Acylated Calcitonin Mimetics

Abstract

Disclosed herein are calcitonin mimetics that are acylated at a lysine residue located at the 11 position or 19 position of the calcitonin mimetic, and the use thereof as medicaments in the treatment of various diseases and disorders, including diabetes, excess bodyweight, excessive food consumption and metabolic syndrome, NASH, alcoholic and non-alcoholic fatty liver disease, the regulation of blood glucose levels, the regulation of response to glucose tolerance tests, the regulation of food intake, and the treatment of osteoporosis and the treatment of osteoarthritis.

Claims

1. A calcitonin mimetic that is a peptide acylated at a lysine residue located at the 11 position of the calcitonin mimetic and/or that is acylated at a lysine residue located at the 19 position of the calcitonin mimetic, wherein the side chain C-amino group of said lysine residue is acylated with an acyl group that is: a C.sub.16 or longer fatty acid with an optional linker, or a C.sub.16 or longer fatty diacid with an optional linker.

2. The calcitonin mimetic of claim 1, wherein the calcitonin mimetic is a peptide of formula (I) (a):
CX.sub.2X.sub.3LSTCX.sub.8LGK.sub.Ac wherein X.sub.2=A, G or S, X.sub.3=N or S, X.sub.8=M, V or α-aminoisobutyric acid (AiB), and wherein K.sub.Ac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group that is: the C.sub.16 or longer fatty acid with an optional linker, or the C.sub.16 or longer fatty diacid with an optional linker.

3. The calcitonin mimetic of claim 1, wherein the calcitonin mimetic is peptide of formula (I) (b):
CX.sub.2X.sub.3LSTCX.sub.8LGX.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18K.sub.Ac wherein X.sub.2=A, G or S, X.sub.3=N or S, X.sub.8=M, V or α-aminoisobutyric acid (AiB), X.sub.11=R, K, T, A or KAc, X.sub.12=L or Y, X.sub.13=S, T, W or Y, X.sub.14=Q, K, R or A, X.sub.15=D, E or N, X.sub.16=L or F, X.sub.17=H or N, X.sub.18=R, K or N, and wherein K.sub.Ac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group that is: the C.sub.16 or longer fatty acid with an optional linker, or the C.sub.16 or longer fatty diacid with an optional linker.

4. The calcitonin mimetic of claim 1, wherein the calcitonin mimetic is a peptide of formula (II):
CX.sub.2X.sub.3LSTCX.sub.8LGX.sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.sub.20X.sub.21X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27GX.sub.29X.sub.30X.sub.31P wherein X.sub.2=A, G or S, X.sub.3=N or S, X.sub.8=M, V or α-aminoisobutyric acid (AiB), X.sub.11=R, K, T, A or K.sub.Ac, X.sub.12=L or Y, X.sub.13=S, T, W or Y, X.sub.14=Q, K, R or A, X.sub.15=D, E or N, X.sub.16=L or F, X.sub.17=H or N, X.sub.18=R, K or N, X.sub.19=K.sub.Ac, L, F or K, X.sub.20=Q, H or A, X.sub.21=T or R, X.sub.22=Y or F, X.sub.23=S or P, X.sub.24=G, K, Q or R, X.sub.25=T, I or M, X.sub.26=S, N, D, G or A, X.sub.27=T, V, F or I, X.sub.29=S, A, P or V, X.sub.30=N, G or E, X.sub.31=A, T or S, wherein either X.sub.11 is K.sub.Ac and/or X.sub.19 is K.sub.Ac, and wherein K.sub.Ac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group that is: a C.sub.16 or longer fatty acid, a C.sub.16 or longer fatty diacid, a linker-C.sub.16 or longer fatty acid, or a linker-C.sub.16 or longer fatty diacid.

5. The calcitonin mimetic of claim 4, wherein the peptide of formula (II) is:
CX.sub.2X.sub.3LSTCX.sub.8LGX.sub.11LX.sub.13X.sub.14X.sub.15LX.sub.17X.sub.18X.sub.19X.sub.20TX.sub.22PX.sub.24TDVGANAP wherein X.sub.2=A, G or S, X.sub.3=N or S, X.sub.8=M, V or AiB, X.sub.11=KAc, R, K, T or A, X.sub.13=T, S or Y, X.sub.14=Q or A, X.sub.15=D or E, X.sub.17=H or N, X.sub.18=R or K, X.sub.19=KAc, L, F or K, X.sub.20=Q, H or A, X.sub.22=Y or F, or X.sub.24=K, Q or R.

6. The calcitonin mimetic of claim 4, wherein X.sub.2 is S and X.sub.3 is N; or X.sub.2 is G and X.sub.3 is N; or X.sub.2 is A and X.sub.3 is S.

7. The calcitonin mimetic of claim 4, wherein X.sub.11 is K.sub.Ac, X.sub.17 is H, X.sub.18 is K, X.sub.19 is L and X.sub.20 is Q or A; or X.sub.11 is K.sub.Ac, X.sub.17 is H, X.sub.18 is R, X.sub.19 is L and X.sub.20 is Q or A; or X.sub.11 is K.sub.Ac, X.sub.17 is N, X.sub.18 is K, X.sub.19 is F and X.sub.20 is H or A; or X.sub.11 is K.sub.Ac, X.sub.17 is N, X.sub.18 is R, X.sub.19 is is F and X.sub.20 is H or A; or X.sub.11 is K.sub.Ac, X.sub.17 is H, X.sub.18 is K, X.sub.19 is is K.sub.Ac and X.sub.20 is Q or A; or X.sub.11 is K.sub.Ac, X.sub.17 is H, X.sub.18 is R, X.sub.19 is is K.sub.Ac and X.sub.20 is Q or A; or X.sub.11 is K.sub.Ac, X.sub.17 is N, X.sub.18 is K, X.sub.19 is K.sub.Ac and X.sub.20 is H or A; or X.sub.11 is K.sub.Ac, X.sub.17 is N, X.sub.18 is R, X.sub.19 is K.sub.Ac and X.sub.20 is H or A.

8. The calcitonin mimetic claim 4, wherein X.sub.2 is S, X.sub.3 is N, X.sub.11 is K.sub.Ac, X.sub.13 is S, X.sub.17 is H, X.sub.18 is K or R, X.sub.19 is L, X.sub.20 is Q or A and X.sub.22 is Y; or X.sub.2 is S, X.sub.3 is N, X.sub.11 is R or K, X.sub.13 is S, X.sub.17 is H, X.sub.18 is K or R, X.sub.19 is K.sub.Ac, X.sub.20 is Q or A and X.sub.22 is Y; or X.sub.2 is A, X.sub.3 is S, X.sub.11 is K.sub.Ac X.sub.13 is S, X.sub.17 is H, X.sub.18 is K or R, X.sub.19 is L, X.sub.20 is Q or A and X.sub.22 is F; or X.sub.2 is A, X.sub.3 is S, X.sub.11 is K.sub.Ac, X.sub.13 is S, X.sub.17 is H, X.sub.18 is K or R, X.sub.19 is K.sub.Ac, X.sub.20 is Q or A and X.sub.22 is F; or X.sub.2 is G, X.sub.3 is N, X.sub.11 is K.sub.Ac, X.sub.13 is T, X.sub.17 is N, X.sub.18 is K or R, X.sub.19 is F, X.sub.20 is H or A and X.sub.22 is F; or X.sub.2 is G, X.sub.3 is N, X.sub.11 is R or K, X.sub.13 is T, X.sub.17 is N, X.sub.18 is K or R, X.sub.19 is K.sub.Ac, X.sub.20 is H or A and X.sub.22 is F.

9-10. (canceled)

11. The calcitonin mimetic of claim 1, wherein the calcitonin mimetic is a 33mer peptide in accordance with formula (III):
CSNLSTCX.sub.6LGX.sub.7LSQDLHRX.sub.8QTYPKX.sub.1TX.sub.5VGANAP; or wherein the calcitonin mimetic is a 35mer peptide in accordance with formula (IV):
CSNLSTCX.sub.6LGX.sub.7LSQDLHRX.sub.8QTYPKX.sub.1X.sub.2X.sub.3TX.sub.5VGANAP; or wherein the calcitonin mimetic is a 36mer peptide in accordance with formula (V):
CSNLSTCX.sub.6LGX.sub.7LSQDLHRX.sub.8QTYPKX.sub.1X.sub.2X.sub.3X.sub.4TX.sub.5VGANAP; or wherein the calcitonin mimetic is a 37mer peptide in accordance with formula (VI):
CSNLSTCX.sub.6LGK.sub.AcLZX.sub.1X.sub.2X.sub.3X.sub.4TX.sub.5VGANAP; wherein each of X.sub.1 to X.sub.4 is any amino acid, with the proviso that at least one of X.sub.1 to X.sub.4 is a basic amino acid residue, and/or at least two of X.sub.1 to X.sub.4 are independently a polar amino acid residue or a basic amino acid residue, and/or at least one of X.sub.1 to X.sub.4 is a Gly residue, and wherein none of X.sub.1 to X.sub.4 is an acidic residue; wherein X.sub.5 is D or N; wherein X.sub.6 is AiB or M; wherein either X.sub.7 is K.sub.Ac and X.sub.8 is L, or X.sub.7 is R or K and X.sub.8 is K.sub.Ac; wherein Z is selected from SQDLHRLSNNFGA, SQDLHRLQTYGAI or ANFLVHSSNNFGA; and wherein K.sub.Ac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group that is: a C.sub.16 or longer fatty acid, a C.sub.16 or longer fatty diacid, a linker-C.sub.16 or longer fatty acid, or a linker-C.sub.16 or longer fatty diacid.

12. The calcitonin mimetic of claim 11, wherein at least one of X.sub.1 or X.sub.4 is a basic amino acid residue.

13. The calcitonin mimetic of claim 11, wherein at least one of X.sub.7 or X.sub.4 is a basic amino acid residue, and at least two of X.sub.1 to X.sub.4 are independently a polar amino acid residue or a basic amino acid residue, and none of X.sub.1 to X.sub.4 is an acidic residue.

14-16. (canceled)

17. The calcitonin mimetic of claim 11, wherein the basic amino acid residue is Arg, His or Lys, and/or the polar amino acid residue is Ser, Thr, Asn, Gln or Cys.

18. The calcitonin mimetic of claim 11, wherein X.sub.1 is selected from Asn, Phe, Val, Gly, lie, Leu, Lys, His or Arg; X.sub.2 is Ala, Asn, His, Leu, Ser, Thr, Gly or Lys; X.sub.3 is Ala, Phe, lie, Ser, Pro, Thr, Gly or Lys; and/or X.sub.4 is Ile, Leu, Gly, His, Arg, Asn, Ser, Lys, Thr or Gln; with the proviso that at least one of X.sub.1 or X.sub.4 is a basic amino acid residue, and/or at least two of X.sub.1 to X.sub.4 are independently a polar amino acid residue and/or a basic amino acid residue, and/or at least one of X.sub.1 to X.sub.4 is a Gly residue.

19. The calcitonin mimetic of claim 18, wherein X.sub.1 is Asn, Gly, lie, His or Arg; X.sub.2 is Asn, Leu, Thr, Gly or Lys; X.sub.3 is from Phe, Pro, lie, Ser, Thr, Gly or Lys; and/or X.sub.4 is from Gly, His, Asn, Ser, Lys, Thr or Gln.

20-21. (canceled)

22. The calcitonin mimetic of claim 1, wherein the linker comprises a glutamic acid residue and/or an oligoethyleneglycol (OEG) amino acid linker comprising one OEG amino acid or two or more OEG amino acids linked together, wherein said OEG amino acid is: ##STR00129## wherein n is from 1 to 10.

23. (canceled)

24. The calcitonin mimetic of claim 22, wherein said OEG amino acid linker further comprises one or more glutamic acid residues linked to the amino terminus or to the carboxyl terminus of the OEG amino acid linker.

25. (canceled)

26. The calcitonin mimetic of claim 22, wherein the OEG amino acid linker is ##STR00130##

27. The calcitonin mimetic of claim 26, wherein the linker is ##STR00131##

28. (canceled)

29. The calcitonin mimetic of claim 1, wherein the acyl group is a C.sub.18 to C.sub.30 fatty acid, a C.sub.18 to C.sub.30 fatty diacid, a linker-C.sub.18 to C.sub.30 fatty acid, or a linker-C.sub.18 to C.sub.30 fatty diacid.

30-31. (canceled)

32. The calcitonin mimetic of claim 1, wherein the peptide is: TABLE-US-00026 CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKTDVGANAP, CSNLSTC(AiB)LGK.sub.AcLSQDLHRLQTYPKTDVGANAP, CGNLSTC(AiB)LGK.sub.AcLTQDLNKFHTFPKTDVGANAP, CSNLSTCVLGK.sub.AcLSQELHKLQTYPRTDVGANAP, CSNLSTCMLGK.sub.AcLSQELHRLQTYPKTDVGANAP, CASLSTCVLGK.sub.AcLSQDLHKLQTFPKTDVGANAP, CASLSTCMLGK.sub.AcLSQDLHKLQTFPKTDVGANAP, CGNLSTCMLGK.sub.AcLSQDLNKFHTFPQTDVGANAP, CSNLSTC(AiB)LGK.sub.AcLANFLVHSSNNFGAILPKTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHSSTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHSSNTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLSNNFGAILSSTNVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYGAILSPKTDVGANAP, CSNLSTCMLGK.sub.AcLANFLVHSSNNFGAILPKTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKILSSTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKGLITTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKNNFGTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKRTTQTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHTTNTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHGGQTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHKKNTDVGANAP, CSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHKKHTDVGANAP, CSNLSTC(AiB)LGRLSQDLHRK.sub.AcQTYPKTDVGANAP, or CSNLSTCMLGRLSQELHRK.sub.AcQTYPKTDVGANAP

33. The calcitonin mimetic of claim 1, wherein the peptide is: TABLE-US-00027 AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKTDVGANAP-NH.sub.2, AcCSNLSTC(AiB)LGK.sub.AcLSQDLHRLQTYPKTDVGANAP-NH.sub.2, AcCGNLSTC(AiB)LGK.sub.AcLTQDLNKFHTFPKTDVGANAP-NH.sub.2, AcCSNLSTCVLGK.sub.AcLSQELHKLQTYPRTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQELHRLQTYPKTDVGANAP-NH.sub.2, AcCASLSTCVLGK.sub.AcLSQDLHKLQTFPKTDVGANAP-NH.sub.2, AcCASLSTCMLGK.sub.AcLSQDLHKLQTFPKTDVGANAP-H2, AcCGNLSTCMLGK.sub.AcLSQDLNKFHTFPQTDVGANAP-NH21 AcCSNLSTC(AiB)LGK.sub.AcLANFLVHSSNNFGAILPKTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHSSTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHSSNTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLSNNFGAILSSTNVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYGAILSPKTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLANFLVHSSNNFGAILPKTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKILSSTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKGLITTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKNNFGTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKRTTQTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHTTNTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHGGQTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHKKNTDVGANAP-NH.sub.2, AcCSNLSTCMLGK.sub.AcLSQDLHRLQTYPKHKKHTDVGANAP-NH.sub.2, AcCSNLSTC(AiB)LGRLSQDLHRK.sub.AcQTYPKTDVGANAP-NH.sub.2, or AcCSNLSTCMLGK.sub.AcLSQELHRLQTYPKTDVGANAP-NH.sub.2, and wherein K.sub.Ac is acylated with a linker-fatty diacid, wherein the fatty diacid is a C.sub.18 to C.sub.22 fatty diacid and the linker is ##STR00132##

34. The calcitonin mimetic of claim 1, wherein the peptide is formulated for enteral administration, for oral administration, for parenteral administration, or for injection.

35. The calcitonin mimetic of claim 34, wherein the peptide is is coated with citric acid particles for oral administration, and wherein the coated citric acid particles increase the oral bioavailability of the peptide.

36. A pharmaceutical composition comprising the calcitonin mimetic of claim 1, and a pharmaceutically acceptable carrier.

37. The pharmaceutical composition of claim 36, wherein the pharmaceutically acceptable carrier comprises N-(5-chlorosalicyloyl)-8-aminocaprylic acid (5-CNAC), sodium salt of 10-(2-Hydroxybenzamido)decanoic acid (SNAD), or sodium salt of N-(8-[2-hydroxybenzoyl]amino)caprylic acid (SNAC) for oral administration.

38-45. (canceled)

46. The calcitonin mimetic of claim 36, further comprising an insulin sensitizer or a weight loss drug.

47. (canceled)

48. A method of treating diabetes (Type I and/or Type II), excess bodyweight, excessive food consumption, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis, or osteoarthritis, poorly regulated blood glucose levels, poorly regulated response to glucose tolerance tests, or poor regulation of food intake, comprising administering an effective amount of the calcitonin mimetic of claim 1 to a patient in need of said treatment.

49. A method of treating diabetes (Type I and/or Type II), excess bodyweight, excessive food consumption, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis, or osteoarthritis, poorly regulated blood glucose levels, poorly regulated response to glucose tolerance tests, or poor regulation of food intake, comprising administering an effective amount of the calcitonin mimetic of claim 1 in combination with metformin or another insulin sensitizer to a patient in need of said treatment.

50. A method of treating an overweight condition comprising administering an effective amount of the calcitonin mimetic of claim 1 in combination with a weight loss drug to a patient in need of said treatment.

Description

DESCRIPTION OF THE FIGURES

[0203] FIG. 1: Comparison of KBP346, KBP347, KBP349, KBP351, KBP352, KBP353 and KBP089 on food intake and body weight. A) Food intake, 0-4 hours. B) Body weight change, 4 hours. C) Food intake, 4-24 hours. D) Body weight change, 24 hours. E) Food intake, 24-49 hours. F) Body weight change, 48 hours.

[0204] FIG. 2: Single dose test of KBP375, KBP376 and KBP377. Single dose was given at t=0 and the effect on food intake and body weight of a single dose 36 nmol/kg of each molecule were monitored for 168 hours and compared head-to-head with a non-acylated benchmark. A) Food intake. B) Body weight change.

[0205] FIG. 3: Dose response test of KBP356, KB358, KBP362, KBP364, KB368 and KBP370. Single dose was given at t=0 and the effect on food intake and body weight of a single dose 36 nmol/kg of each molecule were monitored for 168 hours. A-B) Food intake and Body weight of acylated KBP-066 variants. C-D) Food intake and Body weight of acylated KBP-062 variants. E-F) Food intake and Body weight of acylated KBP-110 variants.

[0206] FIG. 4: Effect of a single high dose KBP372 and KBP356 on food intake and body weight. A) KBP-042A11.03 (KBP372) effect on food intake. B) KBP-042A11.03 (KBP372) effect on body weight. C) KBP-066A11.03 (KBP356) effect on food intake. D) KBP-066A11.03 (KBP356) effect on body weight.

[0207] FIG. 5: 4 hour food intake study for KBP350.

[0208] FIG. 6: Accumulated food intake. A) Accumulated food intake over time. Food intake is monitored once daily for the initial 21 days of the study. n=3-4 cages.+/−SEM. B) Total area under the curve of the data presented in FIG. 9A. n=9-10.+/−SEM.

[0209] FIG. 7: ZDF Body weight during study. A) Body weight of individual rats in grams B) Body weight normalised to vehicle in percent. Body weight is recorded daily throughout the first 21 days, then twice weekly until one week prior to study end (day 62). The body weight of the KBP-066A11.03 group was monitored daily until one week prior to study end (day 62). n=9-10 rats.+/−SEM.

[0210] FIG. 8: ZDF Fasting blood glucose. Fasting blood glucose is measured after 6 h of fasting on day 0, 14, 28, 42 and 62 after study start. n=9-10.+/−SEM.

[0211] FIG. 9: ZDF HbA1c values. A) HbA1c at baseline. B) HbA1c at study end. HbA1c is measured on day −3 from study start. HbA1c is measured at study end, day 62. n=9-10. +/−SEM.

[0212] FIG. 10: Oral glucose tolerance test (OGTT). A) an OGTT over 180 min in male ZDF rats. B) Total area under the curve during the OGTT shown in A). The OGTT is performed after 8 weeks of treatment. The rats were fasted for 11 h prior to time point −30 minutes. Blood glucose levels are measured at time point −30, 0, 15, 30, 60, 120 and 180 minutes. Glucose is administered orally at time point 0 minutes. Blood glucose values above 33.3 mmol*L.sup.−1 were assigned with the upper limit of detection; 33.3 mmol*L.sup.−1. The rats had not been pre-dosed with saline or KBP-066 og KBP-066A on that same day. n=9-10. +/−SEM.

[0213] FIG. 11: Single dose test of KBP-305, KBP-306, KBP-307, KBP-356, KBP-381, KBP-382 and KBP-383. Single dose was given at t=0 and the effect on food intake and body weight of a single dose 3 nmol/kg of each molecule were monitored for 96 hours and compared head-to-head with one another and vehicle to determine the optimal acylation length. A) Acute food intake in grams(g). B) Body weight change in grams(g). n=4 rats per group. Data as +/−SEM.

[0214] FIG. 12: Six-week body weight loss study in HFD SD rats using KBP-066A11 compounds with different acylation length, 0.03, 0.04, and 0.05 acylations. Rats were treated with treated with KBP-066A11.03, KBP-066A11.04, KBP-066A11.05 or vehicle and dosed every 3.sup.rd day with a single s.c. injection of 4 nmol compound/kg. Body weight is recorded daily throughout the study. A) Daily food intake in grams(g) B) Body weight loss of individual rats in grams(g). n=6 rats per group. Data as +/−SEM.

[0215] FIG. 13: Additional parameters from the body weight loss study in HFD SD rats using KBP-066A11 compounds with different acylation length, 0.03, 0.04, or 0.05 acylation. Rats were treated with KBP-066A11.03, KBP-066A11.04, KBP-066A11.05 or vehicle. Rats were dosed every 3.sup.rd day with a single s.c. injection of 4 nmol compound/kg. A) Oral glucose tolerance test. B) Incremental area under the curve of the OGTT. C) Weight of the epididymal WAT at study end in grams(g). D) Weight of the inguinal WAT at study end in grams(g). E) Weight of the perirenal WAT at study end in grams(g). F) Change in body weight at study end from baseline in grams(g). Body weight was recorded daily throughout the study. n=6 rats per group. Data as +/−SEM.

[0216] FIG. 14: Competitive ligand binding assay using radio labelled salmon calcitonin (.sup.125I-sCT) as tracer and conducted 2% serum albumin from two different species, rat (Rattus norvegicus) and man (Homo sapiens). As a tracer 0.25 nM .sup.125I-sCT was used. A) Competitive binding assay conducted in 2% RSA. B) Competitive binding assay conducted in 2% HSA. Data as +/−SEM.

[0217] FIG. 15: Single dose test of KBP-356, KBP-386, KBP-387, KBP-388, KBP-389, and KBP-390 for investigating the acylation position of the KBP-066 backbone. Single dose was given at t=0 and the effect on food intake and body weight of a single dose 3 nmol/kg of each molecule were monitored for 96 hours and compared head-to-head with one another and vehicle to determine the optimal acylation position. A) Acute food intake in grams(g). B) Body weight change in grams(g). n=4 rats per group. Data as +/−SEM.

[0218] FIG. 16: Single dose test of KBP-391, KBP-312, KBP-313, KBP-314, KBP-315, KBP-316, KBP-317, and KBP-318 for investigating acylation position of the KBP-021 backbone. Single dose was given at t=0 and the effect on food intake and body weight of a single dose 3 nmol/kg of each molecule were monitored for 96 hours and compared head-to-head with one another or vehicle to determine the optimal acylation position. A) Acute food intake in grams(g). B) Body weight change in grams(g). n=4 rats per group. Data as +/−SEM.

[0219] FIG. 17: Six-week body weight loss study in HFD SD rats using KBP-066 compounds with same acylation length, 0.03, but different position, A11 and A19. Rats were dosed every 3.sup.rd day with a single s.c. injection of 4 nmol compound/kg KBP-066A11.03 (KBP-356), KBP-066A19.03 (KBP-389) or vehicle. Body weight and food intake was recorded daily throughout the study. A) Daily food intake during the study in grams(g). B) Body weight loss of individual rats in grams(g). n=6 rats per group. Data as +/−SEM.

[0220] FIG. 18: Additional parameters from the body weight loss study in HFD SD rats using KBP-066A11 compounds with different acylation length, 0.03, 0.04, and 0.05 acylations. Rats were treated with KBP-066A11.03, or KBP-066A19.03 or vehicle. Rats were dosed every 3.sup.rd day with a single s.c. injection of 4 nmol compound/kg. A) Oral glucose tolerance test. B) Incremental area under the curve of the OGTT. C) Weight of the epididymal WAT at study end in grams(g). D) Weight of the inguinal WAT at study end in grams(g). E) Weight of the perirenal WAT at study end in grams(g). F) Change in body weight at study end from baseline in grams(g). Body weight is recorded daily throughout the study. n=6 rats per group. Data as +/−SEM.

[0221] FIG. 19: Investigating acylation linker of the KBP-066 backbone using single dose test of KBP-356, KBP-384, and KBP-385. Single dose was given at t=0 and the effect on body weight of a single dose of 4 nmol/kg of each molecule were monitored for 96 hours and compared head-to-head with one another to determine the optimal acylation linker A) Acute food intake in grams(g). B) Body weight change in grams(g). n=4 rats per group. Data as +/−SEM.

EXAMPLES

[0222] The presently disclosed embodiments described in the following Examples, which are set forth to aid in the understanding of the disclosure, should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. In the following examples, the following materials and methods were employed.

[0223] Cells and Cell Lines

[0224] The following cell lines expressing the calcitonin, amylin and CGRP receptors were purchased and cultured according to the manufacturer's instructions. [0225] 1. Calcitonin Receptor (CTR): U2OS-CALCR from DiscoveRx (Cat. No.: 93-0566C3). [0226] 2. Amylin Receptor (AMY-R): CHO-K1 CALCR+RAMP3 from DiscoveRx (Cat. No.: 93-0268C2).

[0227] Chemicals

[0228] Thioflavin T (T3516, Sigma). Assay stock ThT is prepared as a 10 mM solution in 5 mM sodium phosphate pH 7.2. Aliquots are stored, protected from light, at −20° C. Stock ThT is thawed and diluted just prior to use.

[0229] For the tested calcitonin mimetics (hereinafter referred to as “acylated KBPs” or simply “KBPs”), final buffer conditions are 10 mM Tris-HCl pH 7.5.

[0230] The final peptide concentration in the wells should be 100-200 μM, and the final ThT concentration should be 4 μM. ThT is added last (10 μL).

[0231] Animal Models

[0232] In the animal model studies, 12 week healthy Sprague Dawley (SD) rats were used to assess the potency of the acylated KBPs. In some examples they were fed normal chow during prior and during the tests, whereas in other examples, the 12 week healthy SD rats were fed high fat diet (HFD) for eight weeks prior to the test and for the duration of the test.

[0233] Acylated Calcitonin Mimetics

[0234] The following Tables 1a and 1b set out the amino acid sequences of the acylated calcitonin mimetics that have been tested. As used therein:

[0235] 1 acylation means K.sub.Ac-(glutamic acid linker)-(C16 fatty acid [palmitate]);

[0236] 2 acylation means K.sub.Ac-(glutamic acid linker)-(C18 diacid [Octadecanedioic acid]);

[0237] 3 acylation means K.sub.Ac-(2×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C18 diacid [Octadecanedioic acid]).

[0238] 4 acylation means KAc-(2×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C20 diacid [Eicosanedioic acid]).

[0239] 5 acylation means KAc-(2×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C22 diacid [Docosanedioic acid]).

[0240] 6 acylation means KAc-(2×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C16 diacid [Hexadecanedioic acid]).

[0241] 7 acylation means KAc-(3×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C18 diacid [Octadecanedioic acid]).

[0242] 8 acylation means KAc-(1×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C18 diacid [Octadecanedioic acid]).

[0243] 9 acylation means KAc-(2×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C24 diacid [Tetracosanedioic acid]).

[0244] 10 acylation means KAc-(2×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C26 diacid [Hexacosanedioic acid]).

[0245] 11 acylation means KAc-(2×OEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C14 diacid [Tetradecanedioic acid]).

[0246] The tested calcitonin mimetics are based on the following core peptide sequences prior to modification:

TABLE-US-00004 CSNLSTCMLGRLSQDLHRLQTYPKTDVGANAP (KBP089) CSNLSTC(AiB)LGRLSQDLHRLQTYPKTDVGANAP (KBP066)  CGNLSTC(AiB)LGRLTQDLNKFHTFPKTDVGANAP (KBP062) CSNLSTCVLGKLSQELHKLQTYPRTDVGANAP (KBP042) CSNLSTC(AiB)LGRLANFLVHSSNNFGAILPKTDVGANAP (KBP110) CSNLSTCMLGRLSQELHRLQTYPKTDVGANAP (KBP021)

[0247] In Table 1b, the following additional nomenclature is also used:

TABLE-US-00005 Acylated Amino Acid KBP Name Modifier 01 A01 02 A02 03 A03 . . . . . . XX AXX . . . . . . 31 A31 32 A32 Type Acylation Name Addition C16 .01 C18 diacid .02 C18 diacid 2*OEG .03 C20 diacid 2*OEG .04 C22 diacid 2*OEG .05 C16 diacid 2*OEG .06 C18 diacid 3*OEG .07 C18 diacid 1*OEG .08 C24 diacid 2*OEG .09 C26 diacid 2*OEG .10 C14 diacid 2*OEG .11

[0248] Thus, by way of example, the nomenclature KBP-066A11.03 indicates that the peptide consists of the KBP-066 core sequence, modified by substitution at the 11 position with a lysine residue with a C18 diacid 2*OEG acylation.

TABLE-US-00006 TABLE 1 Acylated calcitonin mimetics N- c- KBP term 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 term 346 Ac— C S N L S T C V L G [00008] L S Q E L H K L Q T Y P R T D V G A N A P —NH2 347 Ac— C S N L S T C M L G R L S Q D L H [00009] L Q T Y P K T D V G A N A P —NH2 363 Ac— C S N L S T C M L G [00010] L S Q D L H R L Q T Y P K T D V G A N A P —NH2 Ac— C S N L S T C M L G R [00011] S Q D L H R L Q T Y P K T D V G A N A P —NH2 Ac— C S N L S T C M L G R L S Q D [00012] H R L Q T Y P K T D V G A N A P —NH2 Ac— C S N L S T C M [00013] G R L S Q D L H R L Q T Y P K T D V G A N A P —NH2 Ac— C S N L S T C M L G R L S Q D L H R L Q T Y P K T D V G A N A P K [00014] Ac— C S N L S T C [00015] [00016] G R L S Q D L H R L Q T Y P K T D V G A N A P —NH2 Ac— C S N L S T C [00017] [00018] G R L S Q D L H R L Q T Y P K T D V G A N A P —NH2 Ac— C S N L S T C [00019] L G L S Q D L H R L Q T Y P K T D V G A N A P —NH2 Ac— C S N L S T C [00020] L G [00021] L S Q D L H R L Q T Y P K T D V G A N A P —NH2 Ac— C S N L S T C [00022] L G R L S Q D L H R L Q T Y P K T D V G A N A P K [00023] Ac— C S N L S T C [00024] L G R L S Q D L H R L Q T Y P K T D V G A N A P K [00025] Ac— C G N L S T C [00026] [00027] G R L T Q D L N K F H T F P K T D V G A N A P —NH2 Ac— C G N L S T C [00028] [00029] G R L T Q D L N K F H T F P K T D V G A N A P —NH2 363 Ac— C G N L S T C [00030] L G L T Q D L N K F H T F P K T D V G A N A P —NH2 Ac— C G N L S T C [00031] L G [00032] L T Q D L N K F H T F P K T D V G A N A P —NH2 286 Ac— C G N L S T C [00033] L G R L T Q D L N K P H T F P K T D V G A N A P K [00034] 366 Ac— C G N L S T C [00035] L G R L T Q D L N K F H T F P K T D V G A N A P K [00036] Ac— C S N L S T C [00037] [00038] G R L A N F L V H S S N N F G A I L P K T D V G A N A P —NH2 Ac— C S N L S T C [00039] [00040] G R L A N F L V H S S N N F G A I L P K T D V G A N A P —NH2 368 Ac— C S N L S T C [00041] L G [00042] L A N F L V H S S N N F G A I L P K T D V G A N A P —NH2 369 Ac— C S N L S T C [00043] L G [00044] L A N F L V H S S N N F G A I L P K T D V G A N A P —NH2 STD Ac— C S N L S T C [00045] L G R L A N F L V H S S N N F G A I L P K T D V G A N A P [00046] Ac— C S N L S T C [00047] L G R L A N F L V H S S N N F G A I L P K T D V G A N A P [00048] 372 Ac— C S N L S T C V L G [00049] L S Q E L H K L Q T Y P R T D V G A N A P —NH2 373 Ac— C S N L S T C V [00050] G K L S Q E L H K L Q T Y P R T D V G A N A P —NH2 374 Ac— C S N L S T C V L G K L S Q E L H K L Q T Y P R T D V G A N A P K [00051] 375 Ac— C S N L S T C M [00052] G R L S Q D L H R L Q T Y P K T D V G A N A F —NH2 376 Ac— C S N L S T C M L G [00053] L S Q D L H R L Q T Y P K T D V G A N A P —NH2 377 Ac— C S N L S T C M L G R L S Q D L H R L Q T Y P K T D V G A N A P K [00054] 378 Ac— C A S L S T C V [00055] G K L S Q D L H K L Q T F P K T D V G A N A P —NH2 379 Ac— C A S L S T C V L G [00056] L S Q D L H K L Q T F P K T D V G A N A P —NH2 380 Ac— C A S L S T C V L G K L S Q D L H K L Q T F P K T D V G A N A P K [00057] [00058][00059][00060][00061] indicates data missing or illegible when filed

TABLE-US-00007 TABLE 1b Acylated Calcitonin Mimetics N- KBP Core peptide Acylation term 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 KBP-065 A11.04 Ac— C S N L S T C [00062] L G [00063] L S Q D L H 381 KBP-066 A11.05 Ac— C S N L S T C [00064] L G [00065] L S Q D L H KBP-066 A11.06 Ac— C S N L S T C [00066] L G [00067] L S Q D L H 305 KBP- A11.07 Ac— C S N L S T C [00068] L G [00069] L S Q D L H KGP-065 A11.08 Ac— C S N L S T C [00070] L G [00071] L S Q D L H 307 KBP-066 A11.09 Ac— C S N L S T C [00072] L G [00073] L S Q D L H KBP-064 A11.16 Ac— C S N L S T C [00074] L G [00075] L S Q D L H 305 KBP-066 A11.13 Ac— C S N L S T C [00076] L G [00077] L S Q D L H 354 KBP-066 A09.03 Ac— C S N L S T C [00078] [00079] G R L S Q D L H 356 KBP-066 A11.03 Ac— C S N L S T C [00080] L G [00081] L S Q D L H KBP-068 A12.03 Ac— C S N L S T C [00082] L G R [00083] S Q D L H 387 KBP-064 A16.03 Ac— C S N L S T C [00084] L G R L S Q D [00085] H 348 KBP-064 A16.03 Ac— C S N L S T C [00086] L G R L S Q D L H 399 KBP-064 A19.03 Ac— C S N L S T C [00087] L G R L S Q D L H 399 KBP-066 A19.05 Ac— C S N L S T C [00088] L G R L S Q D L H 390 KBP-064 A24.03 Ac— C S N L S T C [00089] L G R L S Q D L H 358 KBP-066 A32.03 Ac— C S N L S T C [00090] L G R L S Q D L H 312 KBP-021 A09.03 Ac— C S N L S T C M [00091] G R L S Q [00092] L H 391 KBP-021 A11.03 Ac— C S N L S T C M L G [00093] L S Q [00094] L H 393 KBP-035 A11.06 Ac— C S N L S T C M L C [00095] L S Q [00096] L H 394 KBP-021 A11.06 Ac— C S N L S T C M L G [00097] L S Q [00098] L H KBP-021 A11.03 Ac— C S N L S T C M L G R [00099] S Q [00100] L H 314 KBP-021 A16.03 Ac— C S N L S T C M L G R L S Q [00101] [00102] H 315 KBP-021 A19.03 Ac— C S N L S T C M L G R L S Q [00103] L H 316 KBP-021 A19.03 Ac— C S N L S T C M L G R L S Q [00104] L H 395 KBP-021 A19.05 Ac— C S N L S T C M L G R L S Q [00105] L H 217 KBP-021 A24.03 Ac— C S N L S T C M L G R L S Q [00106] L H 318 KBP-021 A32.03 Ac— C S N L S T C M L G R L S Q [00107] L H KBP 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 C-term R L Q T Y P K T D V G A N A P —NH2 381 R L Q T Y P K T D V G A N A P —NH2 R L Q T Y P K T D V G A N A P —NH2 305 R L Q T Y P K T D V G A N A P —NH2 R L Q T Y P K T D V G A N A P —NH2 307 R L Q T Y P K T D V G A N A P —NH2 R L Q T Y P K T D V G A N A P —NH2 305 R L Q T Y P K T D V G A N A P —NH2 354 R L Q T Y P K T D V G A N A P —NH2 356 R L Q T Y P K T D V G A N A P —NH2 R L Q T Y P K T D V G A N A P —NH2 387 R L Q T Y P K T D V G A N A P —NH2 348 [00108] L Q T Y P K T D V G A N A P —NH2 399 R [00109] Q T Y P K T D V G A N A P —NH2 399 R [00110] Q T Y P K T D V G A N A P —NH2 390 R L Q T Y P [00111] T D V G A N A P —NH2 358 R L Q T Y P K T D V G A N A P K [00112] 312 R L Q T Y P K T D V G A N A P —NH2 391 R L Q T Y P K T D V G A N A P —NH2 393 R L Q T Y P K T D V C A N A P —NH2 394 R L Q T Y P K T D V G A N A P —NH2 R L Q T Y P K T D V G A N A P —NH2 314 R L Q T Y P K T D V G A N A P —NH2 315 [00113] L Q T Y P K T D V G A N A P —NH2 316 R [00114] Q T Y P K T D V G A N A P —NH2 395 R [00115] Q T Y P K T D V G A N A P —NH2 217 R L Q T Y P [00116] T D V G A N A P —NH2 318 R L Q T Y P K T D V G A N A P K [00117] [00118][00119][00120][00121][00122][00123][00124][00125][00126][00127] indicates data missing or illegible when filed

[0249] The various acylations have the following chemical structures:

##STR00128##

Initial Acylation Studies (Examples 1-5)

Example 1 (FIGS. 1 & 5)

[0250] Single dose comparative effect of 1 acylated variants at different positions (9 position “A09”, 11 position “A11”, 16 position “A16”, 18 position “A18”, and 32 position “A32”) to a non-acylated Benchmark peptide (KBP-089) on food intake and body weight in 12 week lean SD rats.

TABLE-US-00008 KBP Core Position/Acylation KBP-346 KBP-042 A11/1 acylation KBP-347 KBP-089 A18/1 acylation KBP-349 KBP-089 A11/1 acylation KBP-350 KBP-089 A12/1 acylation KBP-351 KBP-089 A16/1 acylation KBP-352 KBP-089 A9/1 acylation KBP-353 KBP-089 A32/1 acylation

[0251] Rats were single caged four days prior to the test. Rats were randomized by weight into six groups (Vehicle (0.9% NaCl), KBPs (doses: 25 nmol/kg ({circumflex over ( )}100 μg/kg)). They were fasted overnight and then treated with a single dose of peptide or vehicle in the morning using subcutaneous administration. Food intake was monitored in the following intervals (0-4 hours, 4-24 hours, 24-48 hours). Body weight was measured at baseline and at 24 hours and 48 hours post s.c injection.

[0252] Acylation at positions “A09”, “A11” and “A32” with 1 acylation produced a protracted in vivo response (FIG. 1) that merited further testing (see below). Positions 12 (FIG. 5), 16, and 18 returned an unacceptable result and were not advanced into further experiments.

Example 2: β-Arrestin Assay

[0253] PathHunter β-arrestin GPCR assays are whole cell, functional assays that directly measure the ability of a ligand to activate a GPCR by detecting the interaction of β-arrestin with the activated GPCR. Because β-arrestin recruitment is independent of G-protein signaling, these assays offer a powerful and universal screening and profiling platform that can be used for virtually any Gi-, Gs, or Gq-coupled receptor.

[0254] In this system, the GPCR is fused in frame with the small enzyme fragment ProLink™ and co-expressed in cells stably expressing a fusion protein of β-arrestin and the larger, N-terminal deletion mutant of β-gal (called enzyme acceptor or EA). Activation of the GPCR stimulates binding of β-arrestin to the ProLink-tagged GPCR and forces complementation of the two enzyme fragments, resulting in the formation of an active β-gal enzyme. This interaction leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter® Detection Reagents.

[0255] In independent bioassays, CTR and AMY-R cells were treated at the indicated time points with increasing doses of KBPs identified in Tables 2 and 3 below (100, 20, 4, 0.8, 0.16, 0.032 nM and vehicle). The assay was performed in white 384 well plates (Greiner Bio-One, 784080). Cells were seeded 2500 cells per well in 10 μL cell-type specific medium the day prior to the experiment. To quantify the GPCR-mediated β-arrestin recruitment the Pathhunter™ Detection Kit (93-0001, DiscoverX) was used and assay performed accordingly to the manufacturer's instructions.

[0256] The prolonged/protracted response was conducted using the calcitonin receptor (CTR): U2OS-CALCR from DiscoveRx (Cat. No.: 93-0566C3) cell line, and as opposed to the classical three hour output, β-arrestin accumulation was conducted over 3, 6, 24, 48 or 72 hour and then assayed and analyzed. Table 2 (2 acylation) and Table 3 (3 acylation) set out the results of the β-arrestin study.

TABLE-US-00009 TABLE 2 β-arrestin study for the 2 acylation (K.sub.Ac-(glutamic acid linker)-(C18 diacid)) Compound U2OS U2OS CHO-K1 (CTR) (CTR) (AMY-R) Acylated KBPs 3 Acylation β-arrestin β-arrestin Prolonged β-arrestin Fold CTR response Fold Recruitment (10 nM) Recruitment Core Acylation EC50 values tAUC value EC50 values NO Sequence Position/Type (10.sup.−9 M) 0-72 h (10.sup.−09 M) KBP-355 KBP-066 A09/2 31.2 ± 4.4 (3) 147 ± 004 (2) 509 ± 695 (3) KBP-357 KBP-066 A11/2 9.2 ± 1.0 (3) 1576 ± 171 (2) 11.4 ± 5.8 (3) KBP-359 KBP-066 A32/2 40.8 ± 7.2 (3) 1438 ± 003 (2) 96.5 ± 65 (3) KBP-361 KBP-062 A09/2 127.5 ± 45 (3) 136 ± 007 (2) 18.4 (1) KBP-363 KBP-062 A11/2 10.9 ± 7.0 (3) 1581 ± 066 (2) 36.6 ± 31 (2) KBP-365 KBP-062 A32/2 34.6 ± 6.8 (3) 1282 ± 034 (2) 51.9 ± 1.6 (2) KBP-367 KBP-110 A09/2 >1000 (3) 095 ± 020 (3) >1000 (3) KBP-369 KBP-110 A11/2 182 ± 1.2 (3) 537 ± 073 (3) 230 ± 4.3 (3) KBP-371 KBP-110 A32/2 >1000 (3) 109 ± 001 (3) >1000 (3) Table 2: In vitro peptide screening characteristics. AX/2 means position X with a 2 acylation, e.g. A09/2 means acylation at the 9 position with the 2 acylation.

TABLE-US-00010 TABLE 3 β-arrestin study for the 3 acylation (K.sub.Ac-(2xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C18 diacid [Octadecanedioic acid]) Compound U2OS U2OS CHO-K1 Food (CTR) (CTR) (AMY-R) Intake Acylated KBPs 2 Acylation β-arrestin β-arrestin Prolonged β-arrestin Fold CTR response Fold ΔFOOD Recruitment (10 nM) Recruitment Sustained EC50 tAUC EC50 Attenuation Core Acylation values value values (36 nmol/kg) NO Sequence Type (10.sup.−9 M) 0-72 h (10.sup.−09 M) Hours (h) KBP-354 KBP-066 A09/3 4.7 ± 0.6 (3) 260 ± 019 (2) 93.0 ± 26 (3) 4 h KBP-356 KBP-066 A11/3 8.5 ± 0.8 (3) 2512 ± 295 (2) 12.0 ± 4.0 (3) 96 h KBP-358 KBP-066 A32/3 44.2 ± 5.7 (3) 1460 ± 202 (2) 98.6 ± 53 (3) 72 h KBP-360 KBP-062 A09/3 45.2 ± 9.4 (3) 182 ± 006 (2) 83.9 ± 42 (3) 4 h KBP-362 KBP-062 A11/3 13.2 ± 9.6 (3) 1784 ± 330 (2) 14.5 ± 0.2 (2) 72 h KBP-364 KBP-062 A32/3 53.3 ± 8.6 (3) 1322 ± 035 (2) 106 ± 32 (2) 48 h KBP-366 KBP-110 A09/3 >1000 (3) 084 ± 007 (3) >1000 (3) 4 h KBP-368 KBP-110 A11/3 193 ± 2.9 (3) 827 ± 140 (3) 166 ± 43 (3) 72 h KBP-370 KBP-110 A32/3 473 ± 34 (3) 635 ± 077 (3) >1000 (3) 4 h KBP-373 KBP-042 A09/3 96.5 ± 17 (3) 337 (1) 263 ± 7.3 (3) 4 h KBP-372 KBP-042 A11/3 7.8 ± 2.5 (3) 1304 ± 238 (3) 45.6 ± 12 (3) 96 h KBP-374 KBP-042 A32/3 49.2 ± 6.4 (3) 1073 (1) 151 ± 15 (4) 72 h KBP-375 KBP-089 A09/3 56.3 ± 20 (3) 624 (1) 232 ± 27 (4) 4 h KBP-376 KBP-089 A11/3 14.7 ± 2.7 (3) 1395 (1) 25.0 ± 2.2 (4) 96 h KBP-377 KBP-089 A32/3 66.0 ± 36 (3) 1403 (1) 73.1 ± 7.4 (4) 72 h Table 3: In vitro peptide screening characteristics AX/3 means position X with a 3 acylation, e.g. A09/3 means acylation at the 9 position with the 3 acylation.
The β-arrestin studies indicated the following:

[0257] 1) Potency of the acylations in terms of the acylation position on the peptide is as follows: A11>A32>A09.

[0258] 2) The 2 or 3 acylation at the 11 position (A11) is the generally far superior acylation/position combination for every peptide core in terms of activing the calcitonin receptor (CTR), the amylin receptor (AMY-R), prolonged CTR response, and suppressing food intake.

[0259] 3) Acylated KBPs with different cores demonstrate similar potency and patterns in vitro when modified with identical acylations.

Example 3 (FIG. 2)

[0260] Single dose comparative effect of A09 (KBP375), A11 (KBP376) and A32 (KBP377) 3 acylated variants of KBP089 with the non-acylated Benchmark KBP089 on food intake and body weight in 20 week HFD SD rats.

TABLE-US-00011 KBP Core Annotation Position/Acylation KBP-375 KBP-089 KBP-089A09.03 A9/3 acylation KBP-376 KBP-089 KBP-089A11.03 A11/3 acylation KBP-377 KBP-089 KBP-089A32.03 A32/3 acylation

[0261] Rats were single caged four days prior to the test. Rats were randomized by weight into eleven groups (Vehicle (0.9% NaCl), KBPs (doses: 36 nmol/kg (150-157 μg/kg)). They were fasted overnight and then treated with a single dose of peptide or vehicle in the morning using subcutaneous administration. Food intake was monitored in the following intervals (0-4 hours, 4-24 hours, 24-48 hours . . . 144-168 hours). Body weight was measured at baseline and every 24 hours post s.c injection.

[0262] The animal model studies confirmed the results of the β-arrestin study and demonstrated improved efficacy vis-à-vis the naked peptide:

[0263] 1) A11>A32>A09 in terms of benefit of acylation position using KBP-089 as core peptide.

[0264] 2) 2 acylation and 3 acylation are far superior to non-acylated KBP-089 at the dose given in terms of protracted in vivo activity and efficacy.

[0265] The animal model study also showed that acylating at the 9 position reduced the potency of the peptide when compared to the naked peptide, thereby ruling out the 9 position as a position of interest in further studies.

Example 4 (FIG. 3)

[0266] Single dose comparative effect of A11 and A32 3 acylated variants with different peptide core to the respective non-acylated Benchmark KBP (KBP-066, KBP-062 and 7KBP-110) on food intake and body weight in 20 week HFD SD rats.

TABLE-US-00012 KBP Core Annotation Position/Acylation KBP-356 KBP-066 KBP-066A11.03 A11/3 acylation KBP-358 KBP-066 KBP-066A32.03 A32/3 acylation KBP-362 KBP-062 KBP-062A11.03 A11/3 acylation KBP-364 KBP-062 KBP-062A32.03 A32/3 acylation KBP-368 KBP-110 KBP-110A11.03 A11/3acylation KBP-370 KBP-110 KBP-110A32.03 A32/3 acylation

[0267] Rats were single caged four days prior to the test. Rats were randomized by weight into eleven groups (Vehicle (0.9% NaCl), KBPs (doses: 4 nmol/kg ({circumflex over ( )}17 μg/kg), 12 nmol/kg ({circumflex over ( )}50 μg/kg) or 36 nmol/kg ({circumflex over ( )}50 μg/kg)). They were fasted overnight and then treated with a single dose of peptide or vehicle in the morning using subcutaneous administration. Food intake was monitored in the following intervals (0-4 hours, 4-24 hours, 24-48 hours . . . 144-168 hours). Body weight was measured at baseline and every 24 hours post s.c injection.

[0268] The results are as follows:

[0269] 1) The peptide core does not affect the improvement observed by acylating at the 11 or 32 positions.

[0270] 2) A11 is a better acylation site than A32.

Example 5 (FIG. 4)

[0271] Single high dose effect of A11/3 acylated variants of KBP-042 and KBP-066 on food intake and body weight in 20 week HFD SD rats. Rats were single caged four days prior to the test. Rats were randomized by weight into eleven groups (Vehicle (0.9% NaCl), KBPs (doses: 300 nmol/kg ({circumflex over ( )}1000 μg/kg)).

TABLE-US-00013 KBP Core Annotation Position/Acylation KBP-372 KBP-042 KBP-042A11.03 A11/3 acylation KBP-356 KBP-066 KBP-066A11.03 A11/3 acylation
The rats were fasted overnight and then treated with a single dose of peptide or vehicle in the morning using subcutaneous administration. Food intake was monitored in the following intervals (0-4 hours, 4-24 hours, 24-48 hours . . . 188-312 hours). Body weight was measured at baseline and every 24 hours post s.c injection.

[0272] The high dose test using KBP356 and KBP372 demonstrated a superior protracted in vivo efficacy that lasted for days. These acylated peptides are therefore clear candidates for development of a once-weekly peptide therapeutic.

Example 6 (FIGS. 6-10)

[0273] Further work was performed on compound KBP-356 (KBP-066A11.03), which comprises an AiB residue at the 8 position and the preferred acylation at the 11 position of the peptide.

[0274] A chronic study was performed in male ZDF rats. (obese homozygous recessive (fa/fa) strain: 370) (Charles River, USA). Rats were delivered 5 weeks of age. The rats were housed 2-3 per cage.

Chronic Treatment of Male ZDF Rats:

[0275] Rats were delivered to the animal facility of Nordic Bioscience at five weeks of age (DAY −6). Rats were acclimatized for three days. HbA1c and BW was registered (DAY −3). Rats were randomized based on HbA1c (primarily) and BW (secondly) at day 4. The study was initiated at DAY 1.

Dosage Concentrations and Frequency

[0276] Animals were dosed once daily with KBP-066 or saline (vehicle). Dosing with KBP-066A11.03 was performed once every third day. Dosing was administered subcutaneously (SC) around noon.

Saline: Dosage volume was 1 mL/kg.
KBP-066: Dosage volume was 1 mL/kg, Dosage concentration was 5, 50 or 500 μg/kg, and compound concentration was 5, 50 or 500 mg/L. The dose equivalent in nmol/kg is 1.43, 14.3 and 143 nmol/kg, respectively.
KBP-066A11.03: Dosage volume was 1 mL/kg, dosage concentration was 25 nmol/kg, and compound concentration was 25 mmol/L. The dose equivalent in μg/kg is 104 μg/kg.

TABLE-US-00014 Treatment groups in nmol/kg Dosing Dosing Compound Admin. Intervention Compound volume conc. conc. route n Vehicle Saline 1 mL/kg NA NA SC. 10 1.43 nmol/kg KBP-066 1 mL/kg 1.43 nmol/kg 1.43 μmol/L SC. 10 14.3 nmol/kg KBP-066 1 mL/kg 14.3 nmol/kg 14.3 μmol/L SC. 10 143 nmol/kg KBP-066 1 mL/kg 143 nmol/kg 143 μmol/L SC. 10 25.0 nmol/kg KBP-066A11.03 1 mL/kg 25.0 nmol/kg 25.0 μmol/L SC. 10

TABLE-US-00015 Treatment groups in μg/kg Dosing Dosing Compound Admin. Intervention Compound volume conc. conc. route n Vehicle Saline 1 mL/kg NA NA SC. 10 5 μg/kg KB P-066 1 mL/kg 5 μg/kg 5 mg/L SC. 10 50 μg/kg KB P-066 1 mL/kg 50 μg/kg 50 mg/L SC. 10 500 μg/kg KB P-066 1 mL/kg 500 μg/kg 500 mg/L SC. 10 104 μg/kg KBP-066A11.03 1 mL/kg 104 μg/kg 104 mg/L SC. 10
Weekly total dose per treatment group:
5 μg/kg KBP-066 equals to 35 μg/kg/week or 10 nmol/kg/week
50 μg/kg KBP-066 equals to 350 μg/kg/week or 100.4 nmol/kg/week
500 μg/kg KBP-066 equals to 3500 μg/kg/week or 1004 nmol/kg/week
25 nmol/kg KBP-066 equals to 243.4 μg/kg/week or 58.3 nmol/kg/week
Compounds were dissolved in saline and stored at −20° C. Aliquots were thawed immediately prior to administration.

Collection of Test Results

[0277] DAY −3: HbA1c measurement
DAY 1: (first day of study), rats were fasted for 6 h and a BG and blood sample was taken. Dosing was performed subsequently.
DAY 14: Fasting blood glucose (FBG)+blood sample (6 h fasting)
DAY 28: FBG+blood sample (6 h fasting)
DAY 42: FBG+blood sample (6 h fasting)
DAY 57: (gr. 1+2)/58 (gr. 3+4) OGTT with no pre-dosing of KBP-066 or KBP-066A11.03 (11 h fasting). Hb1Ac is measured during the OGTT at t=120 or t=180.
DAY 62: FBG+blood sample (6 h fast)

Food Intake

[0278] Food intake was monitored daily. Body weight was monitored daily for first three weeks, then twice weekly after week three.

Fasting Blood Glucose

[0279] Fasting blood glucose was monitored every two weeks using Accu-Check® Avia monitoring system (Roche Diagnostics, Rotkreuz, Switzerland): Measurement was taken from the tail vein (25 G needle).

HbA1c

[0280] Rats were non-fasted for the first (randomization) and second (after the second OGTT) HbA1c measurement. A single drop of blood was applied to the HbA1c cassette and the HbA1c was measured using a DCA Vantage Analyzer. Dosing of compound or saline was performed subsequently during first and second HbA1c measurement.

Oral Glucose Tolerance Test

[0281] A glucose tolerance test (OGTT) was performed after eight weeks of treatment. Body weight from the day prior was used to calculate glucose dose given. Animals were fasted for 11 h. Heat was applied app. 45 min prior to time point −30 min (see below figure). Animals were pre-dosed with KBP-066, KBP-066A11.03 or saline during the first OGTT but not in the second OGTT, hence (C) in the below figure.

Results

FIG. 6A+B, Accumulated Food Intake

[0282] FIG. 6A shows the accumulated food intake during the course of the study. All treatment groups eat less than the vehicle. Furthermore, higher doses leads to higher reduction in food intake. The acylated KBP-066A11.03 treatment group had the greatest reduction of food intake compared to KBP-066 at all dosages. At study end all treatment groups had a significant reduction in food consumed over the course of the study compared to vehicle, with acylated KBP-066A11.03 treatment showing the greatest reduction in food intake; ˜35% reduction in food intake vis-à-vis vehicle (FIG. 6B).

FIG. 7A+B, Body Weight

[0283] All treatment groups lost body weight over the first three weeks of the study. As the ZDF vehicle rats became progressively sicker and thus failed to maintain their body weight/rate of gain (FIG. 7A) the treatment groups caught up to the vehicle group in terms of weight. The rate of weight gain compared to the vehicle was dose dependent as well as being dependent upon the type of compound used in the treatment group (FIG. 7B). The acylated KBP-066A11.03 treatment group has the slowest regain rate, followed by the 14.3 and 143 nmol/kg groups and with 1.43 nmol/kg as the fastest regainer of weight.

[0284] This shows that acylated KBP-66A11.03 given in a s.c dose regiment once every three days has additional pharmacological benefits over non-acylated KBP-066 given s.c., once daily.

FIG. 8, Fasting Blood Glucose

[0285] As the ZDF vehicle rats became progressively sicker and failed to maintain FBG, all treatment groups attenuate FBG effectively for the duration of the study compared to vehicle. The acylated KBP-066A11.03 treatment was the most effective treatment, only allowing a modest 5 mM increase in FBG during the 62-day study in this super aggressive animal model of type 2 diabetes. The non-acylated KBP-066 reduced FBG in a dose dependent manner, but was not as potent as the acylated treatment group in attenuating FBG. Again, this shows that acylated KBP-66A11.03 has additional pharmacological benefits over non-acylated KBP-066.

FIG. 9, HbA1c at Baseline and Study End

[0286] As expected, HbA1c values at baseline are almost identical prior to onset of diabetes and treatment modalities in male ZDF rats (FIG. 9A). At study end (Day 62), all treatment groups had significantly reduced HbA1c levels compared to vehicle. Interestingly, the acylated KBP-066A11.03 treatment group had the lowest HbA1c values. Furthermore, it was also significantly lower than all the non-acylated KBP-066 treatment groups (FIG. 9B), demonstrating a further advantage of the acylation vis-à-vis the non-acylated equivalent.

FIG. 10, Oral Glucose Tolerance Test (OGTT)

[0287] An oral glucose tolerance test was conducted after eight weeks of treatment and results are illustrated in FIG. 10A. Due to treatment-induced differences in FBG the individual OGTT curves are markedly different. This difference is highlighted in the calculated tAUC values (FIG. 10B). All treatment groups have significantly lower tAUC compared to vehicle. The acylated KBP-066A11.03 treatment group had the lowest tAUC value, and was also significantly lower than two of three the non-acylated KBP-066 treatment groups, and with a p-value of 0.06 when compared to the last group, 143 nmol/kg KBP-066 (FIG. 10B).

[0288] In conclusion, the collective data show that acylated KBP-66A11.03 given in a s.c dose regiment every three days have significantly advantageous additional pharmacological benefits over non-acylated KBP-066 given s.c. once daily in obese and diabetic ZDF rats.

Summary of Results of Examples 1-6

Results of Acylation Studies by Acylation Site

Position A09 (Acylation at the 9 Position of the Peptide)

[0289] Acylation of position A09 with 1 acylation produced a sustained prolonged in vivo activity that merited further testing (FIG. 1A-F).

[0290] Furthermore, acylation of position A09 with 2 and 3 acylations attenuated EC50 on both the CTR and AMYR receptor and produced no prolonged response on the CTR (Table 3-4).

[0291] However, acylation of A09 with 2 and 3 acylations disrupted the previously observed prolonged in vivo efficacy of the core peptide making the potency of the acylated KBP similar to that of vehicle. Hence, they were less potent than the non-acylated core peptide (FIG. 2A-B) in both reducing both food intake and body weight after a single s.c. injection of 36 nmol/kg compound.

Position A09 was Therefore not Given any Further Consideration.

[0292] Position A11 (acylation at the 11 position of the peptide) Acylation of position A11 with the 1 acylation produced a sustained prolonged in vivo activity that merited further testing (FIG. 1A-F).

[0293] Acylation of position A11 with acylations 2 and 3 resulted in the best assayed EC50 value on both the CTR and AMYR receptor, and producing the highest prolonged response values (tAUC) across all core peptides tested (Table 3-4).

[0294] Furthermore, A11/3 acylations improved the in vivo activity of the core peptide significantly compared to the non-acylated core peptide in both reducing food intake (FIG. 2A) and body weight (FIG. 2B) after a single s.c. injection of 36 nmol/kg compound. A11/3 acylations were also better at reducing food intake and body weight than any other acylated positions when using KBP-089 as core peptide (FIG. 2A-B).

[0295] This difference was further underscored in a dose response test (FIG. 3A-F) in which three doses (4 nmol/kg, 12 nmol/kg and 36 nmol/kg) were all superior to the corresponding A32/3 acylated peptides. In terms of potency, the lowest dose of 4 nmol/kg A11/3 had a similar profile to the 36 nmol/kg A32/3 acylated peptides. This was consistently demonstrated for all tested core peptides (FIG. 3A-F).

[0296] To further investigate the potency of the A11 position with the 3 acylation, KBP-042 and KBP-066 acylated at position A11 with the 3 acylation was tested at a high dose (300 nmol/kg) and compared to the non-acylated versions to demonstrate the potential maximum effect of the protracted in vivo efficacy combined with the protracted bio-availability (FIG. 4A-D).

[0297] Acylation 3 at position A11 attenuated food intake for more than 120 hours returning to vehicle food consumption levels after {circumflex over ( )}144 hours for both KBP-042 (FIG. 4A) and KBP-066 (FIG. 4C). Treatment mediated body weight loss peaked after 96 hours and body weight returned to baseline levels after {circumflex over ( )}240 hours for both KBP-042 (FIG. 4B) and KBP-066 (FIG. 4D).

[0298] In conclusion, A11 was the best position tested in terms of preserving ligand potency and maximizing the protracted in vivo efficacy.

Position A12 (Acylation at the 12 Position of the Peptide)

[0299] A12 position with a 1 acylation produced a worse result in vivo in the 4 h food intake study when compared to the vehicle (FIG. 5).

[0300] Thus, position A12 was not a good candidate for acylation, and was not tested further.

Position A16 (Acylation at the 16 Position of the Peptide)

[0301] A16 position with a 1 acylation demonstrated no prolonged activity in vivo (FIG. 1A-F).

[0302] Thus, position A16 was not a good candidate using 1 acylation, and was not tested further.

Position A18 (Acylation at the 18 Position of the Peptide)

[0303] A18 position with a 1 acylation was efficacious across the 4-24 hour testing period, however the observed efficacy was not maintained in the prolonged activity study (KBP-347, 48 h, FIG. 1F).

[0304] Thus, position A18 was not a good candidate using 1 acylation, and was not tested further.

Position A32 (Acylation at the 32 Position of the Peptide)

[0305] A32 position with a 1 acylation demonstrated a prolonged effect in vivo on both food intake and body weight, and was among the best of the tested compounds (FIG. 1A-F).

[0306] Position A32 with acylation 2 and 3 resulted in inferior assayed EC50 values on both the CTR and AMYR receptor compared to position A11. Acylation of position A32 attenuated the CTR mediated prolonged response slightly compared to position A11, but still maintained a prolonged response (Table 3-4).

[0307] Acylations at position A32 improved the in vivo efficacy of a single dose s.c. treatment compared to the non-acylation counterparts for all tested core peptides.

[0308] However, the position was inferior to A11 in all tested 2 and 3 acylations during in vivo studies at equivalent doses (FIG. 2A-B, FIG. 3A-F).

[0309] In conclusion, A32 was a mediocre position in terms of preserving ligand potency and improving in vivo efficacy using 1, 2 and 3 acylations when compared to position A11.

Further Acylation Studies (Examples 7-12)

Example 7: μ-Arrestin and Thioflavin T Assays

[0310] Additional PathHunter β-Arrestin GPCR assays were carried out, using the same protocol as described above in connection with Example 2. In independent bioassays, CTR and AMY-R cells were treated at the indicated time points with increasing doses of KBPs identified in Tables 4.1-4.4 (ranging from 1 μM-0.1 nM and vehicle).

[0311] Thioflavin T assays were also conducted. Thioflavin T (ThT) is a dye widely used for the detection of amyloid fibrils. In the presence of fibrils, ThT has an excitation maximum at 450 nm and enhanced emission at 480 nm, whereas ThT is essentially non-fluorescent at these wavelengths when not bound to amyloid fibrils.

[0312] Thus, ThT in combination with a fluorescent plate reader is an ideal tool for screening large numbers of in vitro samples for the presence of amyloid fibrils. The ThT assay used for the KBPS was a modification of the procedure described by Nielsen et. al. (Nielsen L, Khurana R, Coats A, FrØkjaer S, Brange J, Vyas S, et al. Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry. 2001; 40 (20): 6036-46.1) for measuring insulin fibrillation.

[0313] Fibrillation screening assays were conducted in 384-well plates (Greiner Bio-One, 784080) in sample triplicates with a final volume of 20 μL. The plate is sealed using an optical adhesive film to prevent sample evaporation over the course of the assay.

[0314] The plate is loaded into a fluorescent plate reader, such as a SpectraMax with SoftMax Pro 7.0.2 software, and the template set to 37° C. with excitation wavelength at 450 nm and emission wavelength at 480 nm.

[0315] Plate reader should measure fluorescence every 10 minutes for 24 hours with a five-second plate shake before the first read and a three-second plate shake before all other reads. Alternatively, the plate is read after the following incubation times; 0, 1, 2, 4 and 24 hours.

[0316] Plot relative fluorescence units (RFU) as a function of time. Fibrillation is determined as an increase in RFU over baseline as described by Nielsen et. al.

[0317] In this filing four fibrillations tiers have been defined based on the 18 h fluorescence signal to get a single output that reflects the peptides fibrillation potential: None=<1000 RFU, Low=1000-3000 RFU, Medium=3000-10000, High=>10000

[0318] The results of the Thioflavin T assays are also shown in Tables 4.1-4.4.

TABLE-US-00016 TABLE 4.1 β-arrestin study for different acylations length (KAc-(glutamic acid linker)-(C14 to C26 diacid)) Compounds U2OS CHO-K1 Peptide Food (CTR) (AMY-R) Fibrillation Intake Acylated KBPs β-arrestin β-arrestin Thioflavin T ΔFOOD Fold Fold ΔFluorescence Sustained Recruitment Recruitment 18 h Attenuation Core Acylation EC50 values EC50 values Assay (4 nmol/kg) NO Sequence Type (10.sup.−9 M) (10.sup.−9 M) Score Hours (h) 356 KBP-066 A11.03 3.0 ± 2.4 (23) 7.2 ± 4.7 (26) None (10) 72-96 h 383 KBP-066 A11.04 33.0 ± 10.3 (3) 16.6 ± 2.9 (3) None (3) 96 h 382 KBP-066 A11.05 56.1 ± 10.6 (3) 23.3 ± 4.2 (3) None (3) 96 h 381 KBP-066 A11.06 3.9 ± 0.9 (3) 1.0 ± 0.5 (3) None (3) 24-48 h 307 KBP-066 A11.09 26.6 ± 6.1 (3) 149 ± 127 (4) None (3) 72 h 306 KBP-066 A11.10 65.7 ± 2.9 (3) 82.7 ± 6.6 (4) None (3) 48 h 305 KBP-066 A11.11 2.1 ± 1.0 (3) 0.95 ± 0.2 (4) None (3) 24 h Table 4.1: In vitro peptide screening characteristics

TABLE-US-00017 TABLE 4.2 β-arrestin study for different acylations positions using backbone (KBP-066) and 3 acylation (KAc-(glutamic acid linker)-(C18 diacid)) Compounds U2OS CHO-K1 Peptide Food (CTR) (AMY-R) Fibrillation Intake Acylated KBPs β-arrestin β-arrestin Thioflavin T ΔFOOD Fold Fold ΔFluorescence Sustained Recruitment Recruitment 18 h Attenuation Core Acylation EC50 values EC50 values Assay (4 nmol/kg) NO Sequence Type (10.sup.−9 M) (10.sup.−9 M) Score Hours (h) 354 KBP-066 A09.03 4.7 ± 0.6 (3) * 93.0 ± 26 (3) * None (3) 4 h 356 KBP-066 A11.03 3.0 ± 2.4 (23) 7.2 ± 4.7 (26) None (10) 72-96 h 386 KBP-066 A12.03 56.5 ± 21.1 (3) 98.9 ± 74.8 (3) None (3) 0-4 h 387 KBP-066 A16.03 25.4 ± 9.9 (3) 21.1 ± 9.9 (3) Low (3) 48 h 388 KBP-066 A18.03 22.8 ± 11.6 (3) 34.7 ± 33.8 (3) None (3) 72 h 389 KBP-066 A19.03 9.4 ± 3.2 (3) 6.5 ± 3.1 (3) None (3) 72-96 h 390 KBP-066 A24.03 11.1 ± 2.8 (3) 6.0 ± 3.3 (3) None (3) 72 h 358 KBP-066 A32.03 44.2 ± 5.7 (3) * 98.6 ± 53 (3) * None (3) 72 h Table 4.2: In vitro peptide screening characteristics * Data from original patent filing

TABLE-US-00018 TABLE 4.3 β-arrestin study for different acylations positions using backbone (KBP-021) and 3 acylation (KAc-(glutamic acid linker)-(C18 diacid)) Compounds U2OS CHO-K1 Peptide Food (CTR) (AMY-R) Fibrillation Intake Acylated KBPs β-arrestin β-arrestin Thioflavin T ΔFOOD Fold Fold ΔFluorescence Sustained Recruitment Recruitment 18 h Attenuation Core Acylation EC50 values EC50 values Assay (4 nmol/kg) NO Sequence Type (10.sup.−9 M) (10.sup.−9 M) Score Hours (h) 312 KBP-021 A09.03 16.7 ± 2.9 (3) 1528 ± 1201 (3) Low (3) 4 h 391 KBP-021 A11.03 12.5 ± 10.9 (9) 9.0 ± 2.2 (5) None (3) 72-96 h 393 KBP-021 A11.04 55.1 ± 48.9 (3) 32.8 ± 14.6 (4) Low (3) 72-96 h 394 KBP-021 A11.05 56.9 ± 33.4 (3) 53.9 ± 21.2 (4) Low (3) 24 h 313 KBP-021 A12.03 314 ± 116 (3) 330 ± 124 (4) None (3) 4 h 314 KBP-021 A16.03 183 ± 149 (3) 428 ± 175 (5) None (3) 4 h 315 KBP-021 A18.03 19.2 ± 6.6 (3) 44.9 ± 6.6 (5) Medium (3) 48 h 316 KBP-021 A19.03 2.5 ± 1.4 (3) 6.1 ± 1.8 (5) High (3) 72-96 h 395 KBP-021 A19.05 95.2 ± 95.2 (3) 32.9 ± 12.7 (4) High (3) 72-96 h 317 KBP-021 A24.03 12.8 ± 12.8 (3) 31.5 ± 7.4 (5) None (3) 24 h 318 KBP-021 A32.03 197 ± 83.6 (3) 301 ± 225 (5) Low (3) 4 h Table 4.3: In vitro peptide screening characteristics

TABLE-US-00019 TABLE 4.4 β-arrestin study for different acylations linkers using same backbone (KBP-066) and same acylation(C18 diacid)) Compounds U2OS CHO-K1 Peptide Food (CTR) (AMY-R) Fibrillation Intake Acylated KBPs β-arrestin β-arrestin Thioflavin T ΔFOOD Fold Fold ΔFluorescence Sustained Recruitment Recruitment 18 h Attenuation Core Acylation EC50 values EC50 values Assay (4 nmol/kg) NO Sequence Type (10.sup.−9 M) (10.sup.−9 M) Score Hours (h) 385 KBP-066 A11.07 20.1 ± 11.1 (3) 9.3 ± 1.7 (2) None (3) 72 h 384 KBP-066 A11.08 29.7 ± 29.2 (3) 6.9 ± 1.1 (3) Low (3) 72 h 356 KBP-066 A11.03 3.0 ± 2.4 (23) 7.2 ± 4.7 (26) None (10) 72-96 h Table 4.4: In vitro peptide screening characteristics

Results—Acylation Length

[0319] In terms of in vitro potency as a function of acylation length there was a clear correlation between acylation length and in vitro potency. EC50 values on both the CTR and AMYR by the shortest acylations, 11(C14 diacid) and 6 (C16 diacid), produced the lowest EC50s on both receptors (Table 4.1), whereas the longest acylations, 9 (C24 diacid) and 10 (C26 diacid), produced some of the highest recorded EC50 values on both receptors.

[0320] None of the tested acylated peptides in this series using the KBP-066 backbone had any fibrillation issues.

Results—Acylation Position on the KBP-066 Backbone

[0321] EC50 values on the CTR and AMYR on this series are listed in Table 4.2. In terms of in vitro potency as a function of acylation position on the KBP-066 backbone, three positions stand out as potent dual calcitonin and amylin receptor agonists. All, A19 and A24 all have EC50 values on both receptors in the 5×10.sup.−9 M range as the only ones, whereas all other tested positions are impaired in comparison. The increased potency of A11, A19 and A24 appears to translate into improved in vivo efficacy for the KBP-066 backbone (see FIGS. 15, 17 and 18 described infra). Interestingly, A09 is among the best CTR agonist, but has poor AMYR activity, suggesting that an acylation to close to the N-terminus can disrupt AMYR activity and generate a biased ligand.

[0322] Fibrillation does not appear to be an issue for the KBP-066 backbone at most positions, as only one peptide (KBP-066A16.03 (387)) produced a “Low” score in the ThT assay.

Results—Acylation Position on the KBP-021 Backbone

[0323] EC50 values on the CTR and AMYR for this series are listed in Table 4.3. In terms of in vitro potency as a function of acylation position on the KBP-021 backbone, two positions stand out as potent dual agonists. All and A19 both have EC50 values on both receptors in the 5×10.sup.−9 M range as the only ones, whereas all other tested positions are impaired in comparison. The increased potency of A11 and A19 also appear to translate into improved in vivo efficacy for the KBP-021 backbone (see FIG. 16 described infra).

[0324] Interestingly, fibrillation appears to be an issue for the KBP-021 backbone, where position A19 as the only peptide tested scored a “High” score in the ThT assay despite good potency both in vitro and in vivo. The position next to it “A18” also scored high with a “Medium” score in the ThT assay suggesting the KBP-021 backbone is susceptible to fibrillation when acylated in that area of the backbone.

[0325] Furthermore, longer acylations also appear to increase fibrillation for this backbone, KBP-021, as the 4 and 5 acylation on position A11, scored a “Low” score in the ThT assay, however, this issue did not affect the favoured position A11 with 3 acylation.

Results—Acylation Linker

[0326] EC50 values on the CTR and AMYR for this series are listed in Table 4.4. In terms of in vitro potency as a function of acylation position on the KBP-066 backbone, the OEG-OEG-γGLU linker (356) have an almost 10-fold better EC50 on the CTR compared to OEG-OEG-OEG-γGLU (385) and OEG-γGLU (384), however, all linkers have very similar EC50 in the 5×10.sup.−9 M range on the AMYR.

[0327] In terms of fibrillation, the shortest linker, OEG-γGLU (384), produced a “low” score in the ThT assay, whereas the two other linkers produce a “None” score.

Example 8: (FIG. 11)

[0328] Single dose comparative effect of several acylated variants (3, 4, 5, 6, 9, 10, 11) at the same position and backbone, A11 and KBP-066, respectively, on food intake and body weight in an acute setting in 20-week old SD rats feed HFD for 8 weeks prior to the experiment

TABLE-US-00020 Acylation Position/ KBP Core length Annotation Acylation KBP-356 KBP-066 C18 diacid KBP-066A11.03 A11/3 acylation KBP-383 KBP-066 C20 diacid KBP-066A11.04 A11/4 acylation KBP-382 KBP-066 C22 diacid KBP-066A11.05 A11/5 acylation KBP-381 KBP-066 C16 diacid KBP-066A11.06 A11/6 acylation KBP-307 KBP-066 C24 diacid KBP-066A11.09 A11/9 acylation KBP-306 KBP-066 C26 diacid KBP-066A11.10 A11/10 acylation KBP-305 KBP-066 C14 diacid KBP-066A11.11 A11/11 acylation

[0329] Rats were single caged four days prior to the test. Rats were randomized by weight into eight groups (Vehicle (0.9% NaCl), KBPs (doses: 3 nmol/kg ({circumflex over ( )}10-11 μg/kg)). They were fasted overnight and then treated with a single dose of peptide or vehicle in the morning using subcutaneous administration. Food intake was monitored in the following intervals (0-4 hours, 4-24 hours, 24-48 hours, 48-72 hours, and 72-96 hours). Body weight was measured at baseline and at 4 hour, 24 hours, 48 hours, 72 hours and 96 hours post s.c injection.

FIG. 11 Results—Food Intake and Body Weight

[0330] Acylation 6, 10, 11 are able attenuate food intake and body weight with a peak suppression at 24 hours followed by rebound to vehicle levels. Acylation 9 is able attenuate food intake and body weight with a peak suppression at 48 hours followed by rebound to vehicle levels. Acylation 3 is able attenuate food intake and body weight with a peak suppression at 72 hours followed by rebound to vehicle levels. Acylation 4 and 5 were able to attenuate food intake and body weight with a peak suppression after 96 hours followed by a rebound.

[0331] Hence, acylation 3, 4, and 5 are all prime candidates for acylation length as the initial goal was to suppress food intake and body weight for a minimum of 72 hours as every 3.sup.rd day dosing in rodents appears to translate into once weekly dosing in man.

Example 9 (FIGS. 12, 13 and 14)

[0332] Further work was conducted on the best performers from the acute testing, acylated variants (3, 4, 5), and a study using repeated dosing for comparative effect of the acylations with the same position and backbone, A11 and KBP-066 respectively, was carried out. Food intake and body weight were investigated in a chronic setting (five-week study) in 20-week old SD rats feed HFD for 8 weeks prior to study start.

TABLE-US-00021 Acylation Position/ KBP Core length Annotation Acylation KBP-356 KBP-066 C18 diacid KBP-066A11.03 A11/3 acylation KBP-383 KBP-066 C20 diacid KBP-066A11.04 A11/4 acylation KBP-382 KBP-066 C22 diacid KBP-066A11.05 A11/5 acylation

[0333] Rats were caged two and two and were randomized by weight into treatment groups (Vehicle (0.9% NaCl), KBPs (doses: 3 nmol/kg ({circumflex over ( )}14 μg/kg)). Food intake and body weight were monitored daily for 35 days. At study end, an OGTT was performed followed by animal termination, in which, adipose tissue was taken out and weighed.

Chronic Treatment of Male HFD SD Rats

[0334] Rats were delivered to the animal facility of Nordic Bioscience at twelve weeks of age and immediately put on HFD and fed on it for an additional eight weeks. Prior to study start the rats were randomized based on body weight. The study was initiated at DAY 1.

Dosage Concentrations and Frequency

[0335] Animals were dosed with KBPs once every third day. Dosing was administered subcutaneously (SC) around noon every day. Compounds were dissolved in saline and stored at −20° C. Aliquots were thawed immediately prior to administration.

Saline: Dosage volume was 1 mL/kg.
KBPs: Dosage volume was 1 mL/kg, Dosage concentration was 4 nmol/kg.

[0336] The dose equivalent in μg/kg was {circumflex over ( )}14 μg/kg. Weekly total dose per treatment group: 4 nmol/kg KBP equals to 28 nmol/kg/week or {circumflex over ( )}100 μg/kg/week

Collection of Test Results

[0337] DAY 1: (first day of study dosing was performed
Day 1-35: Daily monitoring of food intake and body weight
DAY 35: Body weight at study end
DAY 35: Oral Glucose tolerance test
DAY 35: Termination+adipose tissue weighed

Oral Glucose Tolerance Test

[0338] A glucose tolerance test (OGTT) was performed after five weeks of treatment. Body weight from the day prior was used to calculate glucose dose given. Animals were fasted for 11 h. Heat was applied app. 45 min prior to time point −30 min (see below figure). Animals were dosed with KBPs or vehicle the day before the OGTT.

White Adipose Tissue (WAT) Weighing

[0339] The entire epididymal and perirenal WAT depot was dissected out and weighed. For Inguinal WAT, a fixed anatomical limited area was dissected out and weighed.

FIG. 12 Results, Food Intake and Body Weight

[0340] FIG. 12 shows the change in food intake and body weight over time during the chronic study as a function of treatment. FIG. 12A shows the dynamic in food intake between acylation 3, 4 and 5, whereas FIG. 12B shows the body weight loss mediated by acylation 3, 4 and 5. It is evident that all three acylations give a significant reduction in body weight after 5 weeks of treatment, however, there are no differences between acylation 3, 4 and 5 in terms on efficacy on body weight.

FIG. 13 Results, OGTT and Adipose Tissue

[0341] FIG. 13 shows the results from the OGTT with the corresponding iAUC (OGTT)(Figure A+B) was well as the weight of three different adipose tissue, epididymal, inguinal and perirenal (FIG. 13C-E) and the body weight as study end (FIG. 13F). Treatment with acylation 3 resulted in a significant reduction in iAUC (OGTT), epididymal WAT size, perinal WAT size, and body weight as study. Treatment with acylation 4 and 5 resulted in a significant reduction in iAUC (OGTT), epididymal WAT size, and body weight at study end, but not in perirenal WAT size. Neither treatment significantly reduced the size of the inguinal WAT. Acylation 3 performed slightly better against vehicle compared to 4/5, but there were no significant differences between treatment groups.

FIG. 14 Results, Competitive 1-125 sCT Ligand Binding

[0342] To investigate whether the improved efficacy in the acute setting of acylation 4 and 5 could be translated to man, a competitive ligand binding assay was conducted to explore acylation binding to serum albumin in rodent and man. FIG. 14 shows the competitive binding of KBP-066A11.03 and KBP-066A11.05 with radio-labelled I-125 salmon calcitonin (NEX423, Perkin Elmer) in 2% serum albumin from rodents (RSA) (FIG. 4A) or 2% serum albumin from humans (HSA)(FIG. 4B). When the assay is conducted 2% HSA there are no differences in EC.sub.50 between acylations 3 and 5. However, when the assay was conducted in RSA, the 5 acylation shifted the IC50 further to the right suggesting a stronger affinity towards the RSA in the assay. Hence, the improved efficacy observed in the acute setting in rodents is most likely a phenomenon unique to rodents and not translational to man.

Example 10 (FIG. 15)

[0343] Single dose comparative effect of 3 acylated variants at different positions (9 position “A09”, 11 position “A11”, 12 position “A12”, 16 position “A16”, 18 position “A18”, 19 position “A19”, and 32 position “A32”) to one another on food intake and body weight in 20 week HFD SD rats.

TABLE-US-00022 Position/ KBP Core Annotation Acylation KBP-354 KBP-066 KBP-066A09.03 A9/3 acylation KBP-356 KBP-066 KBP-066A11.03 A11/3 acylation KBP-386 KBP-066 KBP-066A12.03 A12/3 acylation KBP-387 KBP-066 KBP-066A16.03 A16/3 acylation KBP-388 KBP-066 KBP-066A16.03 A18/3 acylation KBP-389 KBP-066 KBP-066A18.03 A19/3 acylation KBP-390 KBP-066 KBP-066A19.03 A24/3 acylation KBP-358 KBP-066 KBP-066A24.03 A32/3 acylation

[0344] Rats were single caged four days prior to the test. Rats were randomized by weight into eight groups (Vehicle (0.9% NaCl), KBPs (doses: 4 nmol/kg ({circumflex over ( )}10-11 μg/kg)). They were fasted overnight and then treated with a single dose of peptide or vehicle in the morning using subcutaneous administration. Food intake was monitored in the following intervals (0-4 hours, 4-24 hours, 24-48 hours, 48-72 hours, and 72-96 hours). Body weight was measured at baseline and at 4 hour, 24 hours, 48 hours, 72 hours and 96 hours post s.c. injection. Two backbones were tested, KBP-066 and KBP-021.

FIG. 15 Results—Food Intake and Body Weight

[0345] In terms of position on backbone, the KBP-066 results are as follows. At 4 nmol/kg in an acute setting (FIG. 5), position A11 and A19 were the two-best positions at suppressing both food intake for 72 hours and body weight for 96 hours. Third best position was A24, followed by A18 and A16. The least potent position to acylate was A12 which looks like a disadvantageous position to acylate as it appears to somewhat interfere with the DACRA mediated efficacy on both food intake and body weight.

[0346] Based on these data, A11 and A19 are the preferred positions to acylate backbone KBP-066.

FIG. 16 Results—Food Intake and Body Weight

[0347] When using a different backbone, KBP-021, with the same experimental settings as for KBP-066, the position pattern was slightly different.

TABLE-US-00023 Position/ KBP Core Annotation Acylation KBP-312 KBP-021 KBP-021A09.03 A9/3 acylation KBP-391 KBP-021 KBP-021A11.03 A11/3 acylation KBP-313 KBP-021 KBP-021A12.03 A12/3 acylation KBP-314 KBP-021 KBP-021A16.03 A16/3 acylation KBP-315 KBP-021 KBP-021A16.03 A18/3 acylation KBP-316 KBP-021 KBP-021A18.03 A19/3 acylation KBP-317 KBP-021 KBP-021A19.03 A24/3 acylation KBP-318 KBP-021 KBP-021A24.03 A32/3 acylation

[0348] At 3 nmol/kg in an acute setting (FIG. 6), position A11 and A19 were by far the two-best positions at suppressing both food intake for 72 hours and body weight for 96 hours like what was observed for the KBP-066 backbone. Third best position was A18, but it was far inferior when compared to A11 and A19. Position A24 was better than vehicle, but nowhere near what was observed for the KBP-066 backbone. Position A16, A12 and A09 all failed to separate from vehicle on both food intake and body weight suggesting the positions they were acylated were disadvantageous as they inferred with peptide activity.

[0349] However, as the in vitro characteristics table 4.3 shows, A19 and A18 have major issues in terms of fibrillation potential in combination with KBP-021, which make A11 the preferred position to acylate when it comes to backbone KBP-021.

Example 11 (FIGS. 17 and 18)

[0350] Further work was conducted on the best performers from the acute testing, acylated positions (A11 and A19), and a study using repeated doses for comparative effect of the acylations with the same acylation and backbone, namely 3 acylation and KBP-066, respectively.

[0351] Food intake and body weight were investigated in a chronic setting (five-week study) in 20-week old SD rats feed HFD for 8 weeks prior to study start.

TABLE-US-00024 Acylation Position/ KBP Core Position Annotation Acylation KBP-356 KBP-066 A11 KBP-066A11.03 A11/3 acylation KBP-389 KBP-066 A19 KBP-066A19.03 A19/3 acylation

[0352] The experimental protocol as described above in Example 9 was followed. Briefly, rats were caged two and two and were randomized by weight into treatment groups (Vehicle (0.9% NaCl), KBPs (doses: 4 nmol/kg ({circumflex over ( )}14 μg/kg)). Food intake and body weight were monitored daily for 35 days. At study end, an OGTT was performed followed by animal termination in which adipose tissue was taken out and weighed.

FIG. 17 Results—Food Intake and Body Weight for all Vs A19 in a Chronic Setting

[0353] FIG. 17 shows the change in food intake (FIG. 17A) and in body weight (FIG. 17B) over time during the chronic study as a function of treatment. It is evident that A11 and A19 both suppress food intake in a similar fashion and give a significant reduction in body weight after 5 weeks of treatment, however, there is no difference between position A11 and A19 in terms on efficacy on body weight-.

FIG. 18 Results, OGTT and Adipose Tissue

[0354] FIG. 18 shows the results from the OGTT with the corresponding iAUC (OGTT)(Figure A+B) as well as the weight of three different adipose tissue, epididymal, inguinal and perirenal (FIG. 18C-E) and the body weight as study end (FIG. 18F). Treatment with position A11 resulted in a significant reduction in iAUC (OGTT), epididymal WAT size, perirenal WAT size, and body weight as study. Treatment with position A19 resulted in a significant reduction in iAUC (OGTT), epididymal WAT size, and body weight as study, but not perirenal WAT size. Neither treatment significantly reduced the size of the inguinal WAT. Position A11 performed slightly better against vehicle compared to Position A19, but there was no significant difference between treatment groups.

Example 12 (FIG. 19)

[0355] Single dose comparative effect of three acylated linker variants (3, 7 and 8) at the same position and backbone, A11 and KBP-066, respectively, on food intake and body weight in an acute setting in 20-week old SD rats feed HFD for 8 weeks prior to the experiment.

TABLE-US-00025 Acylation Acylation Position/ KBP Core length Linker Annotation Acylation KBP-356 KBP-066 C18 diacid OEG- KBP- A11/3 OEG- 066A11.03 acylation γGLU KBP-385 KBP-066 C18 diacid OEG- KBP- A11/7 OEG- 066A11.07 acylation OEG- γGLU KBP-384 KBP-066 C18 diacid OEG- KBP- A11/8 γGLU 066A11.08 acylation

[0356] Rats were single caged four days prior to the test. Rats were randomized by weight into eight groups (Vehicle (0.9% NaCl), KBPs (doses: 4 nmol/kg ({circumflex over ( )}13-14 μg/kg)). They were fasted overnight and then treated with a single dose of peptide or vehicle in the morning using subcutaneous administration. Food intake was monitored in the following intervals (0-4 hours, 4-24 hours, 24-48 hours, 48-72 hours, and 72-96 hours). Body weight was measured at baseline and at 4 hour, 24 hours, 48 hours, 72 hours and 96 hours post s.c injection.

FIG. 19 Results—Food Intake and Body Weight

[0357] All three linkers tested worked well in an acute setting and all attenuated food intake (FIG. 19A) for up to 72 hours before rebounding. Acylation 3 was slightly better than 7 and 8 after 96 hours. This was evident on the corresponding body weight loss (FIG. 19B) where acylation 3 separate from acylation 7 and 8 early on (24 hours) and continued to separate on two following time points, 72 hours and 96 hours.

[0358] Furthermore, in terms of fibrillation potential (Table 4.4), acylation 8 appear to have some minor tendencies that could complicate further development of compound using that type of acylation.

Summary of Results of Examples 7-12

Acylations Length

[0359] The collected data from FIG. 11-14 and Table 4.1 suggest that the acylations, 3, 4 and 5, in the range of C18 diacid to C22 diacid, are interchangeable when developing acylated peptides for a once weekly dosing regimen in man.

[0360] Hence, C18, C20 and C22 diacid are the preferred length of acylation for this invention.

Acylation Position

[0361] The collected data from FIGS. 15, 17 and 18 demonstrated that A19 may have a slight edge in an acute setting, whereas A11 has a slight edge in a chronic setting when using the KBP-066 backbone and 3 acylation.

[0362] Neither A11 nor A19 had any issues with fibrillation when combined with the KBP-066 backbone (Table 4.2)

[0363] Overall, these data suggest that the acylations position A11 and A19 together with KBP-066, are the two best all-round positions to acylate for development of a once weekly dosing regimen in man.

[0364] Hence A11 and A19 are the preferred positions for acylating KBP-066.

[0365] Similarly, based on Table 4.3 and FIG. 16 it is evident that A11 and A19 are the two best acylation positions when combined with KBP-021.

[0366] However, as A19 is very fibrillation prone in this setting, the A11 with 3 acylation is the preferred acylation position and -length for the KBP-021 backbone based on overall performance.

Acylation Linker

[0367] Based on this test and Table 4.4 it appears that the OEG-OEG-γGLU linker is the optimal linker as shortening it generates potential fibrillation issues and elongating it at best does nothing. Furthermore, As FIG. 19 demonstrated, the OEG-OEG-γGLU linker was also the best performer in an acute setting, in vivo, making it the overall preferred acylation linker.

[0368] In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used in the sense of ‘including’ rather than in to mean ‘consisting of’. All prior teachings acknowledged above are hereby incorporated by reference.