DUAL AGONIST GLP-1 AND NEUROTENSIN FUSION PEPTIDE

Abstract

The present invention relates to a polypeptide comprising a first peptide linked to a second peptide, optionally via a linker molecule, which first peptide comprises an appetite regulating hormone peptide, e.g. glucagon like peptide 1 (GLP-1), such as amino acids 7-37 of the initial GLP-1 product (1-37), and a Neurotensin (NT) like peptide, targeting both the GLP-1 receptor (GLP-1R) and NT receptors (NTR1-3) and display an increased effect on decrease of appetite and food intake and body weight compared to simultaneous administration of both peptides.

Claims

1-30. (canceled)

31. A fusion peptide comprising a first peptide linked to a second peptide, which first peptide comprises the sequence: X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9 wherein X.sub.1 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β hydroxy-histidine, homohistidine, N.sup.α-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; X.sub.2 is A, G, V, L, I, K, S, aminoisobutyric acid (Aib), (1-aminocyclopropyl) carboxylic acid, (1 aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1 aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid; X.sub.3 is E, D or Q; X.sub.4 is G or A; X.sub.5 is T, V, S or I; X.sub.6 is F or Y; X.sub.7 is T, or S; X.sub.8 is S, V, or D; X.sub.9 is S, D, E, N or is not present, or the first peptide comprising the sequence laid out in SEQ ID NO: 40 or SEQ ID NO: 41 or SEQ ID NO: 42, and which second peptide has an amino acid sequence with at least 70% identity with any one of SEQ ID NO:24 to SEQ ID NO:31, or wherein the second peptide is selected from the list consisting of: X.sub.10-X.sub.11-P-X.sub.12-I-L; P-X.sub.10-X.sub.11-P-X.sub.12-I-L; K-P-X.sub.10-X.sub.11-P-X.sub.12-I-L; N-K-P-X.sub.10-X.sub.11-P-X.sub.12-I-L; E-N-K-P-X.sub.10-X.sub.11-P-X.sub.12-I-L; Y-E-N-K-P-X.sub.10-X.sub.11-P-X.sub.12-I-L; L-Y-E-N-K-P-X.sub.10-X.sub.11-P-X.sub.12-I-L; Q-L-Y-E-N-K-P-X.sub.10-X.sub.11-P-X.sub.12-I-L; or E-L-Y-E-N-K-P-X.sub.10-X.sub.11-P-X.sub.12-I-L wherein X.sub.10 is R or K; X.sub.11 is R or K; and X.sub.12 is Y, S, C or T, and wherein the fusion peptide is a dual agonist of both a glucagon like peptide 1 receptor and a neurotensin receptor.

32. The fusion peptide according to claim 31 comprising a first peptide linked to a second peptide, which first peptide comprises the sequence: X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10 wherein X.sub.1 is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, β hydroxy-histidine, homohistidine, N.sup.α-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; X.sub.2 is A, G, V, L, I, K, S, aminoisobutyric acid (Aib), (1-aminocyclopropyl) carboxylic acid, (1 aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylic acid, (1 aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylic acid; X.sub.3 is E, D or Q; X.sub.4 is G or A; X.sub.5 is T, V, S or I; X.sub.6 is F or Y; X.sub.7 is T, or S; X.sub.8 is S, V, or D; X.sub.9 is S, D, E, N or is not present; X.sub.10 is V or the first peptide comprising the sequence laid out in SEQ ID NO: 40 or SEQ ID NO: 41 or SEQ ID NO: 42.

33. The fusion peptide according to claim 31, wherein the first peptide has at least 70% identity with any one of SEQ ID NO:2 to SEQ ID NO: 23 or SEQ ID NO: 34 to SEQ ID NO: 39.

34. The fusion peptide according to claim 31, wherein the second peptide is any of the sequences laid out in SEQ ID NO: 24-30.

35. The fusion peptide according to claim 31, wherein the first peptide is the N-terminus of the fusion peptide.

36. The fusion peptide according to claim 31, wherein the C-terminus of the fusion peptide has the amino acid sequence laid out in SEQ ID NO:30.

37. The fusion peptide according to claim 31, wherein the fusion peptide is a peptide having at least 70% identity with SEQ ID NO:32 or a peptide having at least 70% identity with SEQ ID NO:33.

38. The fusion peptide according to claim 31, wherein the first peptide is linked to the second peptide via a linker molecule.

39. The fusion peptide according to claim 31, wherein the first peptide is H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q (SEQ ID No: 15).

40. The fusion peptide according to claim 39, wherein the second peptide is E-L-Y-E-N-K-P-R-R-P-Y-I-L.

41. The fusion peptide according to claim 31, wherein the first peptide has the sequence H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-K-G-R (SEQ ID No: 2) and the second peptide is E-L-Y-E-N-K-P-R-R-P-Y-I-L.

42. The fusion peptide according to claim 31, wherein the C-terminal end of said fusion peptide is amidated.

43. The fusion peptide according to claim 31, wherein the fusion peptide is pegylated.

44. The fusion peptide according to claim 35, wherein the second peptide is pegylated.

45. The fusion peptide according to claim 40, wherein the second peptide is pegylated and the pegylation site is the lysine of the second peptide.

46. The fusion peptide according to claim 31, wherein the fusion peptide is linked to an albumin binding moiety via a spacer.

47. The fusion peptide according to claim 46, wherein the albumin binding moiety linked via a spacer is attached to said fusion peptide via the s-amino group of a lysine residue.

48. A nucleic acid molecule having a sequence encoding a fusion peptide according to claim 31.

49. A vector comprising the nucleic acid molecule according to claim 48.

50. A host cell comprising the nucleic acid molecule according to claim 48.

51. A pharmaceutical composition comprising the fusion peptide according to claim 31.

52. A method of reducing appetite in a mammal comprising administering the fusion peptide according to claim 31 to the mammal.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0167] FIG. 1 shows the effect of GLP-1, NT and GLP-1/NT combi peptide on food intake.

[0168] FIG. 2 shows the effect of NT alone and NT+NT antagonist on food intake.

[0169] FIG. 3 represents PEGylated and lipidated NT peptides.

[0170] FIG. 4 shows the effect of NT and Liraglutide on body weight, food intake and body composition.

[0171] FIG. 5 shows in-vitro potency and efficacy of PEG-NT on inositol phosphate accumulation and food intake.

[0172] FIG. 6 shows the effect of NT and Liraglutide on selected gene expression levels in the liver.

[0173] FIG. 7. shows how doses of 100 nmol/kg of the shortened GLP-1-NT combination peptides effects food intake in lean mice.

[0174] FIG. 8. shows that a full Peg-NT-GLP-1 combination peptides at different dose levels reduces acute food intake and body weight in diet induced obesity (DIO) mice.

[0175] FIG. 9. shows that a full Peg-NT-GLP-1 (100 nmol/kg) combination peptides reduces cumulative food intake and induces body weight loss relative to both saline and Peg-NT (100 nmol/kg) after sub-chronic treatment in DIO mice.

DESCRIPTION OF EMBODIMENTS

[0176] The term “polypeptide” and “peptide” as used herein means a compound composed of at least six constituent amino acids connected by peptide bonds. The constituent amino acids may be from the group of the amino acids encoded by the genetic code and they may be natural amino acids which are not encoded by the genetic code, as well as synthetic amino acids.

[0177] A peptide or polypeptide will have an amino terminus and a carboxyl terminus. In the context of the invention, the amino terminus and a carboxyl terminus may also be referred to as the N-terminus and the C-terminus, respectively, and corresponding derived forms.

[0178] Natural amino acids, which are not encoded by the genetic code, comprise e.g. hydroxyproline, γ-carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic amino acids comprise amino acids manufactured by chemical synthesis, e.g. D-isomers of the amino acids encoded by the genetic code such as D-alanine and D-leucine, a-aminoisobutyric acid (Aib), a-aminobutyric acid (Abu), tert-butylglycine (Tie), p-aianine, 3-aminomethyl benzoic acid, and anthranilic acid.

[0179] Peptide synthesis may be carried out by methods that are well known to the person skilled in the art. Briefly, peptides may be synthesized on Fmoc protected Rink amide resin or a similar resin suitable for solid phase peptide synthesis. Boc chemistry may be used; alternatively, protection amines can also be accomplished in acetonitrile solution using 4-dimethylaminopyridine (DMAP) as base. The Fmoc strategy using the FastMoc UV protocols employing HBTU (2-(1H-Benzotriazol-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphate) mediated couplings in N-methyl pyrrolidone and UV monitoring of the deprotection of the Fmoc protection group is yet another appropriate method.

[0180] Other coupling reagents besides from HBTU and HATU as described in e.g. Current Opinion in Chemical Biology, 2004, 8:211-221 may also be used.

[0181] The attachment of sidechains and linkers to specific lysine residues on the crude resin bound protected peptide may eventually be introduced in a specific position by incorporation of Fmoc-Lys(Dde)-OH during automated synthesis followed by selective deprotection with hydrazine. Other orthogonal protecting groups may be used on Lysine.

[0182] The first peptide of the polypeptide of the invention may also be referred to using the term “GLP-1 peptide”, and its derived forms, e.g. GLP-1 [7-36] (i.e. SEQ ID NO:2), GLP-1 analogue, GLP-1 derivative or a derivative of a GLP-1 analogue. GLP-1 analogues or derivatives of GLP-1 analogues may also be considered to be appetite regulating hormone peptides, e.g. Exendin-4, GLP-2, Glucagon, VIP, Secretin or PACAP-38 amongst others, or NKA, amylin, or PYY, which would also be suitable.

[0183] In one embodiment the first peptide is an insulinotropic agent.

[0184] The second peptide of the polypeptide of the invention may also be referred to using the term “NT peptide”, and its derived forms, e.g. neurotensin (NT) [1-13] (i.e. SEQ ID NO:24), NT analogue, NT derivative or derivative of a NT analogue.

[0185] The term “analogue” as used herein referring to a polypeptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide. Formulae of peptide analogues and derivatives thereof are drawn using standard single letter abbreviation for amino acids used according to IUPAC-IUB nomenclature.

[0186] The term “derivative” as used herein in relation to a peptide means a chemically modified peptide or an analogue thereof, wherein at least one substituent is not present in the unmodified peptide or an analogue thereof, i.e. a peptide which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters and the like.

[0187] The term “albumin binding moiety” as used herein means a moiety which binds non-covalently to human and/or other animal serum albumin. The albumin binding moiety attached to the polypeptide typically has an affinity (as defined herein above) below 10 μM to human serum albumin and preferably below 1 μM. A range of albumin binding moieties are known among linear and branched lipophilic moieties containing 4-40 carbon atoms, compounds with a cyclopetanophenanthrene skeleton, peptides having 10-30 amino acid residues etc.

[0188] The term “spacer” as used herein means a spacer that separates a peptide and an albumin binding moiety with a chemical moiety which comprises at least 5 non-hydrogen atoms where 30-50% of these are either N or O.

[0189] The term “therapeutic polypeptide” as used herein means a polypeptide which is being developed for therapeutic use, or which has been developed for therapeutic use.

[0190] The term “insulinotropic agent” as used herein means a compound which is an agonist of the human GLP-1 receptor, i.e. a compound which stimulates the formation of cAMP in a suitable medium containing the human GLP-1 receptor. The potency of an insulinotropic agent is determined by calculating the EC50 value from the dose-response curve.

EXAMPLES

Example 1: GLP-1/NT Fusion Peptide Reduces Food Intake Synergistically

[0191] As shown in FIG. 1, both condition 5) (GLP-1/Neurotensin fusion peptide) and condition 4) (Neurotensin+GLP-1) show a lower food intake when compared to condition 2) (Neurotensin) and 3) (GLP-1), where each of the peptides are administrated separately, indicating a synergistic effect of the co-administration. Importantly, condition 5) (Neurotensin/GLP-1 fusion peptide) show a greater effect in lowing the food intake compared to condition 4) (Neurotensin+GLP-1) indicating an improved effect by the fusion peptide, where the peptide is constrained and may activate the two individual receptors (the neurotensin receptor and the GLP-1 receptor) simultaneously.

[0192] Lean eight weeks old C57Bl/6J mice (Janvier) were single housed in metabolic cages (TSE systems) in a temperature controlled room with a 12:12 h light dark cycle (lights on at 6:00) with ad libitum access to tap water and a chow diet (#1310, Altromin). Mice were acclimatized to the cages before peptides were tested.

[0193] For peptide testing, mice were randomized into the following groups according to their food intake during acclimatization:

[0194] 1) Vehicle (saline) n=6

[0195] 2) Neurotensin (30 nmol/kg) n=6

[0196] 3) GLP-1 (30 nmol/kg) n=7

[0197] 4) Neurotensin (30 nmol/kg)+GLP-1 (30 nmol/kg) n=6

[0198] 5) Neurotensin/GLP-1 fusion peptide (30 nmol/kg) n=7

[0199] Peptides were dosed subcutaneously in a volume of 10 ml/kg in the early light phase after overnight fasting in a cross-over design. Food intake was monitored automatically after dosing and the mice were allowed a washout period of 5 days between dosings. As shown in FIG. 1, condition 5) (Neurotensin/GLP-1 fusion peptide) shows a lower food intake when compared to condition 4) (Neurotensin+GLP-1) indicating a synergistic effect of the fusion perpetide despite a 1:2 molar ratio of administered peptide in the two conditions.

Example 2: The GLP-1/NT Fusion Peptide is a Potent NTSR1 Agonist

[0200] The neurotensin receptor (NTS-R1) is described as a primarily Gα.sub.q/11-coupled receptor.

[0201] Human Embryonic Kidney (HEK) 293 cells were cultured at 10% CO.sub.2, 90% humidity and 37° C. in Dulbecco's Modified Eagle Medium with GlutaMAX (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin.

[0202] For inositol phosphate (IP) accumulation assay, HEK293 cells were seeded in poly-D-lysine-coated 96-well-plates (PerkinElmer) the day before transfections, at a density of 30,000 cells per well. Transient transfections were performed using Lipofectamine 2000 (Thermo Fisher) according to the manufacturer's instructions. Cells were transfected with 20 ng DNA (pCMV-hNTSR 1 or empty pCMV) and 0.6 μl Lipofectamine 2000 per well for 5 h. IP assays were performed 48 h after the transfection was started.

[0203] One day after the transfection, HEK293 cells were incubated in cell culture medium containing 5 μCi/ml myo-[2-.sup.3H]inositol. Following 24 h incubation, the cells were washed in HBSS (Gibco) and incubated in 100 μl per well HBSS containing 10 mM LiCl for 30 min at 37° C. The ligands were added and the cells incubated for 45 min at 37° C. The cells were then lysed in 10 mM formic acid for >40 min. 20 μl of the lysate were transferred to a white 96-well plate containing 80 μl of yttrium silicate Scintillation Proximity Assay (YSi-SPA) beads (PerkinElmer). Lyophilized YSi-SPA beads were reconstituted in H.sub.2O (1 g in 10 ml) and then diluted 1:8 before use. Plates were sealed, shaken at maximum speed for 10 min and centrifuged at 400 g for 5 min. After an 8 h delay, γ-radiation was measured in a Packard Top Count NXT scintillation plate reader. Determinations were made in duplicate or triplicate. As shown in FIG. 1B and Table 1, myo-[2-.sup.3H]inositol accumulates similarly in the GLP-1/NT fusion peptide (SEQ ID NO: 45) sample as it does in the NT peptide sample, indicating that the GLP-1/NT fusion peptide acts on the NTSR1 receptor with similar potency to NT.

TABLE-US-00001 TABLE 1 NT GLP1-NT Sigmoidal dose-response (variable slope) Best-fit values Bottom 176 139.4 Top 436.1 3336 LogEC50 −8.603 −8.502 HillSlope 1.11 2.019 EC50 2.494e−009 3.149e−009

[0204] Example 3: FIG. 2 shows the effect of NT alone and NT+NT antagonist on food intake. Graph A shows the effect of saline or NT 3600 nmol/kg on acute 2 h and 4 h food intake in lean mice. Graph B shows the effect of saline, NT 150 nmol/kg, NTS1/NTS2 antagonist SR142948A 692 nmol/kg or NT+SR142948A on acute 30 min food intake in lean rats. Chow fed mice/rats fasted overnight were injected intraperitoneally in the beginning of the light cycle and food returned (n=8-12/group). Food intake was measured continuously after injection in an indirect calorimetry system. Data from both graphs was analyzed using a one-way ANOVA analysis followed by a Tukey's post hoc test. Mean±SEM depicted on graphs. **p<0.01 and ***p<0.001 between groups.

[0205] Example 4: As shown in FIG. 4, P-NT+liraglutide has a significantly greater effect on reducing body weight, food intake and lean or fat body mass in DIO mice relative to either liraglutide or P-NT alone.

[0206] DIO mice fed a high fat high sucrose diet (58% kcal from fat) (for over 4 months before study initiation) were injected subcutaneously right before lights out daily for 6 days, and food intake (FI) and body weight (BW) measured daily (n=6/group). Food intake and body weight were analysed using repeated measures two way ANOVA (A+B) or one-way ANOVA analysis (C+D), both followed by a Tukey's post hoc test. Mean±SEM depicted on graphs. Graph A+B) .sup.##p<0.01, .sup.###p<0.001 and ****p<0.0001 combination treatment versus all other treatment groups. Graphs C+D) *p<0.05, **p<0.01 and ****p<0.0001 between groups.

[0207] Example 5: As shown in FIG. 5, Peg-NT has a greater in-vitro potency and efficacy in an inositol phosphate accumulation assay and in-vivo on food intake, relative to NT. Graph A shows the accumulation of intracellular IP3 upon stimulation with increasing concentrations of Peg-NT in HEK293 cells transfected with the NTS1 receptor. Data has been normalized to maximal NT response (100%) and to vector (pcDNA) response (0%). Graph B shows the effect of NT or Peg-NT on acute food intake in lean mice. Chow fed lean mice fasted overnight were injected subcutaneously in the beginning of the light cycle and food returned. Food intake was measured continuously after injection in an indirect calorimetry system (n=7-8/group). Food intake was analyzed using a one-way ANOVA analysis followed by a Tukey's post hoc test. Mean±SEM depicted on graphs. **p<0.01, ***p<0.001 and ****p<0.0001 between groups.

[0208] Example 6: As shown in table 2 below, P-NT+liraglutide has a greater effect on reducing cholesterol, leptins, insulin, and glucose blood levels in DIO mice relative to either liraglutide or P-NT alone.

[0209] DIO mice fed a high fat high sucrose diet (58% kcal from fat) (for over 4 months before study initiation) were injected subcutaneously right before lights out daily for 6 days (n=6/group). On the day of termination, mice were fasted for 4 h before blood sampling (tail vein) and blood glucose measurement (glucometer) were performed. Blood biochemistry markers were analysed using a one-way ANOVA followed by a Tukey's post hoc test.

TABLE-US-00002 TABLE 2 Plasma P-NT + marker Vehicle P-NT Cholesterol 275.5 ± 32.9 245.9 ± 18.8  226.3 ± 24.3 205.2 ± 17.0 (mg/ Triglycerides 95.1 ± 6.8 82.2 ± 3.7  100.4 ± 7.1  82.2 ± 6.4 (rng/ ) 48.1 ± 4.4 42.3 ± 5.9  35.4 ± 5.2  21.7 ± 1.8* ( /mL) Insulin  6.4 ± 1.2 5.3 ± 0.9  4.9 ± 1.0   2.3 ± 0.8* ( /mL) Glucose  8.3 ± 0.4   6.7 ± 0.4**  8.4 ± 0.3    6.4 ± 0.2** ( ) Values denote mean± SEM. *p < 0.05 difference against vehicle. **p < 0.01 difference against vehicle and P-NT.|

[0210] Example 7: FIG. 6 shows the effect of 6 day NT (396 nmol/kg), liraglutide (8 nmol/kg) or NT+liraglutide treatment on selected hepatic gene expression levels in DIO mice. DIO mice fed a high fat high sucrose diet (58% kcal from fat) (for over 4 months before study initiation) were injected subcutaneously right before lights out daily for 6 days (n=6/group). On the day of termination, mice were fasted for 4 hours before receiving a final injection of peptides. 2 h after the final injection mice were sacrificed and liver samples collected. Gene expression levels were analysed using a one-way ANOVA followed by a Tukey's post hoc test. Mean±SEM depicted on graphs. *p<0.05 and ***p<0.001 between groups.

[0211] Example 8: The following table 3 shows different synthesized peptides of the invention. More particularly, the peptides shown are shortened GLP-1-NT combination peptides±a KKGG linker.

TABLE-US-00003 TABLE 3 Peptide # Peptide sequence 1 HAEGTFTSDRRPYIL-acid 2 HAEGTFTSDKKGGRRPYIL-acid 5 HAEGTFTSDVSSYENKPRRPYIL-acid 6 HAEGTFTSDVSSYKKGGENKPRRPYIL-acid 9 HAEGTFTSDVSSYLEGQELYENKPRRPYIL-acid 10 HAEGTFTSDVSSYLEGQKKGGELYENKPRRPYIL-acid 11 HAEGTFTSDVSSYLEGQAAKELYENKPRRPYIL-acid 12 HAEGTFTSDVSSYLEGQAAKKKGGELYENKPRRPYIL-acid

[0212] The peptides of this table are among the peptides referred to in the following examples.

[0213] Example 9: As shown in FIG. 7, shortened GLP-1-NT combination peptides reduces food intake in lean mice in comparison to saline in chow fed lean mice.

[0214] Chow fed lean mice fasted overnight were injected once SC in the beginning of the light cycle and food returned. Food intake measured continuously after injection in an indirect calorimetry system. Food intake was analysed using repeated measures two way ANOVA followed by a Tukey's post hoc test. A one-way ANOVA followed by a Tukey's post hoc test was performed in the inset in graph B. *p<0.05 peptide 9 vs. saline, .sup.#p<0.05 peptide 10 vs. saline, .sup.##p<0.01 peptide 10 vs. saline, .sup.###p<0.001 peptide 10 vs. saline.

[0215] Example 10: As shown in FIG. 8, a full Peg-NT-GLP-1 (100 nmol/kg) combination peptide at different dose levels reduces acute food intake and body weight in DIO mice relative to both saline and Peg-NT (100 nmol/kg) after sub-chronic treatment in DIO mice.

[0216] Non-fasted DIO mice fed a high-fat high sucrose diet (58% kcal from fat) (for over 4 months before study initiation) were injected with a single dose subcutaneously right before light outs and food intake measured continuously in an indirect calorimetry system (A-B: mean BW±SEM 57.4±1.1 g at study initiation; C-D: mean BW±SEM 48.9±1.5 g at study initiation).

[0217] Example 11: As shown in FIG. 9, a full Peg-NT-GLP-1 (100 nmol/kg) combination peptides reduces cumulative food intake and induces body weight loss relative to both saline and Peg-NT (100 nmol/kg) after sub-chronic treatment in DIO mice.

[0218] Non-fasted DIO mice fed a high-fat high sucrose diet (58% kcal from fat) (for over 4 months before study initiation) were injected with a single dose subcutaneously right before light outs and food intake measured continuously in an indirect calorimetry system (A-B: mean BW±SEM 57.4±1.1 g at study initiation; C-D: mean BW±SEM 48.9±1.5 g at study initiation). Food intake was analysed using repeated measures two way ANOVA (A+C) or one-way ANOVA (B+D) analysis, both followed by a Tukey's post hoc test. Graph A) *p<0.05 saline vs. Peg-NT-GLP1, **p<0.01 saline vs. Peg-NT-GLP1, ***p<0.001 saline vs. Peg-NT-GLP1, ****p<0.0001 saline vs. Peg-NT-GLP1, .sup.#p<0.05 Peg-NT vs. Peg-NT-GLP1, .sup.##p<0.01 Peg-NT vs. Peg-NT-GLP1, .sup.###p<0.001 Peg-NT vs. Peg-NT-GLP1, .sup.####p<0.0001 Peg-NT vs. Peg-NT-GLP-1. Graphs B and D) *p<0.05 vs. saline, ***p<0.001 vs. saline.

Conclusion

[0219] The above embodiments collectively show that the combination of NT and GLP1 analogues reduces acute food intake in mice to a higher degree than monotherapy treatment. Surprisingly, NT and GLP1 combination peptides had an even greater effect on reducing acute food intake in mice than a loose combination of the same. This was unexpected as common knowledge in the field dictates that a loose combination of two peptides should generally have a superior bioavailability for their two sites of actions, than a fusion peptide of the same two peptides. Further, pegylation of NT (Peg-NT) induces a synergistic effect with GLP-1 analogues (in a loose combination or in a full combination peptide) on food intake, body weight and adiposity in sub-chronic treatment settings. Peg-NT and liraglutide combination treatment also gives evidence of inducing a beneficial effect on glycemia and hepatic lipid handling and removal of blood cholesterol. The combination NT and GLP-1 and/or NT and GLP-1 analogues thus represents a novel and promising treatment for obesity and possibly co-morbidities.