determined by mass spectrometry (See Table 4 in Example 5). The above compound enabled HSA-NMU conjugates to be made wherein proportion of conjugates having two molecules of NMU per molecule of HSA is less than 10% or 5% as determined by mass spectroscopy and having three or more molecules of NMU per molecule of HSA is not detectable by mass spectrometry. Compounds that have spacers of similar length but of different composition are expected to provide similar results.
In particular embodiments, the Rn-Zl -peptide precursor molecule is conjugated to a recombinant antibody or Fc fragment in which the TV-terminus or the C-terminus of the antibody or Fc fragment includes a cysteine residue, which provides a functionality for reacting with the iodoacetamide or bromoacetamide reactive group of the Rπ-Zl-peptide precursor molecule to conjugate the Rn-Zl -peptide precursor to the antibody or Fc fragment. In other embodiments, the antibody or Fc fragment is conjugated to the 2λ -peptide precursor molecule via an ETAC reactive group capable for forming a three carbon bridge between two cysteine residues in the antibody or Fc fragment involved in a disulfide bond. Figure 10 provides a scheme for the construction of an Rn-Z^ -peptide precursor molecule comprising an ETAC reactive group.
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 C 1-6 ester, or converted to an amide of formula NRR2 wherein R and R2 are each independently H or C 1-6 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.
Hydroxyl groups of the neuromedin U receptor agonist amino acid side chains may be converted to C I „6 alkoxy or to a C I „6 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 Cl „6 alkyl, Cl ~6 allkoxy, 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. The present invention includes diastereomers as well as their racermc and resolved enantiomerically pure forms. The neuromedin U receptor agonists can contain D -amino acids, L-amino acids, unnatural amino acids, amino acid analogs, rare 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=Alanme; C~Cys=Cysteine; D=Asp=Aspartic Acid; E=Glu=Glutamic Acid; FHPhe^Phenylalanine; G=Gly=Glycine; H=Ηis-Histidine; I=Ile=ϊsoleucine; K=Lys=Lysine; L=Leu=Leuciiie; M=Met=Methionine; N=Asn=Asparagine; P=Pro=Proline; Q=GIn=Glutamine; R=Arg=Arginine; S=Ser=Serine; T=Thr=Threonine; V-Val-Valine; W-Trp=Tryptoρhan; and Y=Tyr=Tyrosine .
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/m2). "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, hyperuricacideraia, 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, emmeniopathy, 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 useful 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, tromethamine, 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, glycollylarsanilate, 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-drag 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; β3-adrenergic receptor agonists; PPARγ receptor agonists; agouti- related protein or analog; angiopoietin-like protein 6 (Angptl6) 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-100641, R483, and LY-300512, and the like and compounds disclosed in WO97/10813, 97/27857, 97/281 15, 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 buformin; 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, C7, 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 midaglizole; 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 LAR5 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- 50I5 and farglitazar (GW-2570/GI-262579), and the like; (13) PPARα/γ 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 (Glaxo Smithkline), E-3030 (Eisai), LY510929 (Lilly), AK109 (Asahi), DRF2655 (Dr. Reddy), DRF8351 (Dr. Reddy), MC3002 (Maxocore), TY515O1 (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-fIuorophenyl- lH-imidazol-5-yl]pyridme, 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 promotors 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) GPRI l 9 (also called RUP3; SNORP 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 palmitøyl transferase inhibitors such as ST 1327, and ST 1326, and the like; (27) Fructose 1,6-bisphospohatase 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-phosphase inhibitors; (31) phosphoenolpyruvate carboxykinase (PEPCK) inhibitors; (32) pyruvate dehydrogenase kinase (PDK) activators; (33) RXR agonists such as MC1036,
CSOOOl 8, 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) lipid 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, pravastatin, 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-VA12 (Forbes Medi-Tech), FM-VP-24 (Forbes Medi-Tech), stanol esters, beta-sito sterol, 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, efiucimibe, pactimibe (KY5O5), 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) anti-oxidants such as probucol, and the like; (9) PP ARa agonists such as beclofibrate, benzafibrate, ciprofibrate, clofibrate, etofibrate, fenofibrate, gemcabene, and gemfibrozil, GW 7647, BM 170744 (Kowa), LY518674 (Lilly), GW590735 (Glaxo Smithkline), KRP-101 (Kyorin), DRFl 0945 (Dr. Reddy), NS-220/R1593 (Nippon Shinyaku/Roche, STl 929 (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 XTCO 179628 (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 JP 10237049, 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), AK105 (Asah Kasei), and the like; (30) Lp-PLA2 inhibitors such as SB480848 (Glaxo Smithkline), 659032 (Glaxo Smithkline), 677116 (GlaxoSmithkline), and the like; (31) other agents which affect lipic composition including ETC1001/ESP31015 (Pfizer), ESP-55016 (Pfizer), AGI1067 (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, esmolol, indenolol, metaprolol, nadolol, nebivolol, penbutolol, pindolol, propanolol, sotalol, tertatolol, tilisolol, and timolol, and the like; (3) calcium channel blockers such as amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, bepridil, cinaldipine, clevidipine, diltiazem, efonidipine, felodipine, gallopamil, isradipine, lacidipine, lemildipine, lercanidipine, nicardipine, nifedipine, nilvadipine, nimodepine, nisoldipine, 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, indoramin, 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 (GlaxoSmithkline), and the like; (2) NE (norepinephrine) transporter inhibitors, such as GW 320659, despiramine, talsupram, and nomifensine; (3) CBl (cannabinoid-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 04/11 1033, WO 04/111034, WO 04/11 1038, 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), RC1291 (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)propanol]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- phenyϊcarbamates (Reidemeister, S. et al., Pharmazie, 55:83-6 (2000)), and proxifan derivatives (Sasse, A. et al., J. Med. Chem.. 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), ATCOl 75/AR224349 (Arena Pharmaceuticals), GW803430 (GlaxoSmithkine), NBI- IA (Neurocrine Biosciences), NGX-I (Neurogen), SNP-7941 (Synaptic), SNAP9847 (Synaptic), T-226293 (Schering Plough), TPI- 136 M 7 (Saitama Medical School/University of California Irvine), and those disclosed WO 01/21169, WO 01/82925, WO 01/87834, WO 02/051809, WO 02/062455 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- 569180A, GW-594884A (GlaxoSmithkline), 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, J. 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 GI-181771 (Glaxo-SmithKline); SR146131 (Sanofi 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, VRl 065 (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 dexfenfiurarnine, 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 59119A, 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-(556,758-tetrahydro-5,5,8J8-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) 11 β HSD-I (1 1-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-triazole, 3-adamantanyl-4,5,6}7,8,9,10,l l,12,3a-decahydro-l,2,4-triazolo[4,3- a][l ljannulene, and those compounds disclosed in WO 01/90091, 01/90090, 01/90092, 02/072084, 04/011410, 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 (Alizyrae/Takeda),
GT389255 (Genzyme/Peptimmune)Triton WRl 339, RHC80267, lipstatin, teasaponin, and diethylumbelliferyl 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 04/1 11004, 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), AC162352 (IC Irmovations/Curis/Amylm), TM30335/TM30338 (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 maslϊnic acid, erythrodiol, ursolic acid uvaol, betulinic acid, betulin, and the like and compounds disclosed in WO 03/01 1267; (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 (Glaxo Smithkline), 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/Daiπippon/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: JV-[3-(4-chlorophenyl)-2(5>-phenyH(5)-methylpropyl]-2-(4- trifluoromethyl»2~pyrimidyloxy)-2-memylpropanamide, iV-f3-(4-chlorophenyl)-2-(3- cyanophenyl)-l-methylpropyl]-2-(5-trifluoromethyl-2-pyridyloxy)-2-methylpropanamide, JV-[3- (4-chlorophenyl)-2-(5-chloro-3-pyridyl)-l-methylpropyl]-2-(5-trifluoromethyl-2-pyridyloxy)-2- methylpropanamide, and pharmaceutically acceptable salts thereof; as well as those in WO05/000809, which includes the following: 3-{ ϊ-[bis(4-chlorophenyl)methyl]azetidin-3- ylidene}-3-(3,5-difluoroρhenyl)-2,2-dimethylpropanerύtriles l-{l-[l-(4- chloroρhenyl)pentyl]azetidin-3-yl}-l-(3,5-difluorophenyl)-2-methylpropan-2-ol. 3 -((S)- (4- chlorophenyl ) { 3 - [( 1 S)- 1 -(3 ,5 -difluorophenyl)-2-hydroxy-2-methylpropyl] azetidin- 1 - yl } methyl)benzonitrile, 3 -((S)~(4-chlorophenyl) { 3 -[( 1 S)- 1 -(3 , 5 -difluorophenyl)-2-fluoro-2- methylpropyl] azetidin- 1 -yl }methyl)benzonitrile , 3 -((4-chlorophenyl) { 3- [ 1 -(3 , 5 -difluorophenyl)- 2 ,2-dimethylpropyl] azetidin- 1 -yl } methyl)benzonitrile, 3 -(( 1 S)- 1 - { 1 - [( S)-(3 -cyanophenyl )(4- cyanophenyl)methyl]azetidin-3-yl}-2-fluoro-2-methylproρyl)-5-fluorobenzonitrile, 3-[(S)-(4- chlorophenyl)(3-{(lS)-2-fluoro-l-t3-fluoro-5-(4H-l,2,4-triazol-4-yl)phenyI]-2- methylpropyl} azetidin- l-yl)methyl]benzonitrile, and 5-((4-chlorophenyJ){3-[(lS)-l-(3,5- difluorophenyl)-2-fluoro-2-methylpropyl] azetidin- 1 -yl } methyl)thiophene-3 -carbonitr ile, and pharamecueitcally acceptable salts thereof; as well as: 3-[(5)-(4-chlorophenyl)(3-{(liS)-2-fluoro- l-[3-fluoro-5-(5-oxo-4,5-dihydro-l,3>4-oxadiazol-2-yl)phenyl]-2-methylpropyl}azetidin-l- yl)methyl]benzonitrile, 3-[(S)-(4-chlorophenyl)(3-{(l S)-2-fluoro- 1 -[3-fluoro-5-(l ,3,4-oxadiazol- 2-yl)pheByl]-2-methylpropyl}azetidin-l-yl)methyl]benzonitrile, 3-[(JS}-(3-{(15)-l-[3-(5-amino- 1 , 3 ,4-oxadiazol-2~yl)~ 5 -fluorophenyl] -2-fktoro-2-methyϊpropyl} azetidin- 1 -yl)(4- chloroρhenyl)rnethyl]benzonitrile5 3 - [(S)-(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- 1 -yl )(4-cyanophenyl)rnethyϊ] benzonitrile, 3 - [(S)-(4-cyanophenyl)(3- { ( 1 iS)-2-fluoro- 1 - [3 - fluoro-5-(l ,3, 4-oxadiazol-2-yI)phenyl]-2~methylpropyl} azetidin- 1 ~yl)methyl] benzonitrile, 3-[(5)- (4-chlorophenyl)(3-{(15)-2-fluoro-l-[3-fluoro-5-(ls2,4-oxadiazol-3-yl)phenyl]-2- methylpropyl}azetidin-l-yl)methyl]benzoniu-ile5 3-[(l,S)-l-(l-{(5)-(4-cyanophenyl)[3-(ls2,4- oxadiazol- 3 -yl)phenyl] -methyl } azetidin-3 -yl)-2-fluoro-2-methylpropyl] -5 -fl uorobenzonitrile, 5 - (3 - { 1 - [ 1 -(diphenyImethyl)azetidin-3 -y 1] -2-fl uoro-2-methylpropyl } - 5 -fluorophenyl )- 1 i/-tetrazoles 5-(3- { 1 -[I -(diρhenylmethyl)azetidin-3-yl]-2-fluoro-2-methylρropyl} -5-fluoroρhenyl)- 1 -methyl- 1 /f-tetrazole , 5 -(3 - { 1 - [ 1 -(diphenylmethyl)azetidin~3 -yl] -2 -fluoro-2-methylproρyl } -5 - fluorophenyl)~2~methyl-2H-tetrazole, 3-[(4-chlorophenyl)(3-{2-fluoro-l-[3-tluoro-5-(2-methyl- 2H-tetrazol- 5 ~yl)phenyl] -2-methylpropyl } azetidin- 1 ~yl)methyl]benzonitrile, 3 - [(4- chlorophenyl)(3-{2-fluoro-l~[3-fluoro-5-(l-methyl-l/i-tetrazol-5-yl)phenyl]-2- methylpropyl } azetidin- 1 -yl)methy 1] benzonitrile, 3- [(4-cyanophenyl)(3 - { 2-fluoro- 1 - [3 -fluoro-5 - ( 1 -methyl- 1 ϋT-tetrazol-5 -yl)pheny 1] -2-methylpropyl } azetidin- 1 -yl)methyl] benzonitri Ie, 3 - [(4- cyanophenyl)(3-{2-fluoro- 1 -[3 -fluoro-5 -(2-methyl-2/ir-tetrazol-5-yl)phenyl] -2- methylpropyl } azetidin- 1 -yl)methyl] benzonitrilef 5-{3-[(5)-{3-[(16)-l-(3 -bromo- 5 -fluoropheny I)- 2-fluoro-2-methylpropyl]azetidin-l-yl}(4-chlorophenyl)methyl]phenyl}-l,3,4-oxadiazol-2(3H)- one, 3 - [( 1 S)- 1 -( 1 - {(5)-(4-chlorophenyl) [3-(5 -oxo-4, 5 -dihydro- 1 ,3 ,4-oxadiazol-2- yl)phenyl]methyl} azetidin-3 -yl)-2-fluoro-2-methylpropyl] -5 -fluorobenzonitrile, 3- [( 1 S)- 1 -( 1 - {(S)-(4-cyanophenyl)[3-(5-oxo~4,5-dihydro-l,3»4-oxadiazol-2-yl)ρhenyl]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-fluoro-2~methylpropyl]-5-fluorobenzonitrile, 3- [( 1 S)- 1 -( 1 - { (5)-(4-chlorophenyl) [3 -( 1 , 3 ,4-oxadiazol-2-yl)phenyl ] methyl } azetidin- 3 -yl)-2-fluoro- 2~methylpropyl]-5-fluorobenzonitrile> 3-((lS)-l-{l-[(S)-[3-(5-amino-l,3,4-oxadiazol-2- yl)phenyl](4-chlorophenyl)methyl]azetidin-3-yl}-2-fluoro~2-methylpropyl)-5-fluorobenzonitrile, 3 -(( 1 S)- 1 - { 1 - [(S)-[3 -(5-amino- 1 ,3 f4~oxadiazol-2-yl)phenyl] (4-cyanophenyl)methyl]azetidin-3 - yl } -2-fluoro-2-methylpropyl)-5 -fluorobenzonitrile, 3 - [( 1 S)- 1 -( 1 - {(5)-(4-cyanophenyl) [3-( 1 ?2,4- oxadiazol-3~yl)phenyl]methyl}azetidin-3-yl)-2-fIuoro-2-methylρroρyl]-5-f1uorobenzonitrile]> 3- [( 1 S)- 1 -( 1 - { (£)-(4-chlorophenyl) [3 ~( 1 ,2 ,4-oxadiazol- 3 -yl)ρheny 1] 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 ,3,4-oxadiazol-2(3/f)-oneJ 5-[3-((S)-(4- chlorophenyl) (3-[(1S)-I -(3 , 5 -difluoroρhenyl)-2-fluoro-2-methyIρroρyI] azetidin- 1 - yl}methyl)phenyl]-l,3,4-oxadiazol-2(3H)-one, 4-{(iS)-{3-[(15)-l-(3,5-difluorophenyl)-2-fluoro- 2-methylρropyl]azetidin-l-yl}[3-(5-oxo-4,5-dIhydro-l,3,4-oxadiazol-2-yl)phenyl]methyl}~ benzonitrile, ACOMPLIA (rimonabant, iV-(l-piperidinyl)-5-(4-chlorophenyl)-l-(2,4- dichloroρhenyl)-4-methylpyrazole-3 -carboxamide, SR 141716A), 3 -(4-chlorophenyl-N' ~(4- chloroρhenyl)sulfonyl-N~methyl-4-phenyl-4,5-dihydro- 1 H-pyrazole- 1 -carboxamide (SLV- 319), taranabant, N-[(lS,2S)-3-(4-CMorophenyl)-2-(3-cyanophenyl)-l-methylpropyl]-2-methyl-2-[[5- (trifluoromethyl)-2-pyridinyl]oxy]ρropanamide, 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)s4'-piperidine]-l '- carboxamide, 3 -oxo-N-(7-trifluoromethylρyrido [3 ,2-b]pyridin-2~yl) spϊro- [isobenzofuran- 1 (3 H) ,4 ' -piperidine]- 1 ' -carboxamide, N- [5 -(3 -fluorophenyl)-2-pyrimidinyl] -3 -oxospiro- [isobenzofuran- 1 (3 H),4' -piperidine] - 1 ' -carboxamide, trans- 3 ' -oxo-N-(5-phenyl-2- pyrimidinyl)spiro[cyclohexane- 1 , 1' (3 ' H)-isobenzofuran] -4-carboxamide, trans-3 ' -oxo-N- [ 1 -(3 - quinolyl)-4-imidazolyl]spiro [cyclohexane- 1,1 ' (3 Η)-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 -fluorophenyl)-2-pyrimidinyl] - 3 -oxospiro [5-azaisobenzofuran- 1 (3 H) , I' - cyclohexane] -4 ' -carboxamide, trans-N- [5 -(2-fluorophenyI)-2-pyrimidiny 1] -3 -oxo spϊro [5 - azaisobenzofuran-l(3H), 1 '-cyclohexane^'-carboxamide, trans-N- [1 -(3 ?5-difluorophenyl)-4- imidazolyl]-3-oxospiro[7-azaisobenzofuran-l(3H), 1 '-cyclohexane]-4'-carboxamide, trans-3-oxo- N-(I -phenyl-4-pyrazolyl)spiro[4-azaisobenzofuran-l (3H), r-cyclohexane]-4'-carboxamide, trans-N- [ 1 -(2-fluorophenyl)-3 -pyrazolyl] -3 -oxospiro [6-azai sobenzofuran- 1 (3 H) , 1 ' -cyclohexane]- 4'-carboxamide, trans-3 -oxo-N-(l -phenyl-3-pyrazolyl)spiro[6-azaisobenzofuran-l(3H),r- cyclohexane] -4 '-carboxamide, trans-3 -oxo-N-(2-phenyl- 1 ,2,3-triazol-4-yl)spiro[6- azaisobenzofαran- 1(3 H)9I '-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-[(458-dimethoxyquinolin-2-yl)carbonyl]-6-(lH-tetrazol-5-yl)sρiro[chroman-2,4'- piperidin]-4-one; (5-{ r-[(4,8-dimethoxyquinolin-2-yl)carbonyl]-4-oxospiro[chroman-254'- piperidin]-6-yl}~2iϊ-tetrazol-2-yl)methyl pivalate; 5-{ l'-[(8-cyclopropyl-4-methoxyquinolin-2- yl)carbonyl]-4-oxospiro[chroman~2,4'-piperidin]-6-yl}nicotinic acid; 1 '-(8-methoxy-4- morpholin~4-yl~2-naphthoyl)-6-(l//-tetrazol-5-yl)spiro[chroman-2,4 -piperidin]-4-one; and 1 '- [(4-ethoxy~8-ethylquinolin-2-yl)carbonyl]-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: l~{4-[(l-ethylazetidin-3-yl)oxy]phenyl}-4-[(4-fluorobenzyl)oxy]pyridin- 2(IH)-OUS, 4- [(4-fluorobenzyl)oxy] - 1 - { 4- [( 1 -isopropylazetidin-3 -yl)oxy] phenyl } pyri din-2( 1 H)- one, 1 -[4-(azetidin-3-yloxy)phenyl]-4-[(5-chloropyridin-2-yl)methoxy]pyridin-2(lH)-one:i 4-[(5- chloropyridin-2-yl)methoxy] - 1 - { 4- [( 1 -ethylazetidin-3 -yl)oxy]phenyl } pyridin-2( 1 H)-one, 4- [(5 - chloropyridin-2-yI)methoxy]-l-{4-[(l-propylazetidin-3-yl)oxy]phenyl}pyridin-2(lH)-one, and 4- [(5-chloropyridin-2-yl)methoxy]- 1 -(4- { [(2S)- 1 -ethyIazetidra-2-yl]raethoxy}phenyl)pyridin- 2(XH)-OUQ, 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-(2,4,5-trifluoroρhenyl)butanoyl]-3-(trifluoromethyl)-5,6>7,8-tetrahydro-l ,2,4- triazolo[4,3-a]pyrazine, or a pharmaceutically acceptable salt thereof.
Specific H3 (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- [(l-cyclobutyl-4-piperidinyl)oxy]phenyl}-2-ethylpyrido[2,3-d]-pyrimidin-4(3H)-one, 3-{4-[(l- cyclobutyl-4-piperidinyl)oxy] phenyl } -2-methylpyrido[4 ,3 -d]pyrimidin-4(3 H)-one? 2-ethyl-3 -(4- { 3 -[(3 S)-3-methylρiperidin- 1 -yljpropoxy} phenyl)pyrido [2,3-d]ρyrimidin-4(3 H)-one 2-methyl~3 - (4- { 3 - [(3 S)-3 -methylpiperidin- 1 -yl] propoxy } phenyl)pyrido [4, 3 -d] pyrimidin-4(3 H)~one, 3 - {4- [(l-cyclobutyl-4-piperidinyl)oxy]ρhenyl}-2,5-dimethyl-4(3H)-quinazolinone, 3-{4-[(l- cyclobutyl-4-piperidinyl)oxy]phenyl}-2-methyl-5-trifluoromethyl-4(3H)-quinazolinonej 3-{4- [(l-cyclobutyl-4-piperidinyl)oxy]phenyl}-5-methoxy-2-methyl-4(3H)-quinazolinone, 3-{4-[(l- cyclobιιtylpiperidin-4-yl)oxy]phenyl } - 5 -fluoro -2-methyl-4(3 H)-quinazolinone, 3-{4-[(l- cyclobutylpiperidin-4-yl)oxy]phenyl}-7-fluoro-2-methyl-4(3H)-quinazolinone, 3-{4-[(l- cyclobutylpiperidin-4-yl)oxy] phenyl }-6-metlioxy-2-methyl-4(3H)-quinazolinone, 3- {4- [(I - cyclobutylpiperidin-4-yl)oxy]phenyl}-6-fluoro-2-methyl-4(3H)-quinazoIinone, 3-{4-[(l- cyclobutylpiperidm-4-yl)oxy3phenyl}-8-fluoro-2-methyl-4(3H)-quin.azolinone, 3-{4-[(l- cyclopentyl-4-piperidinyl)oxy] phenyl } -2-methylpyrido [4,3 -d]pyrimidin-4(3 H)-one, 3 - { 4- [( 1 - cyclobutylpiperidin-4-yl)oxy]phenyl } -6-fluoro-2-methylpyrido [3 ,4-d]pyrimidin-4(3 H)-one, 3 - {4- [( 1 -cyclobutyl-4-piperidinyl)oxy]phenyl } -2-ethylpyrido [4 , 3 -d]pyrimidin-4(3H)-one, 6-methoxy- 2-methyl-3 - {4- [3-( 1 -piρeridϊnyl)ρropoxy]ρhenyl }pyrido [354-d]pyrimidin-4(3H)-one, ό-methoxy- 2-methyl-3 - { 4- [3 -( 1 -pyrrolidinyl)propoxy]ρhenyl } pyrido [3 ,4-d]pyrimi din-4(3 H)-one, 2,5 - dimethyl-3 - {4-[3 -( 1 -pyrrolidinyl)proρoxy]phenyl } -4(3H)-quinazolϊnone, 2-methyl-3 - {4- [3-( 1 - pyrrolidinyl)propoxy]phenyl} -5-trifiuoromethyl-4(3H)-quinazolinone, 5-fluoro-2-methyl-3- {4- [3 -( 1 -piperidinyl)propoxy]phenyl } -4(3 H)-quinazolinone, 6-methoxy-2-methyl-3 - { 4 - [3 -( 1 - piperidmyl)propoxy]phenyl} -4(3 H)-quinazolinones 5-methoxy-2-methyl-3-(4- { 3 - [(3 S)-3- methylpiperidin- 1 -yl]propoxy}phenyl)-4(3H)-quinazolinones 7-methoxy-2-methyl-3-(4- (3-[(3S)- 3-methylpiperidin-l-yl]propoxy}phenyl)-4(3H)-quinazolinone, 2-methyl-3-(4-{3-[(3S)-3- methylpiperidin- 1 -yl]propoxy}phenyl)pyrido[2,3-d]ρyrimidin-4(3H)-one, 5-fluoro-2-methyl-3- (4- {3-[(2R)-2-methylpyrrolidin- 1 -yl]proρoxy}phenyl)~4(3H)-quinazolinone, 2-methyl-3-(4-{3- [(2R)-2-methylpyrrolidin-l-yl]propoxy}phenyl)pyrido[453-d]pyrimidin-4(3H)-one, 6-methoxy-2- methyl-3-(4-{3-[(2R)-2-methylpyrrolidin-l-yl]propoxy}phenyl)-4(3H)-qιtinazolinone, 6- nieihoxy-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-{[l-(3 -ethoxyphenyl)-2-(4-methyiphenyl)- 1 H -imidazol-4-yl] carbonyl } - 1 - piperazinyl)- 1 -naphthoic acid; 3-(4- { [ 1 -(3 -ethoxyphenyl)-2-(2-fluoro-4-methylphenyl)- 1 H - imidazol-4-yl] carbonyl } - 1 -piperazinyl)- 1 -naphthoic acid; 3 -(4- { [ 1 -(3 -ethoxyphenyl)-2-(4- fluorophenyl)- 1 H -imidazol-4-yl] carbonyl } - 1 -piperazinyl)- 1 -naphthoic acid; 3-(4-{[l-(3- ethoxyphenyl)-2-(2,4-difiuorophenyl)~ 1 H -imidazol-4-yl]carbonyl} - 1 -piperazinyl)- 1 -naphthoic acid; and 3-(4-{[l-(2,3-dihydro-l,,4-benzodioxin-6-yl)-2-(4-fluorophenyl)~liϊ-imidazol-4- yl]carbonyl}-l~piperazinyl)-l-naphthoϊc 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 : 1 ) (5S)- 1 '- { [(3Λ, 4R)- 1 -terf-butyl-3 -(2,3 ,4-trifluorophenyl)piperidin~4-yl] carbonyl } -3 - chloro-2-methyl-5- [ 1 -methyl- 1 -( 1 -methyl- 1 H- 1 ,2,4-triazol-5-yl)ethyl]-5/f~spiro[furo [3 ,4-
6]pyridine-7,4'-piperidine] ; 2) (5R)- 1 '-{ [(3 J?,4J?)-1 -ferf-butyl-3-(2,3,4-trifluorophenyl)-piperidin- 4-yl] carbonyl } -3 -chloro-2-methyl-5-[ 1 -methyl- 1 -( 1 -methyl- 1 H- 1 ,2, 4-triazol-5-yl)ethyl]-5iY~ spiro[foro[3,4-δ]pyridine-7,4'-pipeπdine] ; 3) 2-(l '-{ [(35,4J?)-1 -tert-butyl-4-(2,4- difluorophenyϊ)pyrrolidin-3 -yl] carbonyl } -3 -chloro-2-methyl- S/J-spiro [furo [3 t4-b] pyridine-7,4'- piperidin]-5-yl)-2-methylpropanenitrile; 4) l'-{[(35r.4Λ)-l-førf-butyl-4-(2,4- difluorophenyl)pyrrolidin-3 -yl] carbonyl } -3 -chloro-2-methyl- 5 - [ 1 -methyl- 1 -( 1 -methyl- 1 H- 1 ,2 ,4- triazol-5-yl)ethyl]-5/-J-spiro[furo[3,4-&]pyridine-7!4'-piperidine]; 5) N-[(3i?,4i?)-3-({3-chloro-2- methyl-S-fl-methyl-l-tl-methyl-l/f-l^^-triazol-S-y^ethyy-ri^S/f-spiroffuro-tS^-έlpyridine- 7,4t-piperidin]-r-yl}carbonyl)-4-(2,4-difluorophenyl)-cyclopentyl]-N-methyltetrahydro-2/f- pyran-4-amine; 6) 2-[3-chloro-l1-({(li?,2i?)-2-(2f4-difluorophenyl)-4-[methyl(tetrahydro-2Jf/- pyran-4-yl)aniino]-cyclopentyl}-carbonyl)-2-methyl-5/f-spiro[furo[3,4-&]pyridine-7J4'- 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. In 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 al., New Engl. J. Med. 321 : 574 (1989) and a semi-permeable polymeric material (See, for example, Howard, et ah, 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. In 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 container(s) 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 Preparation of several human Serum Albumin (HSA)-NMU conjugates was as follows. Conjugation to Human Serum Albumin (HSA) takes advantage of the presence of the unpaired Cys34 jn the protein primary structure. The site of conjugation on the NMU peptide of the present invention is chosen taking into account the structure of NMU and its interactions with the NMU receptors. Hence, the conjugation was site-specific through the addition of either a maleimido or a haloacetyl functionality and spacer at the JV-terminus of the peptide sequence.
The full-length NMU peptide sequence, or shorter sequences truncated at the N-terminus, can be used as peptide precursors. Spacers between the maleimido or the haloacetyl group and the peptide backbone can be also introduced to minimize the impact on activity on the NMU sequence due to the conjugation to the protein. Synthesis of Neuromedin U (NMU) precursor peptides
Table 1
SEQ
Peptide Sequence ID NO
Maleimidobutiryl-(Ttds)2-FRVDEEFQSPF ASQSRGYFLFRPRN-
5 NMU43 CONH2 (Maleimide linker comparator)
6 NMU79 Iodoacetyl-(Ttds)2-FRVDEEFQSPFASQSRGYFLFRPRN-CONH2
7 NMU 120 Iodoacetyl-(Ttds)2-ASQSRGYFLFRPRN-CONH2
8 NMU 104 Iodoacetyl-(Ttds)2-YFLFRPRN-CONH2
9 NMUI lO Iodoacetyl-(Oxa)24-YFLFRPRN-CONH2
Ttds = 13-amino-4,7,10-trioxa-tridecayl succinamic acid (Oxa)24 = dPEG24™ (Quanta Biodesign Ltd.)
NMU peptide precursors (SEQ ID NOs: 5 to 9) 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 amϊnomethylated polystirene LL (100-200 mesh, 0.41 mmol/g) (Novabiochem) resin derivatized with a modified Rink linker p-[(RfS)-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. 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 equimolar amounts of HBTU (2~(lH-benzotriazole-l-yl)-l,l,3,3-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 (AAPPTEC). 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-diisopropyIethyIamine) solution 2 M in NMP. The side chain protecting groups were: tert-butyl for Asp, GIu, Ser and Tyr; trityl for Asn, and GIn; 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl for Arg.
In the peptide precursor SEQ ID NO; 5, the NMU peptide sequence was modified linking to the N-terminus of the NMU two Ttds spacers (13-amino-457,10-trioxa-tridecayl succinamic acid, NeoMPS Part# FAl 8801) and a maleimidobutiryl reactive group for covalently joining to Cys34 of the HSA. In the peptide precursor SEQ ID NO: 6, the NMU peptide sequence was modified by linking to the N-terminus of the NMU two Ttds spacers and an iodoacetyl reactive group for covalently joining to Cys34 of the HSA. In the peptide precursors SEQ ID NO: 7 and SEQ ID NO: 8> the NMU peptide sequences spanning residues 12-25 and 18-25, respectively, were modified by linking to the N-terminus of the NMU two Ttds spacers and an iodoacetyl reactive group for covalently joining to Cys34 of the HSA. In the peptide precursor SEQ ID NO 9, the NMU peptide sequence spanning residues 18-25 was modified by linking to the N-terminus of NMU an Oxa24 spacer and an iodoacetyl reactive group for covalently joining to Cys34 of the HSA. The N-terminal derivatization with the maleimido group (SEQ ID NO 5) was performed at the end of the peptide assembly by reaction with a four-fold excess of activated 4- maleimidobutyric acid (Fluka, Cat No 63174) over the resin free amino groups for one hour. The 4-maleimidobutyric acid was activated with equimolar amounts of DIPC (1,3- Diisopropylcarbodiimide) and HOBt (Hydroxybenzotriazole) in DMF. The N-terminal derivatization with the iodoacetyl group (for SEQ ID NOs: 6, 7, 8, and 9) was performed at the end of the peptide assembly by reaction with a three-fold excess of iodoacetic anhydride (Aldrich, Cat No 284262) over the resin free amino groups for 15 minutes. The acylation reaction for linking the Oxa24 spacer to the N-terminus was performed for two hours using a two-fold excess of Fmoc-N-amido-dPEG24-acid™ (Quanta Biodesϊgn Ltd., Product No 10313) over the resin amino groups. The Fmoc-N-amido-dPEG24-acid™ was dissolved in DCM
(dichloromethane) and activated with equimolar amounts of HBTU and a two-fold molar excess ofDIEA in DMF.
At the end of the synthesis, the dry peptide-resins were individually treated with 20 mL of the cleavage mixture, 88% TFA, 5% phenol, 2% triisopropylsilane and 5% water (Sole, N. A. and G. Barany, 1992, J. Org. Chem. 57:5399-5403) 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 H2O, 20% acetonitrile, and lyophilized. The crude peptides were purified by reverse-phase HPLC using semi-preparative
ReproSil-Pur C4 column (25 x 200 mm, 15 μm) or Waters X-Bridge C18 (19 x 150 mm, 10 μm) and using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, flow rate 30 mL/min. Analytical HPLC was performed on a Waters UPLC Acquity BEH 130A Cl 8 column (2.1 x 100 mm, 1.7 μra), flow rate 0.4 mL/min, 450C. The purified peptides were characterized by electrospray mass spectrometry on a Micromass LCZ platform.
Conjugation of the NMU peptide precursors to human Serum Albumin (HSA) was as follows. After a preliminary reduction of the thiol on Cys in HSA, or purification of the HSA free of any adducts on Cys34 thiol, the conjugation to the protein was performed under conditions conducive to thioether bond formation between the maleimido or the iodoacetyl group on the NMU precursors and the Cys34 on the HSA. The HSA-NMU conjugates were then isolated through size exclusion chromatography (SEC) and characterized by ESI Q-ToF Hybrid System (Q-Star XL, Applied Biosystems).
Preparation of the HSA-NMU43 conjugate was as follows.
In a first step, the Cys34 in the HSA was reduced. In order to cleave off any thiol adduct on the Cys34 of the protein, an aliquot of 200 μL (50 mg) of human serum albumin (Cortex, Cat. No. CP0925U; 25% solution) was diluted with 2.3 niL PBS pH 7.4 (20 mg HSA/mL; 300 μM) and the resulting mixture was reacted with three molar equivalent of dithiothreitol (25 μL; 0.1 M solution of DTT, Aldrich 45,777-9) for one hour at room temperature. The crude reaction mixture was loaded on a PD-10 pre-packed desalting column (GE Healthcare, cat.17-0851-01) and eluted with 3.5 mL of PBS pH 6.5. The concentration of HSA was determined by UV absorbance at 280 nm (ε~ 33 500 M~l cirri). The percentage of the fully reduced Cys34 sulfhydryl group in the sample was determined by mass spectroscopy on a Q-Star XL Applied Biosystems spectrometer and was estimated as 90-92% (MW 66440) together with a major contamination of 8-10% of HSA Cys34 of higher oxidation species (MW 66472). Conjugation of the NMU peptide precursor to the HSA was by means of a maleimido reactive group to make a comparator molecule. 2.1 mL of the solution of Cys- reduced HSA (14.3 mg/niL; 215 μM in PBS pH 6.5) was reacted with 1.2 molar equivalent of the NMU peptide precursor (SEQ ID NO: 5) (2.1 mg of peptide in 0.6 mL in PBS pH 6.5) for 18 hours at room temperature. The resulting HSA-NMU peptide conjugate was purified by size exclusion chromatography (SEC) using a High-Load 26/60, Superdex 75 prep, grade (Amersham Biosciences) in PBS pH 7.4, flow rate 2.5 mL/min. The purified HSA-NMU conjugate was characterized by ESI Q-ToF Mass spectrometry: found MW 70291 Da; calculated MW 70290 Da. The conjugate net content (17 mg) was determined by UV absorbance at 280 nm (total HSA content) and by mass spectrometry (HSA-NMU conjugate relative content). Figure IA shows the structure of the NMU43 peptide precursor and the HSA-NMU43 conjugate.
Preparation of HSA-NMU79 conjugate was as follows. Purification of rHSA was by IEX chromatography. In order to isolate a free thiol Cys34 albumin sample, an aliquot (30 mg) of recombinant human serum albumin (rHSA, Recombumin® Novozymes) was purified by ion exchange (IEX) chromatography on a Resource Q anion exchange column 6 mL (Amersham Biosciences, Cat Nr. 640262) and using as buffer eluents (A) 25 mM phosphate pH 7.0 and (B) 125 mM phosphate pH 7.0, flow rate 2.5 mL/min. The following multistep gradient was applied: after isocratic step at 0% B for 2 column volumes (CV), eluent B was raised to 20% over one CV followed by isocratic step for two CV, then to 40% over two CV and subsequently to 100% over one CV. The fractions containing the free thiol Cys34 rHSA protein were pooled and buffer exchanged through PD-10 pre-packed desalting column (GE Healthcare, cat.17-0851-01). Elution was performed using a 50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5 buffer. Concentration was determined by UV absorbance at 280 nrn and a sample was analyzed by ESI Q-ToF Mass spectrometry.
Conjugation of the NMU peptide precursor to the HSA was by means of an iodoacetamide reactive group. 3.5 mL of a solution of IEX purified rHSA (6.25 mg/mL; 95 μM in 50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5 buffer) was reacted with 1.3 molar equivalent of the NMU peptide precursor (SEQ ID NO: 6) (1.75 mg; 455 nmol resuspended in 100 μL H2θ) for 24 hours at room temperature. ESI Q-ToF mass spectrometry analysis showed
70 % completed reaction. A 0.75 molar equivalent excess (0.98 mg; 250 nmol) of the same NMU peptide precursor was added to reaction solution and the mixture was kept reacting for another 24 hours at room temperature. ESI Q-ToF mass spectrometry analysis showed > 98% completed reaction. The resulting rHSA-NMU peptide conjugate was purified by size exclusion chromatography (SEC) using a High-Load 26/60, Superdex 75 prep, grade (Amersham Biosciences) in PBS pH 7.4, flow rate 2.5 mL/min. The purified rHSA-NMU conjugate was characterized by ESI Q-ToF Mass spectrometry: found MW 70164 Da; calculated MW 70166 Da. The conjugate net content (12 mg) was determined by UV absorbance at 280 nm. Figure IB shows the structure of the NMU79 peptide precursor and the HSA-NMU79 conjugate.
Preparation of HSA-NMU 120 conjugate was as follows.
2.5 mL of a solution of IEX purified rHSA (6.00 mg/mL; 90 μM in 50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5 buffer) was reacted with 1.6 molar equivalent of the NMU peptide precursor (SEQ ID NO: 7) (0.945 mg; 300 nmol resuspended in 100 μL H2O) for 24 hours at room temperature. ESI Q-ToF mass spectrometry analysis showed 95 % completed reaction. The resulting crude rHSA-NMU peptide conjugate was purified by size exclusion chromatography (SEC) using a High-Load 26/60, Superdex 75 prep, grade (Amersham Biosciences) in PBS pH 7.4, flow rate 2.5 mL/min. The purified rHSA-NMU conjugate was characterized by ESI Q-ToF Mass spectrometry: found MW 68779 Da; calculated MW 68784 Da. The conjugate net content (10.3 mg) was determined by UV absorbance at 280 nm. Preparation of HSA-NMUl 04 conjugate was as follows.
3.5 mL of a solution of IEX purified rHSA (2.3 mg/mL; 35 μM in 50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5 buffer) was reacted with 1.2 molar equivalent of the NMU peptide precursor (SEQ ID NO: 8) (0.275 mg; 145 nmol in 100 μL H2θ) for 24 hours at room temperature. ESI Q-ToF mass spectrometry analysis showed 60 % completed reaction. A 1.1 molar equivalent excess (140 nmol) of the same NMU peptide precursor was added to reaction solution and the mixture was kept reacting for another 24 hours at room temperature. ESI Q-ToF mass spectrometry analysis showed > 98% completed reaction. The resulting crude rHSA-NMU peptide conjugate was purified by size exclusion chromatography (SEC) using a High-Load 26/60, Superdex 75 prep, grade (Amersham Biosciences) in PBS pH 7.4, flow rate 2.5 niL/min. The purified rHSA-NMU conjugate was characterized by ESI Q-ToF Mass spectrometry: found MW 68192 Da; calculated MW 68198 Da. The conjugate net content (4.25 mg) was determined by UV absorbance at 280 nm.
Preparation of HSA-NMUl 10 conjugate was as follows.
2.3 mL of a solution of IEX purified rHSA (6.20 mg/mL; 220 μM in 50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 8.5 buffer) was reacted with 1.3 molar equivalent of the NMU peptide precursor (SEQ ID NO: 9) (0.7 mg; 280 nmol in 100 μL H2O) for 24 hours at room temperature. ESI Q-ToF mass spectrometry analyses showed 30 % completed reaction. More NMUl 10 (0.56 mg, 220 nmole, one molar equivalent), dissolved in 100 μL H2O was added to the reaction solution and kept reacting for 24 hours at room temperature. MS analysis showed 60 % completed reaction. Reaction was left another 24 hours at room temperature without further addition of peptide precursor (total reaction time 72 hours). ESI Q-ToF mass spectrometry analysis showed > 90% completed reaction. The resulting crude rHSA-NMU peptide conjugate was purified by size exclusion chromatography (SEC) using a High-Load 26/60, Superdex 75 prep, grade (Amersham Biosciences) in PBS pH 7.4, flow rate 2.5 mL/mϊn. The purified rHSA- NMU conjugate was characterized by ESI Q-ToF Mass spectrometry: found MW 68718 Da; calculated MW 68721 Da. The conjugate net content (10 mg) was determined by UV absorbance at 280 nm.
EXAMPLE 2
In vitro activity of human Serum Albumin (HSA)-NMU conjugate at NMU receptors was assayed. The results showed that the has-NMU conjugate activity at the NMURl and NMUR2 was similar to the activity of native NMU. The NMU receptors signal primarily through. Gαq/1 1 proteins; therefore FLIPR, a calcium mobilization assay, was used to measure functional activity using cell lines expressing the human and mouse NMU receptors.
FLIPR assay was performed as follows.
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-Iysine 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, 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/peptϊde addition. Eight to twelve point dose responses were tested on NMUR-expressing cell lines using FLIPR with 1 μM 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 the HSA-NMU conjugates constructed in Example 1 in the FLIPR assay for human receptors (Table 2) and mouse receptors (Table 3) are presented. EC50s are reported in nM values. Percent activity refers to the maximum response at 1 μM compared to the hNMU response at the same concentration. The tables show that the HSA-NMU43 and HSA-NMU79 conjugates exhibited agonist activity comparable to native NMU at both the mouse and human NMURl and NMU2 receptors.
Figure imgf000047_0001
Figure imgf000048_0001
EXAMPLE 3 The efficacy of the HSA-NMU43 conjugate was investigated in a series of feeding experiments. The results showed that the HS A-NMU43 conjugate significantly reduced food intake in diet-induced obese mice.
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% 129Sό/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 Lp, 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 I)5 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;/? values < 0.05 were reported as significant and are denoted with an asterisk).
Figures 2A and 2B show that acute peripheral administration of HSA-NMU43 significantly reduced food intake in diet-induced obese mice for two days post dose. At the highest dose tested, food intake was reduced by 80% relative to vehicle treated animals. On day 2 post dose of HSA-NMU, food intake was reduced by 40%. Significant reductions in body weight were also observed. Figures 3 A, 3B, and 3C illustrate the finding that the anorectic effects of HSA- NMU43 are mediated by the contribution of both the NMURl and NMUR2 receptors. Acute administration of HSA-NMU43 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 Nmur2-deficient mice. The data indicate that HS A-NMU43 evokes reductions in food intake on day 2 primarily through the NMUR2 receptor.
EXAMPLE 4 The efficacy of the HSA-NMU79 conjugate was investigated in a series of feeding experiments. The results showed that the HSA-NMU79 conjugate significantly reduced food intake in diet-induced obese mice.
NMURl knockout (Nmurl-/-) mice were generated using standard homologous recombination techniques as described in the previous example. Ad libitum fed male diet- induced obese mice were weighed and dosed either Lp. 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 I)5 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 4 A and 4B shows that NMU79 can reduce food intake for up to three days following a single administration. As shown, NMU79 dose-dependently reduced food intake with a minimal efficacious dose of lmpk for overnight food intake. The effects on glucose excursion of HSA-conjugated NMU analogs were evaluated in an oral glucose tolerance test (OGTT). C57BL/6 diet-induced obese mice were dosed 30 minutes before the onset of the dark cycle with Vehicle (PBS), 0.1, 0.3 or 1 mpk of NMU79. Food was removed at time of dosing. The following day (abiut 18 hours-post dose), baseline blood glucose was measured (T=O). A glucose challenge (given orally at 2 g/kg) was then administered. Blood samples were taken after 15, 30, 60, 90, and 120 minutes and glucose levels were determined with the glucose oxidase method.
The results are shown in Figures SA and 5B. Figure SA shows that the glucose levels at multiple time points after the challenge were reduced at 0.3 and lmpk NMU79. Figure 5B shows the area under the curve (AUC) for the time course of the study. No effect on glucose excursion was observed at 0.1 mpk NMU79.
EXAMPLE 5 In the this example, different length spacers with a haloacetyl functional group for conjugating NMU to HSA were evaluated. The conjugation conditions were as reported for the preparation of HSA-NMU79 conjugate in Example 1. The spacers tested were (Ttds)2, (Ttds)3, and Txa-(Ttds)2- As shown in Figures 6A-D and Table 4, the presence of spacer comprising two or three Ttds units ((Ttds)2 or (Ttds)3)) yielded a homogeneous conjugate bearing mostly only one copy of NMU peptide precursor per molecule of HSA, as targeted (most likely at the free Cys34 of HSA) (Figure 6 A and 6B).
In contrast, substitution of a Ttds spacer with a different spacer or addition of an additional spacer to the terminus of the Ttds spacer, for example, tranexamic acid at the terminus of the (Ttds)2 spacer, as well as not using any spacer, appears to impair the conjugation reaction bringing about formation of a heterogeneous conjugate displaying double (30%) and triple (5%) conjugation of the NMU peptide molecule to HSA (other potential conjugation sites being the surface exposed amino groups of the protein) (Figure 6C and 6D). This difference may be an effect of steric hindrance in. that the haloacetyl group at the N-terminus of NMU peptide sequence might not be able to insert into the HSA Cys34's pocket unless a sufficiently long and flexible spacer exists between the functional reactive group and the N-terminus of NMU.
The results suggest that an appropriate spacer between the haloacetyl function and the NMU peptide sequence is needed to optimize the reactivity and specificity towards HSA's Cys34 in the conjugation step. In particular, a spacer comprising two, three, or more Ttds units allows conjugation of NMU to HSA to be achieved with good yields, yielding a highly pure conjugate in which there is essentially one NMU molecule per molecule of HSA, or wherein at least about 95% of the HSA molecules have one molecule of NMU attached thereto. In current embodiments, it is desired to provide conjugates wherein there is one NMU molecule per molecule of HSA, or wherein at least about 95% of the HSA molecules have one molecule of NMU attached thereto.
Figure imgf000050_0001
% of incorporation estimated by mass spec reconstruction analysis
EXAMPLE 6
This example shows that the acetamide-linked HSA-NMU conjugate is more stable in serum plasma than a maleimido-linked HSA-NMU conjugate. The HSA-NMU conjugate in which NMU was conjugated to Cys34 of HSA using a maleimido reactive group (HSA-NMU43) was compared to the HSA-NMU conjugate in which NMU was conjugated to Cy$34 of HSA using an iodoacetamide reactive group (HSA-NMU79). 100 μL of each HSA-NMU conjugate (3 mg/mL in PBS, pH 7.4) was added 10 μL of mouse plasma to give a final concentration of 10% and the mixture incubated at 370C. Aliquots were withdrawn at different times and analyzed by LC-MS.
LC-MS analysis of HSA-NMU43 incubated in mouse plasma shows (i) a progressive decrease in the amount of intact conjugate, (ii) the appearance of the unconjugated HSA free-Cys34 species, and (iii) the appearance of mouse serum albumin-NMU adduct (Figure 7). The apparent ti/2 was about 20 hours. The results suggest degradation of the conjugate due to the cleavage of the 3-thiosuccinimidyl ether linkage, likely through a retro-Michael mechanism, with subsequent regeneration of the peptide precursor with a maleimido reactive group followed by partial alkylation of the Cys thiol group on mouse serum albumin to form MSA-NMU43 conjugates. The instability of the alkyl-maleimido linkage has been also recently reported in literature for anticancer immunocoηjugates (Alley et al, Bioconjugate Chem., 19 (3): 759 (2008). In contrast to the NMU-HSA43 conjugate, the HSA-NMU79 conjugate was stable over the same time period with no evidence of by-products formed upon conjugate degradation (Figure 8). The apparent ty?_ was greater than 48 hours. In light of these results, the HSA-NMU conjugate linked via a thioether acetamide linkage has improved plasma stability over HSA- NMU conjugated linked via a 3-thiosuccinimidyl ether linkage.
EXAMPLE 7
This example provides a prophetic example of conjugating NMU79 to an Fc fragment of an antibody that avoids denaturing the internal disulfide bonds in the Fc fragment. A recombinant Fc fragment is provided that has an iV-terminal cysteine residue (CysFc). Conjugation of the NMU79 peptide precursor to the CysFc is by means of an iodoacetamide or bromoacetamide reactive group. About 3.5 mL of a solution of IEX purified CysFc (about 6.25 mg/mL; 95 μM in 50 mM Tris, 150 mM NaCl, 5 niM EDTA5 pH 8.5 buffer) is reacted with 1 ,3 molar equivalent of the NMU79 (1.75 mg; 455 nmol resuspended in 100 μL H2O) for 24-48 hours at room temperature. ESI Q-ToF mass spectrometry analysis can be used to estimate the percent completed of the reaction at various time points and the reaction terminated when a sufficient quantity of conjugate has been produced. The resulting CysFc™ NMU79 peptide conjugate can be purified by size exclusion chromatography (SEC) or Protein A affinity chromatography. The purified CysFc-NMU79 conjugate can be characterized by ESI Q- ToF Mass spectrometry.
EXAMPLE 8
This example provides another prophetic example for conjugating NMU precursors having iodoacetamide or bromoacetamide reactive groups to the cysteine groups of Fc fragments prepared from antibodies.
In general, following partial reduction of an IgG (IgGl isotype) by DTT or TCAP, NMU precursors having iodoacetamide or bromoacetamide reactive groups can be conjugated to the now available reduced cysteine residues. However, the resulting peptide-conjugated IgG is much less susceptible to Papain cleavage, the standard protocol to generate Fc fragments from full antibodies. If the antibody is first cleaved by papain and the Fc fragment thus generated is then partially reduced and the reduced cysteine residues conjugated to NMU, stability of the Fc fragment is usually compromised.
To overcome these limitations, a conjugation protocol based on Equilibrium transfer alkylating cross-link (ETAC) can be used. Liberatore et ai, Bioconjugate Chem. 1: 36- 50 (1990) describes this alternative conjugation strategy that retains the covalent disulfide linkages of antibodies or Fc-fragment by creating disulfide bridges (See Figure 9 for a schematic of the synthesis of ETAC). ETAC reagents are three-functionalized reagents, two of which react with free Cys thiols generating a covalent bridge leaving one function available for conjugation. In the reaction, temporarily reduced intra- or inter-molecular covalent bonds are re-established thus maintaining their stabilizing role in the diraeric Fc fragment (or a complete IgG).
In one embodiment shown in Figure 10, ETAC (4-[2,2-bis[(p-toϊylsulfonyl)- methyl] acetyl]benzoic acid; 1.) is reacted with iV-hydroxysuccinimide (NHS) as described in Shaunak et al:, Nature Chem. Biol. 2: 312-313 (2006). Briefly, as an example, under an argon atmosphere, a stirred suspension of ϊ. (2 g, 4 mmol), NHS (0.483 g, 4.2 mmol) and anhydrous dichloromethane (5 ml, Aldrich) is cooled using an ice bath. Neat 1 ,3-diisopropylcarbodiimide (657 μL, 4.2 mmol, DIPC) is then added dropwise. After 1.5 hours, a further 60 μL of DIPC is added, and after 3 hours, the reaction mixture is passed through a non-absorbent cotton wool filter. The homogeneous filtrate is diluted with dichloromethane (about 30 mL), washed with water (2 x 15 mL) and dried with magnesium sulfate. Filtration under gravity and removal of volatiles under vacuum provides an ETAC reagent with an active NHS ester (ETAC-NHS ester, a). Compound 2 is reacted with H2N-(Ttds)2~NMU peptide to produce BTAC-NH-
(Ttds)2-NMU peptide (3) as described in Shaunak et al. (ibid.). Briefly, as an example, under an argon atmosphere, a stirred solution of 2 (75 mg, 125 μmol) in four mL of anhydrous dichloro- methane is cooled using an ice bath. To this is added H2N-(Ttds)2-NMU peptide which has been dissolved in five mL of anhydrous dichloromethane. The reaction solution is stirred for 48 hours. Volaliles are removed under vacuum and the crude solid bis-sulfone ETAC-NH-(Ttds)2-
NMU peptide (3) is redissolved in acetone (15 mL) with gentle warming. The flask containing the stirred solution is then placed in an ice bath to precipitate the desired product 3 which is isolated using a # 3 sintered glass funnel and washed with chilled acetone (about 30 mL).
The ETAC-NH-(Ttds)2~NMU peptide (3) can be then be conjugated to the Fc fragment as described in Liberatore et at (op. cit.) and shown in Figure 9.
In an alternative embodiment, the bis-sulfone ETAC-NH-(Ttds)2-NMU peptide (3) is converted to a mono-sulfone (4) by incubating overnight in phosphate buffered saline (PBS) at pH 7.8 (See Shaunak et ah, (op. cit). Compound 4 can then be conjugated to the Fc fragment following procedures described in Shaunak et al. (ibid.), Balan et al. (Bioconjugate Chem. 18: 61-76 (2007)), Brocchini et al. (Adv. Drug Del. Rev. 60: 3-12 (2008)), and U.S. Pub. Application No. 20060210526. In a variation of the ETAC method, a modified ETAC compound, Methyl-Ketone
(MKE), has been prepared. This compound is linked to the antibody as a bridge between two cysteine residues forming a disulfide linkage where it provides a stable ketone-functionality to which a peptide can be conjugated at a later stage. On one side this approach stabilizes the antibody or antibody-fragment by re-establishing covalent bonds. However, it also offers the additional advantage that large amounts of "conjugation-ready" MKE modified antibodies (or fragments thereof) can be prepared, stored away and subsequently conjugated when needed.
Synthesis of Methyl Ketone-ETAC (MKE) is as follows. MKE has been generated using the synthetic pathway shown in Figure 10. The reagents were (a) Ethylene glycol, £>-toluenesulfonic acid (cat.) in toluene, reflux 16 hours; (b) LiAlH4 in THF dry, six hours; (c) SOCI2 neat, two hours; then add compound 3 or (d) DCC follow by addition of compound 3. MKE modification of partially reduced IgGs has been successfully performed., the modified IgG has been cleaved by papain and analyzed for the presence of Fc cross-Unking MKE moieties.
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 a neuromedin U receptor agonist in which neuromedin U or an analog thereof is conjugated to cysteine residue 34 of human serum albumin by a non-maleimido or non-succinimidyl linkage or a pharmaceutically acceptable salt thereof.
2. The composition of claim 1 , wherein the neuromedin U receptor agonist has the formula
Figure imgf000054_0001
3. A composition comprising the formula (I)
pr.Z2-Zl-peptide~Z3
wherein the peptide has the amino acid sequence Xl-χ2_χ3_χ4.χ5_χ6_χ7_χ8_ χ9-χlθ-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-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 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; Zl is a spacer; Z2 is one or more non-maleimido (3- thiosuccinimidyl ether ) or non-succinimidyl linkages; and 7? is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group and pharmaceutically acceptable salts thereof.
4. The composition of claim 3, wherein the peptide has the amino acid sequence Xl-X2.x3.x4-x5.x6.x7.x8.x9.Xl0.xl l.xl2.xl3-Xl4.xl5.xl6-Xl7-Xl8.phe- Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO:2), wherein amino acids 1 to 17 can be any amino acid or absent.
5. The composition of claim 3, wherein 2λ is a spacer comprising one to four units of Ttds (13-amino-4,7,10-trϊoxa-tridecayl succinamic acid).
6. The composition of claim 3, wherein Zl is a spacer comprising two units of Ttds (13-amino-4J510-trioxa-tridecayl succinamic acid).
7. The composition of claim 3, wherein the Z2 is a thioether acetamide linkage.
8. The composition of claim 3, wherein the carrier protein is selected from the group consisting of human serum albumin, immunoglobulin, lactoferrin, Fab fragment, scFv, and Fc fragment.
9. A compound comprising the formula
Rn-Zl -peptide-Z3
wherein the peptide has the amino acid sequence Xl-χ2.χ3.χ4.χ5_χ6.χ7.χ8.χ9-χlθ.χl L Xl2.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 X 18 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 X21 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 X24 1S 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 a spacer; RA is one or more non-maleimido (3-thiosuccinimidyl ether ) or non-succinimidyl reactive groups; and Z3 is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group.
10. The compound of claim 9, wherein the peptide has the amino acid sequence Xl-X2-X3.x4.x5.x6.x7.x8-x9-Xl0-xn-xl2-Xl3-Xl4-xl5.xl6-xl7-xl8.phe- Leu-Phe-Arg-Pro-Arg-Asn (SEQ ID NO:2), wherein amino acids 1 to 17 can be any amino acid or absent.
11. The compound of claim 9 wherein Rn is iodoacetamide, bromoacetamide, vinyl sulfonate, ETAC, or variant thereof.
12. The compound of claim 9 wherein Z Hs a spacer comprising two units of Ttds (13 -amino-4,7, 1 O-trioxa-tridecayl succinamic acid).
13. The compound of claim 9, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 10.
14. A method for treating a metabolic disorder in an individual comprising: administering to the individual a therapeutically effective amount of a composition comprising a neuromedin U receptor agonist in which neuromedin U or an analog thereof is conjugated to cysteine residue 34 of human serum albumin using a non-maleimido linker or non-succinimidyl or pharmaceutically acceptable salt thereof.
15. The method of claim 14, wherein the neuromedin U receptor agonist has the formula
Figure imgf000056_0001
16. 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 (I)
pr-Z2-Zl~peptide-Z3
wherein the peptide has the amino acid sequence Xl-X2-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 Xl 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 Xl9 is Ala, Trp, Tyr, Phe, GIu, Nva, NIe or an aromatic amino acid; amino acid X20 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 is Arg, Lys, Harg, Ala, or Leu; amino acid X23 is Pro, Ser, Sar, Ala or Leu; amino acid X24 [s 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; Zl is a spacer; Z2 is one or more non-maleimido (3- thiosuccinimidyl ether) or non-succinimidyl linkages; and Z3 is NH2 or an optionally present protecting group that, if present, is joined to the C-terminal carboxy group.
17. The method of claim 16, wherein the peptide has the amino acid sequence χl-χ2-χ3_χ4.χ5mχ6-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.
18. The method of claim 16, wherein Z^ is a spacer comprising one to four units of Ttds (13-amino-4,7,10-trioxa-tridecayl succinamic acid).
19. The method of claim 16, wherein Zl is a spacer comprising two units of Ttds (13 -amino-4,7, 10-trioxa-tridecayl succinamic acid).
20. The method of claim 16, wherein the Z2 is an thioether acetamide linkage.
21. The method of Claim 16, wherein the metabolic disorder is obesity.
22. The method of claim 16, wherein the carrier protein is selected from the group consisting of human serum albumin, immunoglobulin, lactoferrin, Fab fragment, scFv, and Fc fragment.
23. The use of the composition of any one of Claims 1-13 in the manufacture of a medicament for treatment of a metabolic disorder.
24. The use of the composition of any one of Claims 1 - 13 in the manufacture of a medicament for treatment of obesity.
25. The use of the composition of any one of Claims 1 -13 in the manufacture of a medicament for treatment of type II diabetes.
26. A pharmaceutical composition comprising the neuromedin U receptor agonist of any one of Claims 1-8 and a pharmaceutically acceptable carrier.
PCT/US2010/035260 2009-05-27 2010-05-18 Neuromedin u receptor agonists WO2010138343A1 (en)

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WO2018109540A1 (en) 2016-12-13 2018-06-21 Instituto De Medicina Molecular Methods of treating diseases associated with ilc2 cells
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WO2012050227A1 (en) 2010-10-13 2012-04-19 Takeda Pharmaceutical Company Limited Peptide derivative
EP2842965A1 (en) * 2013-09-03 2015-03-04 Gubra ApS Neuromedin U analogs comprising serum albumin binding amino acid residue
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