OA20012A

OA20012A - Acylated Calcitonin Mimetics.

Info

Publication number: OA20012A
Application number: OA1202100072
Authority: OA
Inventors: Kim Henriksen; Kim V ANDREASSEN; Nina SONNE; Morten Asser Karsdal
Original assignee: Key Bioscience Ag
Priority date: 2018-08-22
Filing date: 2019-08-22
Publication date: 2021-08-31

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.

Description

Acylated Calcitonin Mimetics

The présent invention relates to acylated mimetics of calcitonin, and extends to their use as médicaments in the treatment of various diseases and disorders, including, but not limited to diabètes (Type I and Type II), excess bodyweight, excessive food consumption and metabolic syndrome, non-alcoholic steatohepatitis (NASH), alcoholic and non-alcoholic fatty liver disease, the régulation of blood glucose levels, the régulation of response to glucose tolérance tests, the régulation of food intake, the treatment of osteoporosis and the treatment of osteoarthritis.

Worldwide, there are about 250 million diabetics and the number is projected to double in the next two décades. Over 90% of this population suffers from type 2 diabètes mellitus (T2DM). It is estimated that only 50-60% of persons affected with T2DM or in stages preceding overt T2DM are currently diagnosed.

T2DM is a heterogeneous disease characterized by abnormalities in carbohydrate and fat metabolism. The causes of T2DM are multi-factorial and include both genetic and environmental éléments that affect β-cell function and insulin sensitivity in tissues such as muscle, liver, pancréas and adipose tissue. As a conséquence impaired insulin sécrétion is observed and paralleled by a progressive décliné in β-cell function and chronic insulin résistance. The inability of the endocrine pancréas to compensate for peripheral insulin résistance leads to hyperglycaemia and onset of clinical diabètes. Tissue résistance to insulinmediated glucose uptake is now recognized as a major pathophysiologic déterminant of T2DM.

A success criterion for an optimal T2DM intervention is the lowering of blood glucose levels, which can be both chronic lowering of blood glucose levels and increased ability to tolerate high glucose levels after food intake, described by lower peak glucose levels and faster clearance. Both of these situations exert less strain on β-cell insulin output and function.

Type I diabètes is characterised by a loss of the ability to produce insulin in response to food intake and hence an inability to regulate blood glucose to a normal physiological level.

The physical structure of bone may be compromised by a variety of factors, including disease and injury. One of the most common bone diseases is osteoporosis, which is characterized by low bone mass and structural détérioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures, particularly of the hip, spine and wrist. Osteoporosis develops when there is an imbalance such that the rate of bone résorption exceeds the rate of bone formation. Administering an effective amount of an anti-resorptive agent, such as calcitonin, has shown to prevent résorption of bone.

Inflammatory or degenerative diseases, including diseases of the joints, e.g. osteoarthritis (OA), rheumatoid arthritis (RA) or juvénile rheumatoid arthritis (JRA), and including inflammation that results from autoimmune response, e.g. lupus, ankylosing spondylitis (AS) or multiple sclerosis (MS), can lead to substantial loss of mobility due to pain and joint destruction. Cartilage that covers and cushions bone within joints may become degraded over time thus undesirably permitting direct contact of two bones that can limit motion of one bone relative to the other and/or cause damage to one by the other during motion of the joint.

Subchondral bone just beneath the cartilage may also dégradé. Administering an effective amount of an anti-resorptive agent, such as calcitonin, may prevent résorption of bone.

Calcitonins are highly conserved over a wide range of species. Full-length native calcitonin is 32 amino acids in length. The sequences of examples of natural calcitonins are set out below:

Salmon CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP

Eel CSNLSTCVLGKLSQELHKLQTYPRTDVGAGTP

Chicken CASLSTCVLGKLSQELHKLQTYPRTDVGAGTP

Mouse CGNLSTCMLGTYTQDLNKFHTFPQTSIGVEAP

Rat CGNLSTCMLGTYTQDLNKFHTFPQTSIGVGAP

Horse CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP

Canine-1 CSNLSTCVLGTYSKDLNNFHTFSGIGFGAETP

Canine-2 CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP

Porcine CSNLSTCVLSAYWRNLNNFHRFSGMGFGPETP

Human CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP

Synthetic variants of natural calcitonins having modified amino acid sequences which are intended to provide improved properties are disclosed in WO2013/067357 and WO 2015/071229.

However, peptides, such as calcitonin and calcitonin mimetics, typically hâve poor absorption, distribution, metabolism and excrétion properties, with rapid clearance and short half-life. Accordingly, peptide drugs typically require daily parentéral administration. Daily administration of treatment through subcutaneous (s.c.) injections is currently not an optimal method of administration, as it poses as an inconvenience to individual patients, and may cause nonadherence to treatment plan to avoid the discomfort associated with daily injections. As such, a once weekly drug using s.c. injections would increase the quality of life for the patients in question and further assist to adhérence of treatment plan.

There are numerous approaches known in the art for attempting to improve the in-vivo half-life of peptide drugs. Such approaches include improving proteolytic stability (by, e.g., protecting the N- and C-termini, replacing amino acids with D-amino acids or unnatural amino acids, cyclising the peptide, etc.) and reducing rénal clearance (by, e.g., conjugating the peptide to macromolecules, such as large polymers, albumin, immunoglobulins, etc.). However, it is also known in the art that making such modifications to drug peptides can be deleterious in terms of, for example, reduced drug potency and unpredictable adverse side reactions, such as drug sensitisation. As such, it is not possible to predict whether such modifications necessarily would improve the therapeutic profile of a peptide drug.

Accordingly, developing peptide drugs that require only once-weekly administration is a challenging prospect.

One approach to improving the pharmacokinetic and pharmacodynamie properties of peptide drugs is to acylate the peptide. Trier et al (PhD thesis, 2016, Acylation of Therapeutic Peptides, DTU; available for download from http://orbit.dtu.dk/files/127682557/PhD_thesis_Sofie_Trier.pd f) studied the effect of acylating two therapeutic peptides, namely glucagon-like peptide 2 (GLP2) and salmon calcitonin (sCT) , with acyl groups of varying length (Cs-Cie) · Whilst the effects of acylating GLP2 were found to be largely predictable based on previous observations on similar peptides, the effects observed when acylating sCT were found to be unpredictable. For example, Trier et al found that acylating sCT (at various positions on the peptide backbone) consistently caused a substantial loss in receptor potency (60-80% loss), whereas receptor potency was retained for GLP2 following acylation. Accordingly, whilst Trier et al. did uncover some useful properties associated with acylating sCT (particularly with regard to short chain (Cs) acylations), it was also clear that there were numerous unpredictable and significantly disadvantageous effects associated with acylating sCT, most notably a significant loss in receptor potency. An additional noteworthy point is that the studies of Trier et al. focused on acylating the 18 position (Lysl8) of salmon calcitonin. This is because previous studies aiming at improving the efficacy of salmon calcitonin identified the 18 position as being the superior position for modification (in that instance by PEGylation, not acylation). In those studies it was found that PEGylating the Lysl8 position of sCT resulted in better efficacy than the analogous peptides modified at the Cysl or Lysll positions (Youn et al, J. Control. Release, 2006, 334-342).

Summary of the Invention

The présent inventons hâve found that acylating calcitonin mimetics at a lysine residue located at the 11 position of the calcitonin mimetics or at a lysine residue located at the 19 position of the calcitonin mimetics, in particular with certain spécifie acyl moieties, results in a surprising improvement in the efficacy of the peptide vis-àvis the équivalent non-acylated peptide, as well as increasing the duration of action of the peptide. Similarly, it was found that the greatest improvement in efficacy of the calcitonin mimetic corresponded to acylation at the 11 or 19 position, whereas acylating the 18 position produced an inferior resuit, contrary to the findings in Youn et al. As such, the présent inventors hâve developed potent novel acylated calcitonin mimetics that may only need to be administered once weekly, rather than once daily.

Accordingly, in one aspect, the présent invention provides a calcitonin mimetic that is 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. The side chain ε-amino group of said lysine residue is acylated with an acyl group selected from any one of the following: a C16 or longer fatty acid with an optional linker; or a C16 or longer fatty diacid with an optional linker.

As used herein, calcitonin mimetic means a peptide that activâtes the calcitonin receptor (i.e. a calcitonin receptor agonist), and preferably also activâtes the amylin receptor (i.e. a dual amylin and calcitonin receptor agonist).

In certain preferred embodiments, the calcitonin mimetic is from 32 to 37 amino acids in length. Most preferably the calcitonin mimetic is 32 amino acids in length.

In one preferred aspect, in which the calcitonin mimetic is acylated at a lysine residue located at the 11 position, the présent invention relates to a calcitonin mimetic of formula (I)(a ):

CX2X3LSTCX8LGKAC...

wherein

X₂ = A, G or S

X₃ = N or S

Xs = Μ, V or α-aminoisobutyric acid (AiB) and wherein Kac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following:

Ci6 or longer fatty acid with an optional linker, or

Ci6 or longer fatty diacid with an optional linker.

In another preferred aspect, in which the calcitonin mimetic is acylated at a lysine residue located at the 19 position, the présent inventive relates to a calcitonin mimetic of formula (I)(b):

CX2X3LSTCX8LGX11X12X13X14X15X16X17X18KAC...

wherein
X₂ = A, G	or S
X₃ = N or	S
X₈ = Μ, V	or α-aminoisobutyric acid (AiB)

X11	= R, K, T, A or Kac (preferably R, K, or	Kac, most
preferably R	or K)
X12 X13	= L or Y ( = S, T, W	^most or Y	preferably L) (preferably T, S or Y)
X14 X15 X16 X17	= Q, K, R Q) = D, E or = L or F ( = H or N	or A (preferably Q or A, most N (preferably D or E) most preferably L)	preferably

Xi8 = R, K or N (preferably R or K) and wherein Kac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following:

Ci6or longer fatty acid with an optional linker, or

Ci6 or longer fatty diacid with an optional linker.

Preferably, the calcitonin mimetic of formula (I)(a) or (I)(b) is from 32 to 37 amino acids in length, preferably 32, 33, 35, 36 or 37 amino acids in length. Most preferably, the calcitonin mimetic of formula (I)(a) or (I)(b) is 32 amino acids in length.

In a preferred aspect of the invention, the calcitonin mimetic is a 32mer calcitonin mimetic of formula (II): CX2X3LSTCX8LGX11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27GX29X30X31P wherein

X2 =	A, G or S
X3 =	N or S
x₈ =	Μ, V or α-aminoisobutyric acid (AiB)
X11 =	K_Ac, R, K, T or A (most preferably Kac, R or K)
X12 =	L or Y
X13 =	S, T, W or Y
X14 =	Q, K, R or A
X15 =	D, E or N
X16 =	L or F
X17 =	H or N
X18 =	R, K or N
X19 =	Kac, L, F or K (most preferably Kac, L or F)
X20 =	Q, H or A
X21 =	T or R
X22 =	Y or F
X23 =	S or P

X₂4 = G, K, Q orR

X₂₅ = T, I orM

X26 = S, N, D, Gor A

X27 = T, V, F orI

X29 = S, A, P orV

X30 = N, G orE

X31 = A, T or S (most preferably A or T) wherein either Xn is Kac and/or X19 is Kac (such that either Xn is Kac and X19 is L, F or K, preferably L or F; or X11 is R, K, T or A, preferably R or K, and X19 is Ka_c; or Xn is Kac and X19 is Kac) , and wherein Kac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following:

Ci6 or longer fatty acid,

Ci6 or longer fatty diacid,

Iinker-Ci6 or longer fatty acid, or linker-Cie or longer fatty diacid.

Preferably, the 32mer calcitonin mimetic of formula (II) is :

CX2X3LSTCX8LGX11LX13X14X15LX17X18X19X20TX22PX24TDVGANAP

wherein

X₂ = A, G or S

X3 = N or S

X₈ = Μ, V or AiB

X11 = Kac, R, K, T or A (most preferably Kac, R or K)

X13 = T, S or Y

X14 = Q or A (most preferably Q)

X15 = D or E

X17 = H or N

X18 = R or K

X19 = Kac, L, F or K (most preferably Kac, L or F)

X20 = Q, H or A

X22 = Y or F

X24 = K, Q or R wherein either Xn is Kac and/or X19 is Kac, and wherein Kac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following:

Ci6 or longer fatty acid,

Ci6 or longer fatty diacid,

Iinker-Ci6 or longer fatty acid, or

Iinker-Ci6 or longer fatty diacid.

Preferably, X2 is S and X3 is N; or X2 is G and X3 is N; or X2 is A and X3 is S.

Preferably, X13 is S or T, most preferably S.

Preferably, X24 is R or K.

In a preferred embodiment,

- Xn is Kac, X17 is H, Xis is K, X19 is L and X20 is Q or

A; or

- Xn is Kac, X17 is H, Xis is R, X19 is L and X20 is Q or

A; or

- Xn is Kac, X17 is N, Xis is K, X19 is F and X20 is H or

A; or

- Xn is Kac, X17 is N, Xis is R, X19 is F and X20 is H or

A; or

- Xn is R or K, X17 is H, Xis is K, X19 is Kac and X20 is Q or A; or

- Xn is R or K, X17 is H, Xis is R, X19 is Kac and X20 is Q or A; or

- Xn is R or K, X17 is N, Xis is K, X19 is Kac and X20 is H or A; or

- Xn is R or K, X17 is N, Xis is R, X19 is Kac and X20 is H or A.

In a preferred embodiment, X2 is S, X3 is N, Xn is Kac, X13 is S, X17 is H, Xis is K or R, X19 is L, X20 is Q or A and

X22 is Y; or X₂ is S, X₃ is N, Xn is R or K, X13 is S, X17 is

H, Xi8 is K or R, X19 is Kac, X20 is Q or A and X22 is Y. In a preferred embodiment, X₂ is A, X3 is S, X11 is Kac, X13 is S,

X17 is H, Xi8 is K or R, X19 is L, X20 is Q or A and X22 is F;

or X2 is A, X3 is S, Xn is R or K, X13 is S, X17 is H, Xn is K or R, X19 is Kac, X20 is Q or A and X22 is F. In a preferred embodiment, X₂ is G, X3 is N, Xn is Kac, X13 is T, X17 is N, Xis is K or R, X19 is F, X20 is H or A and X22 is F; or X2 is G, X₃ is N, Xn is R or K, X13 is T, X17 is N, Xis is K or R, X19 is

Kac, X20 is H or A and X22 is F.

In another preferred aspect, the invention relates to a calcitonin mimetic, wherein the calcitonin mimetic is a 33mer peptide in accordance with formula (III): CSNLSTCX₆LGX7LSQDLHRX₈QTYPKXiTX₅VGANAP (III) or wherein the calcitonin mimetic is a 35mer peptide in accordance with formula (IV):

CSNLSTCX₆LGX7LSQDLHRX₈QTYPKXiX2X3TX₅VGANAP ( IV) or wherein the calcitonin mimetic is a 36mer peptide in accordance with formula (V):

CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3X4TX5VGANAP (V) or wherein the calcitonin mimetic is a 37mer peptide in accordance with formula (VI):

CSNLSTCX6LGKACLZX1X2X3X4TX5VGANAP (VI ) wherein each of Xi to X4 is any amino acid, with the proviso that at least one of Xi to X4 is a basic amino acid residue, and/or at least two of Xi to X4 are independently a polar amino acid residue or a basic amino acid residue, and/or at least one of Xi to X4 is a Gly residue, and wherein none of Xi to X4 is an acidic residue;

wherein X5 is D or N;

wherein Xg is AiB or M;

wherein either X7 is Kac and Xg is L, or X7 is R or K and X₈ is K_Ac, wherein Z is selected from SQDLHRLSNNFGA, SQDLHRLQTYGAI or ANFLVHSSNNFGA; and wherein Kac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following:

Ci6 or longer fatty acid,

Oie or longer fatty diacid, linker-Cig or longer fatty acid, or Iinker-Ci6 or longer fatty diacid.

Preferably, at least one of Xi or X4 of formulae (III)(VI) is a basic amino acid residue. Preferably still, at least one of Xi or X4 is a basic amino acid residue, and at least one more of Xi to X4 is independently a polar amino acid residue or a basic amino acid residue, and none of Xi to X4 is an acidic residue. Preferably still, at least three of Xi to X4 are independently a polar amino acid residue or a basic amino acid residue, and none of Xi to X4 is an acidic residue. More preferably, ail of Xi to X4 are independently a polar amino acid residue or a basic amino acid residue, and none of Xi to X4 is an acidic residue. Most preferably, ail of Xi to X4 are independently a polar amino acid residue or a basic amino acid residue, at least three of Xi to X4 are basic amino acid residues, and none of Xi to X4 is an acidic residue.

The basic amino acid residues may be any natural or unnatural amino acid residues with basic side chains, and may be selected from, but are not limited to, Arg, His or Lys. The polar amino acid residues may be any natural or unnatural amino acid residues with polar uncharged side chains, and may be selected from, but are not limited to, Ser, Thr, Asn, Gin or Cys. As used herein, the term acidic residue refers to any natural or unnatural amino acid residue that has an acidic side chain, such as, for example, Glu or Asp.

In a preferred embodiment, Xi is selected from Asn, Phe, Val, Gly, Ile, Leu, Lys, His or Arg;

X2 is selected from Ala, Asn, His, Leu, Ser, Thr, Gly or Lys ;

X3 is selected from Ala, Phe, Ile, Ser, Pro, Thr, Gly or Lys; and/or

X4 is selected from Ile, Leu, Gly, His, Arg, Asn, Ser, Lys, Thr or Gin;

with proviso that at least one of Xi or X4 is a basic amino acid residue, and/or at least two of Xi to X4 are independently a polar amino acid residue and/or a basic amino acid residue, and/or at least one of Xi to X4 is a Gly residue.

In a preferred embodiment, Xi is selected from Asn, Gly, Ile, His or Arg;

X2 is selected from Asn, Leu, Thr, Gly or Lys;

X3 is selected from Phe, Pro, Ile, Ser, Thr, Gly or Lys; and/or

X4 is selected from Gly, His, Asn, Ser, Lys, Thr or Gin;

Peptides of the invention in accordance with formulae (III)-(V), supra, may comprise one or more of the following conservative substitutions:

- Asp residue at position 15 of the peptide is substituted with Glu;

- Arg residue at position 18 of the peptide is substituted with Lys; and/or

- Lys residue at position 24 of the peptide is substituted with Arg.

Peptides of the invention in accordance with formulae (VI), supra, wherein the Z component of the peptide of formula (VI) is SQDLHRLSNNFGA or SQDLHRLQTYGAI, may comprise one or more of the following conservative substitutions:

- Asp residue at position 15 of the peptide is substituted with Glu; and/or

- Arg residue at position 18 of the peptide is substituted with Lys.

In ail aspects of the invention, the linker preferably 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:

and wherein n is from 1 to 10, preferably 1 to 5, preferably to 3, preferably 1 or 2, and most preferably 1.

The OEG amino acid linker may preferably comprise one OEG amino acid or two to six OEG amino acids linked together. More preferably, the OEG amino acid linker comprises one OEG amino acid, or two to three OEG amino acids linked together. Most preferably, the OEG amino acid linker comprises two OEG amino acids linked together. The OEG amino acid linker may further comprise one or more glutamic acid residues linked to the amino terminus or to the carboxyl terminus of the OEG amino acid linker. Preferably, the OEG amino acid linker is selected from any one of the following:

O^OH

Preferably, the OEG amino acid linker is:

H O^O V H

In a preferred embodiment, the acyl group is selected from Ci8 or longer fatty acid, Cis or longer fatty diacid, Iinker-Ci8 or longer fatty acid, or linker-Cis or longer fatty diacid. Preferably, the acyl group is selected from any one of the following:

Ci8 to C30 fatty acid, preferably Cis to C22 fatty acid,

Ci8 to C30 fatty diacid, preferably Cis to C22 fatty diacid, linker-Cis to C30 fatty acid, preferably linker-Cia to C22 fatty acid, or linker-Cis to C30 fatty diacid, preferably linker-Cis to C22 fatty acid.

Preferably, the Cis fatty diacid is octadecanedioic acid (CAS No. 871-70-5).

In a preferred embodiment, Kac is acylated with a linkerfatty diacid, wherein the fatty diacid is a Cis to C22 fatty

H O^O Y H O diacid and the linker is JL O H

Preferably, the Cis fatty diacid is octadecanedioic acid.

Preferably, the calcitonin mimetic of the invention is selected from any one of the following:

CSNLSTCMLGKacLSQDLHRLQTYPKTDVGANAP

CSNLSTCMLGKacLSQELHRLQTYPKTDVGANAP

CSNLSTCVLGKacLSQELHKLQTYPRTDVGANAP

CASLSTCVLGKacLSQDLHKLQTFPKTDVGANAP

CGNLSTCMLGKacLSQDLNKFHTFPQTDVGANAP

CSNLSTC (AiB) LGKacLSQDLHRLQTYPKTDVGANAP

CGNLSTC (AiB) LGKacLTQDLNKFHTFPKTDVGANAP

CSNLSTC (AiB) LGKacLANFLVHSSNNFGAILPKTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKHTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKHSSTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKHSSNTDVGANAP

CSNLSTCMLGKacLSQDLHRLSNNFGAILSSTNVGANAP

CSNLSTCMLGKacLSQDLHRLQTYGAILSPKTDVGANAP

CSNLSTCMLGKacLANFLVHSSNNFGAILPKTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKILSSTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKGLITTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKNNFGTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKRTTQTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKHTTNTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKHGGQTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKHKKNTDVGANAP

CSNLSTCMLGKacLSQDLHRLQTYPKHKKHTDVGANAP

CSNLSTC (AiB) LGRLSQDLHRKacQTYPKTDVGANAP

CSNLSTCMLGRLSQELHRKacQTYPKTDVGANAP wherein Kac is as defined supra. The amino acid residue in the 8 position of the above peptides is, where not already the case, optionally substituted with AiB.

AcCSNLSTCMLGKacLSQDLHRLQTYPKTDVGANAP-NH₂

AcCSNLSTC (AiB) LGKa_cLSQDLHRLQTYPKTDVGANAP-NH₂

AcCGNLSTC (AiB) LGKa_cLTQDLNKFHTFPKTDVGANAP-NH₂

AcCSNLSTCVLGKacLSQELHKLQTYPRTDVGANAP-NH₂

AcCSNLSTCMLGKacLSQELHRLQTYPKTDVGANAP-NH₂

AcCASLSTCVLGKacLSQDLHKLQTFPKTDVGANAP-NH₂

AcCGNLSTCMLGKacLSQDLNKFHTFPQTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKHTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKHSSTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKHSSNTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLSNNFGAILSSTNVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYGAILSPKTDVGANAP-NH₂AcCSNLSTCMLGKacLANFLVHSSNNFGAILPKTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKILSSTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKGLITTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKNNFGTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKRTTQTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKHTTNTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKHGGQTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKHKKNTDVGANAP-NH₂AcCSNLSTCMLGKacLSQDLHRLQTYPKHKKHTDVGANAP-NH₂AcCSNLSTC (AiB) LGKa_cLANFLVHSSNNFGAILPKTDVGANAP-NH₂AcCSNLSTC (AiB) LGRLSQDLHRKacQTYPKTDVGANAP-NH₂AcCSNLSTCMLGRLSQELHRKacQTYPKTDVGANAP-NH₂ wherein Kac is acylated with a linker-fatty diacid, and wherein the fatty diacid is a Cis to C22 fatty diacid and the linker is

H 0^0

Y H O

JL o H O .

Preferably, the Cis fatty diacid is octadecanedioic acid. The amino acid residue in the 8 position of the above peptides is, where not already the case, optionally substituted with AiB. In the above peptides, Ac indicates that the N-terminus of the peptide is acetylated, and -NH2 indicates that the C-terminus of the peptide is amidated.

The calcitonin mimetic of the invention may be formulated for enterai administration. For example, the calcitonin mimetic may be formulated in a pharmaceutical composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increase the oral bioavailability of the peptide. Alternatively, or in addition to, the calcitonin mimetic may be formulated with a carrier for oral administration. An exemplary carrier may comprise 5-CNAC, SNAD, or SNAC. The calcitonin mimetic of the invention may also be formulated for parentéral administration. For example, the calcitonin mimetic may be formulated for injection.

The présent invention also relates to a pharmaceutical composition comprising a calcitonin mimetic as described supra.

The présent invention also relates to a calcitonin mimetic as described supra for use as a médicament. In that regard, the calcitonin mimetic may be for use in treating diabètes (Type I and/or Type II), excess bodyweight, excessive food consumption, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis, or osteoarthritis, poorly regulated blood glucose levels, poorly regulated response to glucose tolérance tests, or poor régulation of food intake. The calcitonin mimetic may also be administered in conjunction with metformin or another insulin sensitizer.

The peptides of the invention may be acylated at its Nterminal or otherwise modified to reduce the positive charge of the first amino acid and independently of that may be amidated at its C-terminal.

The peptide may be formulated for administration as a pharmaceutical and may be formulated for enterai or parentéral administration. Preferred formulations are injectable, preferably for subcutaneous injection, however the peptide may be formulated with a carrier for oral administration, and optionally wherein the carrier increases the oral bioavailability of the peptide. Suitable carriers include ones that comprise 5-CNAC, SNAD, or SNAC.

Optionally, the peptide is formulated in a pharmaceutical composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increase the oral bioavailability of the peptide.

The invention includes a peptide of the invention for use as a médicament. The peptide may be for use in treating diabètes (Type I and/or Type II), excess bodyweight, excessive food consumption, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis, or osteoarthritis, poorly regulated blood glucose levels, poorly regulated response to glucose tolérance tests, or poor régulation of food intake. In particular, the peptides may be used to lower an undesirably high fasting blood glucose level or to lower an undesirably high HbAlc or to reduce an undesirably high response to a glucose tolérance test. The peptides of the invention may also be used for producing a decrease in liver triglycérides and/or for reducing fat accumulation in the liver of a subject.

The peptides of the invention may be produced using any suitable method known in the art for generating peptides, such as synthetic (Chemical) and recombinant technologies. Preferably, the peptides are produced using a synthetic method. Synthetic peptide synthesis is well known in the art, and includes (but is not limited to) solid phase peptide synthesis employing various protecting group strategies (e.g. using Fmoc, Boc, Bzl, tBu, etc.).

In some embodiments, the N-terminal side of the calcitonin mimetics discussed supra is modified to reduce the positive charge of the first amino acid. For example, an acetyl, propionyl, or succinyl group may be substituted on cysteine-1. Alternative ways of reducing positive charge include, but are not limited to, polyethylene glycol-based PEGylation, or the addition of another amino acid such as glutamic acid or aspartic acid at the N-terminus. Alternatively, other amino acids may be added to the Nterminus of peptides discussed supra including, but not limited to, lysine, glycine, formylglycine, leucine, alanine, acetyl alanine, and dialanyl. As those of skill in the art will appreciate, peptides having a plurality of cysteine residues frequently form a disulfide bridge between two such cysteine residues. Ail such peptides set forth herein are defined as optionally including one or more such disulphide bridges, particularly at the Cysl-Cys7 locations. Mimicking this, the cysteines at positions 1 and 7 may jointly be replaced by an α-aminosuberic acid linkage. While calcitonin mimetics of the present disclosure may exist in free acid form, it is preferred that the C-terminal amino acid be amidated. Applicants expect that such amidation may contribute to the effectiveness and/or bioavailability of the peptide. Synthetic Chemical methods may be employed for amidating the C-terminal amino acid. Another technique for manufacturing amidated versions of the calcitonin mimetics of the present disclosure is to react precursors (having glycine in place of the C-terminal amino group of the desired amidated product) in the presence of peptidylglycine alphaamidating monooxygenase in accordance with known techniques wherein the precursors are converted to amidated products in reactions described, for example, in US4708934 and EP0308067 and EP0382403.

Production of amidated products may also be accomplished using the process and amidating enzyme set forth by Consalvo, et al in US7445911; Miller et al, US2006/0292672; Ray et al, 2002, Protein Expression and Purification, 26:249-259; and Mehta, 2004, Biopharm. International, July, pp. 44-46.

The production of the preferred amidated peptides may proceed, for example, by producing glycine-extended precursor in E. coli as a soluble fusion protein with glutathione-Stransferase, or by direct expression of the precursor in accordance with the technique described in US6103495. Such a glycine extended precursor has a molecular structure that is identical to the desired amidated product except at the Cterminus (where the product terminâtes --X--NH2, while the precursor terminâtes --X-gly, X being the C-terminal amino acid residue of the product). An alpha-amidating enzyme described in the publications above catalyzes conversion of precursors to product. That enzyme is preferably recombinantly produced, for example, in Chinese Hamster Ovary (CHO) cells), as described in the Biotechnology and Biopharm. articles cited above.

Free acid forms of peptide active agents of the présent disclosure may be produced in like manner, except without including a C-terminal glycine on the precursor, which precursor is instead the final peptide product and does not require the amidation step.

Except where otherwise stated, the preferred dosage of the calcitonin mimetics of the présent disclosure is identical for both therapeutic and prophylactic purposes. Desired dosages are discussed in more detail, infra, and differ depending on mode of administration.

Except where otherwise noted or where apparent from context, dosages herein refer to weight of active compounds (i.e. calcitonin mimetics) unaffected by or discounting pharmaceutical excipients, diluents, carriers or other ingrédients, although such additional ingrédients are desirably included. Any dosage form (capsule, tablet, injection or the like) commonly used in the pharmaceutical industry for delivery of peptide active agents is appropriate for use herein, and the terms excipient, diluent, or carrier includes such non-active ingrédients as are typically included, together with active ingrédients in such dosage form in the industry. A preferred oral dosage form is discussed in more detail, infra, but is not to be considered the exclusive mode of administering the active agents of the présent disclosure.

The calcitonin mimetics of the présent disclosure can be administered to a patient to treat a number of diseases or disorders. As used herein, the term patient means any organism belonging to the kingdom Animalia. In an embodiment, the term patient refers to vertebrates, more preferably, mammals including humans.

Accordingly, the présent disclosure includes the use of the peptides in a method of treatment of type I diabètes, Type II diabètes or metabolic syndrome, obesity, or of appetite suppression, or for mitigating insulin résistance, or for reducing an undesirably high fasting sérum glucose level, or for reducing an undesirably high peak sérum glucose level, or for reducing an undesirably high peak sérum insulin level, or for reducing an undesirably large response to a glucose tolérance test, or for treating osteoporosis, or for treating osteoarthritis, or for treating non-alcoholic steatohepatitis (NASH), or for treating alcoholic fatty liver disease, or for producing a decrease in liver triglycérides, or for reducing fat accumulation in the liver of a subject.

There are a number of art-recognized measures of normal range for body weight in view of a number of factors such as gender, âge and height. A patient in need of treatment or prévention regimens set forth herein include patients whose body weight exceeds recognized norms or who, due to heredity, environmental factors or other recognized risk factor, are at higher risk than the general population of becoming overweight or obese. In accordance with the présent disclosure, it is contemplated that the calcitonin mimetics may be used to treat diabètes where weight control is an aspect of the treatment.

In an embodiment, the method includes enterai administration to a patient in need thereof for treatment of a said condition of a pharmaceutically effective amount of any one of the peptides described herein.

In an embodiment, the method includes parentéral administration to a patient in need thereof for treatment of a said condition of a pharmaceutically effective amount of any one of the peptides described herein. For parentéral administration (including intraperitoneal, subcutaneous, intravenous, intradermal or intramuscular injection), solutions of a peptide of the présent disclosure in either sesame or peanut oil or in aqueous propylene glycol may be employed, for example. The aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonie. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes. The préparation of ail these solutions under stérile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. For parentéral application, examples of suitable préparations include solutions, preferably oily or aqueous solutions as well as suspensions, émulsions, or implants, including suppositories. Peptides may be formulated in stérile form in multiple or single dose formats such as being dispersed in a fluid carrier such as stérile physiological saline or 5% saline dextrose solutions commonly used with inj ectables.

Said method may include a preliminary step of determining whether the patient suffers from a said condition, and/or a subséquent step of determining to what extent said treatment is effective in mitigating the condition in said patient, e.g. in each case, carrying out an oral glucose tolérance test or a resting blood sugar level.

Oral enterai formulations are for ingestion by swallowing for subséquent release in the intestine below the stomach, and hence delivery via the portai vein to the liver, as opposed to formulations to be held in the mouth to allow transfer to the bloodstream via the sublingual or buccal routes.

Suitable dosage forms for use in the présent disclosure include tablets, mini-tablets, capsules, granules, pellets, powders, effervescent solids and chewable solid formulations. Such formulations may include gelatin which is preferably hydrolysed gelatin or low molecular weight gelatin. Such formulations may be obtainable by freeze drying a homogeneous aqueous solution comprising a calcitonin mimetic and hydrolysed gelatin or low molecular weight gelatin and further processing the resulting solid material into said oral pharmaceutical formulation, and wherein the gelatin may hâve a mean molecular weight from 1000 to 15000 Daltons. Such formulations may include a protective carrier compound such as 5-CNAC or others as disclosed herein.

Whilst oral formulations such as tablets and capsules are preferred, compositions for use in the présent disclosure may take the form of syrups, élixirs or the like and suppositories or the like. Oral delivery is generally the delivery route of choice since it is convenient, relatively easy and generally painless, resulting in greater patient compliance relative to other modes of delivery. However, biological, Chemical and physical barriers such as varying pH in the gastrointestinal tract, powerful digestive enzymes, and active agent imperméable gastrointestinal membranes, makes oral delivery of calcitonin like peptides to marnais problematic, e.g. the oral delivery of calcitonins, which are long-chain polypeptide hormones secreted by the parafollicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish, originally proved difficult due, at least in part, to the insufficient stability of calcitonin in the gastrointestinal tract as well as the inability of calcitonin to be readily transported through the intestinal walls into the blood stream.

Suitable oral formulations are however described below.

Treatment of Patients

In an embodiment, a calcitonin mimetic of the présent disclosure is administered at adéquate dosage to maintain sérum levels of the mimetic in patients between 5 picograms and 1000 nanograms per milliliter, preferably between 50 picograms and 500 nanograms, e.g. between 1 and 300 nanograms per milliliter. The sérum levels may be measured by any suitable techniques known in the art, such as radioimmunoassay or mass spectrometry. The attending physician may monitor patient response, and may then alter the dosage somewhat to account for individual patient metabolism and response. Near simultaneous release is best achieved by administering ail components of the présent disclosure as a single pill or capsule. However, the disclosure also includes, for example, dividing the required amount of the calcitonin mimetic among two or more tablets or capsules which may be administered together such that they together provide the necessary amount of ail ingrédients. Pharmaceutical composition, as used herein includes but is not limited to a complété dosage appropriate to a particular administration to a patient regardless of whether one or more tablets or capsules (or other dosage forms) are recommended at a given administration.

A calcitonin mimetic of the présent disclosure may be formulated for oral administration using the methods employed in the Unigene Enteripep® products. These may include the methods as described in US Patent No. 5,912,014, US Patent No. 6,086,918, US Patent No. 6,673,574, US Patent No. 7,316,819, US Patent No. 8,093,207, and US Publication No. 2009/0317462. In particular, it may include the use of conjugation of the compound to a membrane translocator such as the protein transduction domain of the HIV TAT protein, co-formulation with one or more protease inhibitors, and/or a pH lowering agent which may be coated and/or an acid résistant protective vehicle and/or an absorption enhancer which may be a surfactant.

In an embodiment, a calcitonin mimetic of the présent disclosure is preferably formulated for oral delivery in a manner known in U.S. Patent Publication No. 2009/0317462.

In an embodiment, a calcitonin mimetic of the présent disclosure may be formulated for enterai, especially oral, administration by admixture with a suitable carrier compound. Suitable carrier compounds include those described in US Patent No. 5,773,647 and US Patent No. 5866536 and amongst these, 5-CNAC (N-(5-chlorosalicyloyl)-8-aminocaprylic acid, commonly as its disodium sait) is particularly effective. Other preferred carriers or delivery agents are SNAD (sodium sait of 10-(2-Hydroxybenzamido)decanoic acid) and SNAC (sodium sait of N-(8-[2-hydroxybenzoyl]amino)caprylic acid). In an embodiment, a pharmaceutical composition of the présent disclosure comprises a delivery effective amount of carrier such as 5-CNAC, i.e. an amount sufficient to deliver the compound for the desired effect. Generally, the carrier such as 5-CNAC is présent in an amount of 2.5% to 99.4% by weight, more preferably 25% to 50% by weight of the total composition.

In addition, WO 00/059863 discloses the disodium salts of formula I

wherein

R¹, R², R³, and R⁴ are independently hydrogen, -OH, -NR⁶R⁷, halogen, C1-C4 alkyl, or C1-C4 alkoxy;

R⁵ is a substituted or unsubstituted C2-C16 alkylene, substituted or unsubstituted C2-C16 alkenylene, substituted or unsubstituted C1-C12 alkyl(arylene), or substituted or unsubstituted aryl (C1-C12 alkylene); and R⁶ and R⁷ are independently hydrogen, oxygen, or C1-C4 alkyl; and hydrates and solvatés thereof as particularly efficacious for the oral delivery of active agents, such as calcitonins, e.g. salmon calcitonin, and these may be used in the présent disclosure.

Preferred enteric formulations using optionally micronised 5-CNAC may be generally as described in WO2005/014031.

The compound may be formulated for oral administration using the methods employed in the Capsitonin product of Bone Medical Limited. These may include the methods incorporated in Axcess formulations. More particularly, the active ingrédient may be encapsulated in an enteric capsule capable of withstanding transit through the stomach. This may contain the active compound together with a hydrophilic aromatic alcohol absorption enhancer, for instance as described in WO02/028436. In a known manner the enteric coating may become permeable in a pH sensitive manner, e.g. at a pH of from 3 to 7. WO2004/091584 also describes suitable formulation methods using aromatic alcohol absorption enhancers.

The compound may be formulated using the methods seen in the Oramed products, which may include formulation with omega-3 fatty acid as seen in WO2007/029238 or as described in US5,102,666.

Generally, the pharmaceutically acceptable salts (especially mono or di sodium salts), solvatés (e.g. alcohol solvatés) and hydrates of these carriers or delivery agents may be used.

Oral administration of the pharmaceutical compositions according to the disclosure can be accomplished regularly, e.g. once or more on a daily or weekly basis; intermittently, e.g. irregularly during a day or week; or cyclically, e.g. regularly for a period of days or weeks followed by a period without administration. The dosage form of the pharmaceutical compositions of the presently disclosed embodiments can be any known form, e.g. liquid or solid dosage forms. The liquid dosage forms include solution émulsions, suspensions, syrups and élixirs. In addition to the active compound and carrier such as 5-CNAC, the liquid formulations may also include inert excipients commonly used in the art such as, solubilizing agents e.g. éthanol; oils such as cottonseed, castor and sesame oils; wetting agents; emulsifying agents; suspending agents; sweeteners; flavourings; and solvents such as water. The solid dosage forms include capsules, soft-gel capsules, tablets, caplets, powders, granules or other solid oral dosage forms, ail of which can be prepared by methods well known in the art. The pharmaceutical compositions may additionally comprise additives in amounts customarily employed including, but not limited to, a pH adjuster, a preservative, a flavorant, a taste-masking agent, a fragrance, a humectant, a tonicifier, a colorant, a surfactant, a plasticizer, a lubricant such as magnésium stéarate, a flow aid, a compression aid, a solubilizer, an excipient, a diluent such as microcrystalline cellulose, e.g. Avicel PH 102 supplied by FMC corporation, or any combination thereof. Other additives may include phosphate buffer salts, citric acid, glycols, and other dispersing agents. The composition may also include one or more enzyme inhibitors, such as actinonin or epiactinonin and dérivatives thereof; aprotinin, Trasylol and Bowman-Birk inhibitor. Further, a transport inhibitor, i.e. a [rho]glycoprotein such as Ketoprofin, may be présent in the compositions of the présent disclosure. The solid pharmaceutical compositions of the instant disclosure can be prepared by conventional methods e.g. by blending a mixture of the active compound, the carrier such as 5-CNAC, and any other ingrédients, kneading, and filling into capsules or, instead of filling into capsules, molding followed by further tableting or compression-molding to give tablets. In addition, a solid dispersion may be formed by known methods followed by further processing to form a tablet or capsule. Preferably, the ingrédients in the pharmaceutical compositions of the instant disclosure are homogeneously or uniformly mixed throughout the solid dosage form.

Alternatively, the active compound may be formulated as a conjugate with said carrier, which may be an oligomer as described in US2003/0069170, e.g.

O , Il compound----[-C-----(CH₂)₇(OC₂H₄)₇OCH₃]₂

Such conjugates may be administered in combination with a fatty acid and a bile sait as described there.

Conujugates with polyethylene glycol (PEG) may be used, as described for instance in Mansoor et al.

Alternatively, active compounds may be admixed with nitroso-N-acetyl-D,L-penicillamine (SNAP) and Carbopol solution or with taurocholate and Carbapol solution to form a mucoadhesive émulsion.

The active compound may be formulated by loading into chitosan nanocapsules as disclosed in Prego et al (optionally PEG modified as in Prego Prego C, Torres D, Fernandez-Megia E, Novoa-Carballal R, Quihoâ E, Alonso MJ.) or chitosan or PEG coated lipid nanoparticles as disclosed in Garcia-Fuentes et al. Chitosan nanoparticles for this purpose may be iminothiolane modified as described in Guggi et al. They may be formulated in water/oil/water émulsions as described in Dogru et al. The bioavailability of active compounds may be increased by the use of taurodeoxycholate or lauroyl carnitine as described in Sinko et al or in Song et al. Generally, suitable nanoparticles as carriers are discussed in de la Fuente et al and may be used in the présent disclosure.

Other suitable strategies for oral formulation include the use of a transient permeability enhancer (TPE) System as described in WO2005/094785 of Chiasma Ltd. TPE makes use of an oily suspension of solid hydrophilic particles in a hydrophobie medium to protect the drug molécule from inactivation by the hostile gastrointestinal (GI) environment and at the same time acts on the GI wall to induce perméation of its cargo drug molécules.

Further included is the use of glutathione or compounds containing numerous thiol groups as described in US2008/0200563 to inhibit the action of efflux pumps on the mucous membrane. Practical examples of such techniques are described also in Caliceti, P. Salmaso, S., Walker, G. and Bernkop-Schnürch, A. (2004) 'Development and in vivo évaluation of an oral insulin-PEG delivery System.' Eur. J. Pharm. Sci., 22, 315-323, in Guggi, D., Krauland, A.H., and Bernkop-Schnürch, A. (2003) 'Systemic peptide delivery via the stomach: in vivo évaluation of an oral dosage form for salmon calcitonin'. J. Control. Rel. 92,125-135, and in Bernkop-Schnürch, A., Pinter, Y., Guggi, D., Kahlbacher, H., Schôffmann, G., Schuh, M., Schmerold, I., Del Curto, M.D., D'Antonio, M., Esposito, P. and Huck, Ch. (2005) 'The use of thiolated polymers as carrier matrix in oral peptide delivery' - Proof of concept. J. Control. Release, 106, 2633.

The active compound may be formulated in seamless microspheres as described in WO2004/084870 where the active pharmaceutical ingrédient is solubilised as an émulsion, microemulsion or suspension formulated into mini-spheres; and variably coated either by conventional or novel coating technologies. The resuit is an encapsulated drug in presolubilised form which when administered orally provides for predetermined instant or sustained release of the active drug to spécifie locations and at spécifie rates along the gastrointestinal tract. In essence, pre-solubilization of the drug enhances the predictability of its kinetic profile while simultaneously enhancing permeability and drug stability.

One may employ chitosan coated nanocapsules as described in US2009/0074824. The active molécule administered with this technology is protected inside the nanocapsules since they are stable against the action of the gastric fluid. In addition, the mucoadhesive properties of the System enhances the time of adhesion to the intestine walls (it has been verified that there is a delay in the gastrointestinal transit of these Systems) facilitating a more effective absorption of the active molécule.

Methods developed by TSR1 Inc. may be used. These include Hydrophilic Solubilization Technology (HST) in which gelatin, a naturally derived collagen extract carrying both positive and négative charges, coats the particles of the active ingrédient contained in lecithin micelles and prevents their aggregation or dumping. This results in an improved wettability of hydrophobie drug particles through polar interactions. In addition, the amphiphilic lecithin reduces surface tension between the dissolution fluid and the particle surface.

The active ingrédient may be formulated with cucurbiturils as excipients.

Alternatively, one may employ the GIPET technology of Merrion Pharmaceuticals to produce enteric coated tablets containing the active ingrédient with an absorption enhancer which may be a medium chain fatty acid or a medium chain fatty acid dérivative as described in US2007/0238707 or a membrane translocating peptide as described in US7268214.

One may employ GIRES™ technology which consists of a controlled-release dosage form inside an inflatable pouch, which is placed in a drug capsule for oral administration. Upon dissolution of the capsule, a gas-generating System inflates the pouch in the stomach. In clinical trials the pouch has been shown to be retained in the stomach for 16-24 hours.

Alternatively, the active may be conjugated to a protective modifier that allows it to withstand enzymatic dégradation in the stomach and facilitate its absorption. The active may be conjugated covalently with a monodisperse, short-chain methoxy polyethylene glycol glycolipids dérivative that is crystallized and lyophilized into the dry active pharmaceutical ingrédient after purification. Such methods are described in US5438040 and at www.biocon.com.

One may also employ a hepatic-directed vesicle (HDV) for active delivery. An HDV may consist of liposomes (^150 nm diameter) encapsulating the active, which also contain a hepatocyte-targeting molécule in their lipid bilayer. The targeting molécule directs the delivery of the encapsulated active to the liver cells and therefore relatively minute amounts of active are required for effect. Such technology is described in US2009/0087479 and further at www.diasome.corn.

The active may be incorporated into a composition containing additionally a substantially non-aqueous hydrophilic medium comprising an alcohol and a cosolvent, in association with a medium chain partial glyceride, optionally in admixture with a long-chain PEG species as described in US2002/0115592 in relation to insulin.

Alternatively, use may be made of intestinal patches as described in Shen Z, Mitragotri S, Pharm Res. 2002 Apr; 19(4) : 391-5 'Intestinal patches for oral drug delivery'.

The active may be incorporated into an erodible matrix formed from a hydrogel blended with a hydrophobie polymer as described in US Patent No. 7189414.

Suitable oral dosage levels for adult humans to be treated may be in the range of 0.05 to 5mg, preferably about 0.1 to 2.5mg.

The frequency of dosage treatment of patients may be from one to four times weekly, preferably one to two times weekly, and most preferably once weekly. Treatment will desirably be maintained over a prolonged period of at least 6 weeks, preferably at least 6 months, preferably at least a year, and optionally for life.

Combination treatments for relevant conditions may be carried out using a composition according to the présent disclosure and separate administration of one or more other therapeutics. Alternatively, the composition according to the présent disclosure may incorporate one or more other therapeutics for combined administration.

Combination thérapies according to the présent disclosure include combinations of an active compound as described with insulin, GLP-2, GLP-1, GIP, or amylin, or generally with other anti-diabetics. Thus combination thérapies including co-formulations may be made with insulin sensitizers including biguanides such as Metformin, Buformin and Phenformin, TZD's (PPAR) such as Balaglitazone,

Pioglitazone, Rivoglitazone, Rosiglitazone and Troglitazone, dual PPAR agonists such as Aleglitazar, Muraglitazar and Tesaglitazar, or secretagogues including sulphonylureas such as Carbutamide, Chloropropamide, Gliclazide, Tolbutamide, Tolazamide, Glipizide, Glibenclamide, Glyburide, Gliquidone, Glyclopyramide and Glimepriride, Meglitinides/glinides (K+) such as Nateglinide, Repaglinide and Mitiglinide, GLP-1 analogs such as Exenatide, Lixisenatide, Liraglutide, Semaglutide, dulaglutide and Albiglutide, DPP-4 inhibitors such as Alogliptin, Linagliptin, Saxagliptin, Sitagliptin and Vildagliptin, insulin analogs or spécial formulations such as (fast acting) Insulin lispro, Insulin aspart, Insulin glulisine, (long acting) Insulin glargine, Insulin detemir), inhalable insulin - Exubra and NPH insulin, and others including alpha-glucosidase inhibitors such as Acarbose, Miglitol and Voglibose, amylin analogues such as Pramlintide, SGLT2 inhibitors such as Dapagliflozin, Empagliflozin, Remogliflozin and Sergliflozin as well as miscellaneous ones including Benfluorex and Tolrestat.

Further combinations include co-administration or coformulation with leptins. Leptin résistance is a wellestablished component of type 2 diabètes; however, injections of leptin hâve so far failed to improve upon this condition. In contrast, there is evidence supporting that amylin, and thereby molécules with amylin-like abilities, as the salmon calcitonin mimetics, are able to improve leptin sensitivity. Amylin/leptin combination has shown a synergistic effect on body weight and food intake, and also insulin résistance [Kusakabe T et al].

A further preferred combination therapy includes coformulation or co-administration of the peptides of the invention with one or more weight loss drugs. Such weight loss drugs include, but are not limited to, lipase inhibitors (e.g. pancreatic lipase inhibitors, such as Orlistat), appetite suppressing amphétamine dérivatives (e.g.

Phentermine), Topiramate, Qysmia® (Phentermine/Topiramate combination), 5-HT2C receptor agonists (e.g. Locaserin), Contrave® (naltrexone/bupropion combination), glucagon-like peptide-1 [GLP-1] analogues and dérivatives (e.g. Liraglutide, semaglutide), sarco/endoplasmic réticulum (SR) Ca²⁺ ATPase (SERCA) inhibitors (e.g. sarcolipin), Fibroblast growth factor 21 [FGF-21] receptor agonists (e.g. analogs of FGF-21), and β3 adreno receptor agonists (e.g. Mirabegron). Such combinations may be used to treat an overweight condition, such as obesity.

Description of the Figures

Figure 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. Figure 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 molécule were monitored for 168 hours and compared head-to-head with a non-acylated benchmark. A) Food intake. B) Body weight change.

Figure 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 molécule were monitored for 168 hours. A-B) Food intake and Body weight of acylated KBP-066 variants. C20012

D) Food intake and Body weight of acylated KBP-062 variants. E-F) Food intake and Body weight of acylated KBP-110 variants.

Figure 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.

Figure 5: 4 hour food intake study for KBP350.

Figure 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 Figure 9A. n=9-10. +/- SEM.

Figure 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.

Figure 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.

Figure 9: ZDF HbAlc values. A) HbAlc at baseline. B) HbAlc at study end. HbAlc is measured on day -3 from study start. HbAlc is measured at study end, day 62. n=9-10. +/- SEM.

Figure 10: Oral glucose tolérance 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 20012

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-¹ were assigned with the upper limit of détection; 33.3 mmol*L-¹. The rats had not been pre-dosed with saline or KBP-066 og KBP—066A on that same day. n=9-10. +/- SEM.

Figure 11: Single dose test of KBP-305, KBP-306, KBP-307, KBP356, 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 molécule were monitored for 96 hours and compared head-to-head with one another and vehicle to détermine 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.

Figure 12: Six-week body weight loss study in HFD SD rats using KBP-066A11 compounds with different acylation length, .03, .04, and .05 acylations. Rats were treated with treated with KBP-066A11.03, KBP-066A11.04, KBP-066A11.05 or vehicle and dosed every 3^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.

Figure 13: Additional parameters from the body weight loss study in HFD SD rats using ΚΒΡ-066Ά11 compounds with different acylation length, .03, .04, or .05 acylation. Rats were treated with KBP-066A11.03, KBP-066A11.04, KBP-066A11.05 or vehicle. Rats were dosed every 3^rd day with a single s.c. injection of 4 nmol compound/kg. A) Oral glucose tolérance test. B) Incrémental 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.

Figure 14: Compétitive ligand binding assay using radio labelled salmon calcitonin (¹²⁵I-sCT) as tracer and conducted 2% sérum albumin from two different species, rat (Rattus norvegicus) and man (Homo sapiens) . As a tracer 0.25 nM ¹²⁵IsCT was used. A) Compétitive binding assay conducted in 2% 10 RSA. B) Compétitive binding assay conducted in 2% HSA. Data as +/- SEM.

Figure 15: Single dose test of KBP-356, KBP-386, KBP-387, KBP388, KBP-389, and KBP-390 for investigating the acylation position of the KBP-066 backbone. Single dose was given at t=0 15 and the effect on food intake and body weight of a single dose 3 nmol/kg of each molécule were monitored for 96 hours and compared head-to-head with one another and vehicle to détermine the optimal acylation position. A) Acute food intake in grams(g). B) Body weight change in grams(g). n=4 rats per 20 group. Data as +/- SEM.

Figure 16: Single dose test of KBP-391, KBP-312, KBP-313, KBP314, 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 25 a single dose 3 nmol/kg of each molécule were monitored for 96 hours and compared head-to-head with one another or vehicle to détermine 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.

Figure 17: Six-week body weight loss study in HFD SD rats using KBP-066 compounds with same acylation length, .03, but different position, Ail and A19. Rats were dosed every 3^rd day with a single s.c. injection of 4 nmol compound/kg KBP066A11.03 (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.

Figure 18: Additional parameters from the body weight loss study in HFD SD rats using KBP-066A11 compounds with different acylation length, .03, .04, and .05 acylations. Rats were treated with KBP-066A11.03, or KBP-066A19.03 or vehicle. Rats were dosed every 3^rd day with a single s.c. injection of 4 nmol compound/kg. A) Oral glucose tolérance test. B) Incrémental 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.

Figure 19: Investigating acylation linker of the KBP-066 backbone using single dose test of KBP-356, KBP-384, and KBP385. Single dose was given at t=0 and the effect on body weight of a single dose of 4 nmol/kg of each molécule were monitored for 96 hours and compared head-to-head with one another to détermine 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

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 complété disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the présent disclosure nor are they intended to represent that the experiments below are ail or the only experiments performed. Efforts hâve been made to ensure accuracy with respect to numbers used (e.g. amounts, température, etc.) but some experimental errors and déviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, température is in degrees Centigrade, and pressure is at or near atmospheric. In the following examples, the following materials and methods were employed.

Cells and Cell Lines

The following cell lines expressing the calcitonin, amylin and CGRP receptors were purchased and cultured according to the manufacturer's instructions.

1. Calcitonin Receptor (CTR): U2OS-CALCR from DiscoveRx (Cat. No.: 93-0566C3).

2. Amylin Receptor (AMY-R): CHO-K1 CALCR + RAMP3 from DiscoveRx (Cat. No.: 93-0268C2).

Chemicals

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.

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.

The final peptide concentration in the wells should be

100-200 μΜ, and the final ThT concentration should be 4 μΜ.

ThT is added last (10pL).

Animal models

In the animal model studies, 12 week healthy Sprague Dawley (SD) rats were used to assess the potency of the 10 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.

Acylated calcitonin mimetics

The following Tables la and lb set out the amino acid sequences of the acylated calcitonin mimetics that hâve been tested. As used therein:

acylation means Kac-(glutamic acid linker)-(C16 fatty 20 acid [palmitate]);

acylation means Kac^-(glutamic acid linker)-(C18 diacid [Octadecanedioic acid]);

acylation means Kac^-(2xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C18 25 diacid [Octadecanedioic acid]).

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

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

acylation means KAc-(2xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(016 diacid [Hexadecanedioic acid]).

acylation means KAc-(3xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(018 diacid [Octadecanedioic acid]).

acylation means KAc-(lxOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(018 diacid [Octadecanedioic acid]).

acylation means KAc-(2x0EG amino acids linked together with a glutamic acid residue attached to N-terminus)-(024 diacid [Tetracosanedioic acid]).

acylation means KAc-(2xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(026 diacid [Hexacosanedioic acid]).

acylation means KAc-(2xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(014 diacid [Tetradecanedioic acid]).

The tested calcitonin mimetics are based on the following core peptide sequences prior to modification: CSNLSTCMLGRLSQDLHRLQTYPKTDVGANAP (KBP08 9) CSNLSTC(AiB)LGRLSQDLHRLQTYPKTDVGANAP (KBP066) CGNLSTC(AiB)LGRLTQDLNKFHTFPKTDVGANAP (KBP062) CSNLSTCVLGKLSQELHKLQTYPRTDVGANAP (KBP042)

CSNLSTC(AiB)LGRLANFLVHSSNNFGAILPKTDVGANAP (KBP110) CSNLSTCMLGRLSQELHRLQTYPKTDVGANAP (KBP021)

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

Acylated	KBP Name
	Amino
5	Acid	Modifier
	01	A01
	02	A02
	03	A03
10	XX	AXX
	31	A31
	32	A32
15	Type	Name
	Acylation	Addition
	C16	. 01
	C18 diacid	. 02
	018 diacid 2*OEG	. 03
20	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
25	C24 diacid 2*OEG	. 09
	C26 diacid 2*OEG	. 10
	014 diacid 2*OEG	. 11

Thus, by way of example, the nomenclature KBP-066A11.03 30 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 1. Acylated calcitonin mimetics

cterm

-NH2

tn; 4

CM

-NH2

1

a

-NH2

en

CM

Pi

Z

KD en

<

tn en

Z

m

C

c

en en

Z

0

CM en

eu

Z

>

i—I en

<

c

C

<

C

<

C

<

Q

θ

Z

s

Z

o CM

<

Z

00 CM

0

Z

CM

>

Z

KO CM

Q

a

Q

l-l

H

1—1

l-l

I—1

l-l

m CM

Eh

Z

Eh

En

Eh

H

Z

<

S

Z

0

en CM

Z

ÊH

Z

[jz

Z

CM CM

X

Z

X

Z

i^-1 CM

Eh

Z

EH

Eh

H

EH

Eh

En

Eh

Z

H

Z

O CM

CX

ex

O

ex

O

ex

o

Z

CO

0

on r-1

Z

1-4

Z

CO

0

00

Z

rH f

Z

>

KO i^-1

Z

rH

Z

m ri

z

Q

pL|

Z

bz

Z

S

o

CX

ex

O

ex

O

Z

en

0

CO

0

CO

Z

<

CM

Z

rH

Z

ri ri

rH

Z

rH

Z

en

CM

Z

en

CM

Z

en

CM

Z

O

0

Λ

Z

rH

Z

en

CM

Z

ïi » en

CM

Z

cn^

CM

Z

00

J>

S

s

S

Z

s

X

x

X

r*

o

0

Q

o

0

o

Q

0

o

0

o

0

o

0

o

KO

Eh

H

Z

H

Z

m

0

en

CO

0

en

CO

en

CO

0

CO

0

Z

en

Z

is

Z

CM

0

CO

0

CO

0

CO

0

ri

O

o

0

o

Q

o

0

O

o

Q

0

o

Q

O

o

Q

0

Q

o

Nterm

υ

C)

ύ

U

ύ

ù

ύ

ΰ

ύ

ù

ύ

ù <

U

ύ <

ύ C

ΰ

ù

U

ù

ΰ

ù

ΰ

ΰ <

ύ

ù

KBP

346

347

349

350

351

352

353

354

355

356

357

CO lO

359

360

361

3 62

363

364

365

366

367

368

369

370

371

-NH2

en

-NH2

-NH2 rW, -¼ Λ à*· ⁴¹ \

-NH2

—T 1 en

Kar- (qlutamic acid linker)-(Cie fatty acid [palmitate] )________________________.______________________________

Kac-(qlutamic acid linker)-(Ci₈ diacid [Octadecanedioïc acid] )__

Kac-(2xOEG amino acids linked together with a glutamic acid residue attached to N-termmus) - (Ci₈ diacid rOctadecanedioic acid])________________________________

x Aminoisobutyric acid (AiB); CAS No. 62-57 7 ----------------------1

Z

eu

Z

<

Z

<

c

<

o

0

>

Q

H

Eh

H

Eh

H

(X

Di

Z

CL

eu

Z

X

Z

H

Eh

H

Eh

H

Eh

H

Oi

O

O<

CX

O

ex

Z

1-4

Z

W

M

Z

Q

ex

o

ex

0

en

Z

z

Z

en

Z

w

Z

en

Z

0

Z

d

k

Z

en

Z

>

S

Z

>

o

0

o

0

Eh

En

H

Eh

H

Eh

H

0

CO

en

0

Z

en

0

en

ω

en

C

<

c>

o

0

U <

ύ

ù

ΰ

ù

ΰ

ΰ C

ù

ΰ

rd

CM

372 I

373

374

375

376

377

CO

en

380

1

<0 U d φ e π s c •d a o d O <0

C-term	-NH2	-NH2	-NH2	-NH2	-NH2	-NH2	-NH2	-NH2	-NH2	\| -NH2 \|
en en
en cm	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
en rd		C	<		<	<	<	<	<	<
en o	Z	Z		Z	Z	Z	Z	Z	Z	Z
cm σ»		c	c	C		<			c	c
CM CO	0	0	0	0	0	0	0	0	0	ο
cm r-	>	>	>	>	>	>	>	>	>	>
CM	Q	Q	Q	Q	Q	Q	Q	Q	Q	Q
cm m	H	Eh	H	H	Eh	H	H	H	H	Eh
CM	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
cm en	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
CM CM	X	X	X	X	X	X	X	X	X	X
CM rd	Eh	H	H	H	Eh	H	H	H	Eh	Eh
CM O	O	ex	O	ex	ex	ex	ex	ex	O	ex
i—l Oï	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
rd CO	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
rd F*	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
rd	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
rd IG	Q	Q	Q	Q	Q	Q	Q	Q	Q	Q
rd	ex	ex	ex	ex	ex	ex	ex	ex	ex	O<
rd en	en	en	en	en	en	en	en	ω	en	0
rd cm	Z	Z	Z		Z	Z	Z	Z	Z	Z
rd rd		'in⁴	U>'		»*	Ch	o ^:ri	¹ r<	Z	en
rd O	0	0	0	0	0	0	0	0	O	0
Λ	Z	Z	Z	Z	Z	Z	Z	Z	en	Z
CO	X	X	X	X	X:	X	X	X	X	X
Γ-	0	0	o	o	O	o	o	o	O	O
	Eh	H	Eh	H	H	H	H	H	H	H
IC	en	en	en	0	en	en	en	en	en	0
	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
en	Z	Z	Z	Z	Z	Z	Z	Z	Z	Z
CM	en	en	ω	en	en	en	en	en	0	0
	0	0	o	O	o	O	o	O	O	O
Nterm	ù	ù	ΰ	û	ύ <	ΰ	ù	ΰ	ô	ù
c
•ri -P «J	1.04		o	1.07	.1.08	50 ’ Γ	.1. IC	.1.11	)9.01	li.o:
>1 U	S				<	^l<	'c
3ore •ptide	IP-066	i	ÎP-066	iP-066	IP-066	----------------------------------------------------------------------------------------------------------------------------1 5P-066	IP-066	3P-066	3P-066	3P-066
u Oi	LU		LU	LU
KBP	383	CM 00	381	385	384	307	306	305	354	356

-NH2

en ' te

-NH2

en X

Amnoisobutyric acid (AiB) ; CAS No. 62-57-7

linked together with a glutamic acid residue attached to N-terminus)-(C18 diacid

ds linked together with a glutamic acid residue attached to N-terminus)-(C20 diacid ) ___________________________________________________________________________________________________________________________________________________________________________________

KAc-(2xOEG ami nn acids linked together with a glutamic acid residue attached to N-terminus)-(C22 diacid [Docosanedioic acid])

KAc-(2xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(Cl6 diacid [Hexadecanedioic acid])

KAc-(3xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C18 diacid [Octadecanedioic acid])

X

CL

X

x

<

Z

Pt

<

0

>

Q

X

H

Eh

H

X

Eh

X

Eh

X

m

X

«T

X

CL

X

H

Eh

H

X

H

Eh

H

X

O

ex

CX

C»

O

ex

ι-4

Z

X

M

m

X

m

%

X

Pi

en

X

34

X

Z

X

Z

X

Z

X

en

X

<n

X

Q

K

M

t3-

ω

H

w

ω

X

CX

O<

ex

O

ex

O

ex

co

CO

co

CO

co

CO

co

CO

co

1

Z

X

<n*

X

x

X

Pi

X

en

m

X

x

X

0

X

m

X

x

X

z

Z

s

Z

a

Z

a

Z

O

0

H

Eh

X

H

X

Eh

X

H

Eh

X

x

X

x

X

co

CO

co

CO

co

CO

co

CO

co

X

x

X

Z

s

Z

z

Z

co

CO

co

CO

co

O

0

KAc-(2xOEG amino acids [Octadecanedioic acid])

ΰ <

ό

ύ <

ù

ύ <

O fi

ΰ fi

ό i

ό fi

ù fi

U fi

ΰ fi

ύ

ù fi

ΰ

ù

KAc-(2xOEG amino aci [Eicosanedioic acid]

A12.03 |

A16.03

A18.03

A19.03

A19.05

A24.03

A32.03

A09.03

Ail.03

Ail.04

Ali.05

A12.03

A16.03

A18.03

A19.03

A19.05

A24.03

A32.03

KBP-066 |

KBP-066

KBP-021

CO

CO co

389

399

390

CO

312

391

393

394

313

314

315

316

395

317

318

X

An

m

Γ*

•H Ο m •H Ό

O 4J

Ό H U m

H •o

O P

0) Λ O <0 d

d) rl ω 0) M û •H O «d •H Ό m Φ U 3 -p o G u m

rc o

-H

U

Cn

C

-H S O i4 O -H (C

O rc

O <p φ

d) > <C 47

0) £4 O -H cC

Φ 44 -P

Φ

H

O (fl m B O

-H M rC >

Φ 44 H

Ό Ή

O <c -H Ό xT

O I

G

O

I

O ω o i

O ω o

OEG-OEG-yGlu-Cl6 diacid (i.e. the 6 acylation)

c o

-H

O 05

Φ

0EG-0EG-0EG-yGlu-C18 diacid (i.e. the 7 acylation)

OEG-OEG-yGlu-C24 diacid (i.e. the 9 acylation)

INITIAL ACYLATION STUDIES (Examples 1-5)

Example 1 (Figures 1 & 5)

Single dose comparative effect of 1 acylated variants at different positions (9 position A09, 11 position Ail, 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.

KBP	Core	Position/Acylation
KBP-346	KBP-042	Ail/ 1 acylation
KBP-347	KBP-089	A18/ 1 acylation
KBP-349	KBP-089	Ail/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

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 (^Λ100 pg/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 (04hours, 4-24 hours, 24-48 hours). Body weight was measured at baseline and at 24 hours and 48 hours post s.c injection.

Acylation at positions A09, Ail and A32 with 1 acylation produced a protracted in vivo response (Figure 1) that merited further testing (see below). Positions 12 (Figure 5), 16, and 18 returned an unacceptable resuit and were not advanced into further experiments.

Example 2: β-Arrestin Assay

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 βarrestin 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 Gqcoupled receptor.

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 délétion mutant of β-gal (called enzyme accepter or EA). Activation of the GPCR stimulâtes binding of β-arrestin to the ProLink-tagged GPCR and forces complémentation 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® Détection Reagents.

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 pL cell-type spécifie medium the day prior to the experiment. To quantify the GPCRmediated β-arrestin recruitment the Pathhunter™ Détection Kit (93-0001, DiscoverX) was used and assay performed accordingly to the manufacturer's instructions.

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) 5 set out the results of the β-arrestin study.

Table 2.

β-arrestin study for the 2 acylation (Kac^-(glutamic acid linker)-(C18 diacid))

Compound	U2OS (CTR)	U2OS (CTR)	CHO-K1 (AMY-R)
Acylated KBPs 3 Acylation	β-arrestin	β-arrestin	β-arrestin
Fold Recruitment	Prolonged CTR response (10 nM)	Fold Recruitment
NO	Core Sequence	Acylation Position/Type	EC50 values (10 ⁹ M)	tAUC value 0-72h	EC50 values (ΙΟ ⁰⁹ M)
KBP-355	KBP-066	A09/2	31.2 ±4.4 (3)	147 ±004(2)	509 ±695 (3)
KBP-357	KBP-066	All/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	All/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	All/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 3. β-arrestin study for the 3 acylation (Kac~(2xOEG amino acids linked together with a glutamic acid residue attached to N-terminus)-(C18 diacid [Octadecanedioic acid])

Compound	U20S (CTR)	U20S (CTR)	CHO-K1 (AMY-R)	Food Intake
Acylated KBPs 2 Acylation	β-arrestin	β-arrestin	β-arrestin	ÛFOOD
Fold Recruitment	Prolonged CTR response (10 nM)	Fold Recruitment	Sustained Atténuation (36 nmol/kg)
NO	Core Sequence	Acylation Type	EC50 values (ΙΟ⁹ M)	tAUC value 0-72h	EC50 values (ΙΟ⁰⁹ M)	Hours (h)
KBP-354	KBP-066	A09/3	4.7±0.6 (3)	2601019 (2)	93.0 + 26(3)	4h
KBP-356	KBP-066	All/3	8.510.8 (3)	2512 + 295 (2)	12.0 + 4.0 (3)	96h
KBP-358	KBP-066	A32/3	44.215.7 (3)	1460 1 202(2)	98.6153 (3)	72h
KBP-360	KBP-062	A09/3	45.219.4(3)	182 1 006(2)	83.9 + 42 (3)	4h
KBP-362	KBP-062	All/3	13.219.6(3)	1784 1 330 (2)	14.5 + 0.2 (2)	72h
KBP-364	KBP-062	A32/3	53.318.6 (3)	1322 1 035 (2)	106 1 32 (2)	48h
KBP-366	KBP-110	A09/3	>1000 (3)	084 1 007(3)	>1000 (3)	4h
KBP-368	KBP-110	All/3	19312.9 (3)	827 1140 (3)	166143 (3)	72h
KBP-370	KBP-110	A32/3	473 1 34 (3)	635 + 077(3)	>1000 (3)	4h
KBP-373	KBP-042	A09/3	96.5117 (3)	337 (1)	263 + 7.3 (3)	4h
KBP-372	KBP-042	All/3	7.812.5 (3)	13041238 (3)	45.6 + 12(3)	96h
KBP-374	KBP-042	A32/3	49.216.4(3)	1073 (1)	151115 (4)	72h
KBP-375	KBP-089	A09/3	56.3 + 20(3)	624 (1)	232+ 27 (4)	4h
KBP-376	KBP-089	All/3	14.712.7(3)	1395 (1)	25.0 + 2.2(4)	96h
KBP-377	KBP-089	A32/3	66.0136(3)	1403 (1)	73.1 + 7.4 (4)	72h

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 acylation._______________________________

The β-arrestin studies indicated the following:

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

2) The 2 or 3 acylation at the 11 position (Ail) 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.

3) Acylated KBPS with different cores demonstrate similar potency and patterns in vitro when modified with identical acylations.

Example 3 (Figure 2)

Single dose comparative effect of A09 (KBP375), Ail (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.

KBP	Core	Annotation	Position/Acylation___
KBP-375	KBP-089	KBP-089A09.03	A9 / 3 acylation
KBP-376	KBP-089	KBP-089A11.03	Ail / 3 acylation __
KBP-377	KBP-089	KBP-089A32.03	A32 / 3 acylation

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

The animal model studies confirmed the results of the βarrestin study and demonstrated improved efficacy vis-à-vis the naked peptide:

1) Ail > A32 > A09 in terms of benefit of acylation position using KBP-089 as core peptide.

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

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 (Figure 3)

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

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

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 (^Λ 17 pg/kg), 12 nmol/kg (^Λ50 pg/kg)or 36 nmol/kg (^Λ150 pg/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 (0-4hours, 4-24 hours, 24-48 hours ... 144-168 hours) . Body weight was measured at baseline and every 24 hours post s.c injection.

The results are as follows:

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

2) Ail is a better acylation site than A32.

Example 5 (Figure 4)

Single high dose effect of All/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 (^Λ1000 gg/kg)).

KBP	Core	Annotation	Position/Acylation
KBP-372	KBP-042	KBP-042A11.03	Ail / 3 acylation
KBP-356	KBP-066	KBP-066A11.03	Ail / 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 (0-4hours, 4-24 hours, 24-48 hours ... 188-312 hours). Body weight was measured at baseline and every 24 hours post s.c injection.

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 (Figures 6-10)

Further work was performed on compound KBP-356 (KBP066Ά11.03), which comprises an AiB residue at the 8 position and the preferred acylation at the 11 position of the peptide.

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

Chronic treatment of male ZDF rats :

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

Dosage concentrations and frequency

Animais were dosed once daily with KBP-066 or saline (vehicle). Dosing with KBP-066A11.03 was performed once every 5 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 pg/kg, and compound concentration was 5, 50 or 10 500 mg/L. The dose équivalent in nmol/kg is 1.43, 14.3 and

143 nmol/kg, respectively.

ΚΒΡ-066Ά11.03: Dosage volume was 1 mL/kg, dosage concentration was 25 nmol/kg, and compound concentration was 25 mmol/L. The dose équivalent in pg/kg is 104 pg/kg. 15

Treatment groups in nmol/kg

Intervention	Compound	Dosing volume	Dosing conc.	Compound conc.	Admin. 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 pmol/L	SC.	10
14.3 nmol/kg	KBP-066	1 mL/kg	14.3 nmol/kg	14.3 pmol/L	SC.	10
143 nmol/kg	KBP-066	1 mL/kg	143 nmol/kg	143 pmol/L	SC.	10
25.0 nmol/kg	KBP- 066A11.03	1 mL/kg	25.0 nmol/kg	25.0 pmol/L	SC.	10

Treatment groups in pg/kg

Intervention	Compound	Dosing volume	Dosing conc.	Compound conc.	Admin. route	n
Vehicle	Saline	1 mL/kg	NA	NA	SC.	10
5 pg/kg	KBP-066	1 mL/kg	5 pg/kg	5 mg/L	SC.	10
50 pg/kg	KBP-066	1 mL/kg	50 pg/kg	50 mg/L	SC.	10
500 pg/kg	KBP-066	1 mL/kg	500 pg/kg	500 mg/L	SC.	10
104 pg/kg	KBP- 066A11.03	1 mL/kg	104 pg/kg	104 mg/L	SC.	10

Weekly total dose per treatment group:

pg/kg KBP-066 equals to 35 pg/kg/week or 10 nmol/kg/week 50 pg/kg KBP-066 equals to 350 pg/kg/week or 100.4 nmol/kg/week

500 pg/kg KBP-066 equals to 3500 pg/kg/week or 1004 nmol/kg/week nmol/kg KBP-066 equals to 243.4 pg/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

DAY -3: HbAlc measurement

DAY 1: (first day of study), rats were fasted for 6h 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 (llh fasting). HblAc is measured during the OGTT at t=120 or t=180.

DAY 62: FBG + blood sample (6 h fast)

Food intake

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

Fasting Blood Glucose

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 (25G needle).

HbAlc

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

Oral Glucose Tolérance Test

A glucose tolérance test (OGTT) was performed after eight weeks of treatment. Body weight from the day prior was used to calculate glucose dose given. Animais were fasted for 11 h. Heat was applied app. 45 min prior to time point -30 min (see below figure). Animais were pre-dosed with KBP-066, KBP066A11.03 or saline during the first OGTT but not in the second OGTT, hence (C) in the below figure.

OGTT chart

-30 0 15 30 60 120 minutes

B	B	B B	B	B
BG	BG	BG BG	BG	BG
(C)	G
B =	blood	sample (EDTA), app.	200-300	pL

BG = blood glucose.

G = glucose (oral. 1g glucose/kg BW, 2 mL/kg)) C = compound (or saline) (SC.)

RESULTS

Figure 6A+B, Accumulated food intake

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

Figure 7A+B, Body weight

Ail 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 (Figure 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 dépendent as well as being dépendent upon the type of compound used in the treatment group (Figure 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.

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

Figure 8, Fasting blood glucose

As the ZDF vehicle rats became progressively sicker and failed to maintain FBG, ail 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 diabètes. The non-acylated KBP-066 reduced FBG in a dose dépendent 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.

Figure 9, HbAlc at baseline and study end

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

Figure 10, Oral Glucose Tolérance Test (OGTT)

An oral glucose tolérance test was conducted after eight weeks of treatment and results are illustrated in Figure 10A. Due to treatment-induced différences in FBG the individual OGTT curves are markedly different. This différence is highlighted in the calculated tAUC values (Figure 10B). Ail treatment groups hâve 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 (Figure 10B).

In conclusion, the collective data show that acylated KBP66A11.03 given in a s.c dose régiment every three days hâve 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) Acylation of position A09 with 1 acylation produced a sustained prolonged in vivo activity that merited further testing (Figure 1A-F).

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).

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 (Figure 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 considération.

Position Ail (acylation at the 11 position of the peptide)

Acylation of position Ail with the 1 acylation produced a sustained prolonged in vivo activity that merited further testing (Figure 1A-F).

Acylation of position Ail with acylations 2 and 3 resulted in the best assayed EC50 value on both the CTR and 20 AMYR receptor, and producing the highest prolonged response values (tAUC) across ail core peptides tested (Table 3-4).

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

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

To further investigate the potency of the Ail position with the 3 acylation, KBP-042 and KBP-066 acylated at position Ail 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 (Figure 4A-D).

Acylation 3 at position Ail attenuated food intake for more than 120 hours returning to vehicle food consumption levels after ^Λ144 hours for both KBP-042 (Figure 4A) and KBP066 (Figure 4C). Treatment mediated body weight loss peaked after 96 hours and body weight returned to baseline levels after ^Λ240 hours for both KBP-042 (Figure 4B) and KBP-066 (Figure 4D).

In conclusion, Ail 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)

A12 position with a 1 acylation produced a worse resuit In vivo in the 4h food intake study when compared to the vehicle (Figure 5).

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)

A16 position with a 1 acylation demonstrated no prolonged activity in vivo (Figure 1A-F).

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)

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, 48h, Figure 1F).

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)

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 (Figure 1A-F).

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

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

However, the position was inferior to Ail in ail tested 2 and 3 acylations during in vivo studies at équivalent doses (Figure 2A-B, Figure 3A-F).

In conclusion, A32 was a médiocre position in terms of preserving ligand potency and improving in vivo efficacy using 1, 2 and 3 acylations when compared to position Ail.

FURTHER ACYLATION STUDIES (Examples 7-12) Example 7: β-Arrestin and Thioflavin T Assays

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 ΙμΜ-O.l nM and vehicle).

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

Thus, ThT in combination with a fluorescent plate reader is an idéal 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, Fr0kjaer S, Brange J, Vyas S, et al. Effect of environmental factors on the kinetics of insulin fibril formation: élucidation of the molecular mechanism.

Biochemistry. 2001; 40 (20): 6036—46.1) for measuring insulin fibrillation.

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

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 émission wavelength at 480 nm.

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 ail other reads. Alternatively, the plate is read after the following incubation times; 0, 1, 2, 4 and 24 hours.

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.

In this filing four fibrillations tiers hâve been defined based on the 18h 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

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

Table 4.1. β-arrestin study for different acylations length (KAc-(glutamic acid linker)~(C14 to C26 diacid))______________

Compounds	U2OS (CTR)	CHO-K1 (AMY-R)	Peptide Fibrillation	Food Intake
Acylated KBPs	β-arrestin	β-arrestin	Thioflavin T	AFOOD
Fold Recruitment	Fold Recruitment	AFIuorescence 18h Assay	Sustained Atténuation (4 nmol/kg)
NO	Core Sequence	Acylation Type	EC50 values ¢10 ⁹ M)	EC50 values (10⁹ M)	Score	Hours (h)
356	KBP-066	Ail.03	3.0 ± 2.4 (23)	7.2 ±4.7 (26)	None (10)	72-96h
383	KBP-066	Ail.04	33.0 ± 10.3 (3)	16.6 ±2.9 (3)	None (3)	96h
382	KBP-066	Ail.05	56.1 ±10.6 (3)	23.3 ±4.2 (3)	None (3)	96h
381	KBP-066	Ail.06	3.9 ±0.9 (3)	1.0 ±0.5 (3)	None (3)	24-48h
307	KBP-066	Ail.09	26.6 ±6.1 (3)	149 ±127(4)	None (3)	72h
306	KBP-066	Ail.10	65.7 ±2.9 (3)	82.7 ±6.6 (4)	None (3)	48h
305	KBP-066	Ail.11	2.1 ±1.0 (3)	0.95 ± 0.2 (4)	None (3)	24h
Tant4.1; in vitro peptide screening characteristics

Table 4.2. β-arrestin study for different acylations positions using backbone (KBP-066) and 3 acylation (KAc5 (glutamic acid linker)-(C18 diacid))_____________

Compounds	U20S (CTR)	CHO-K1 (AMY-R)	Peptide Fibrillation	Food Intake
Acylated KBPs	β-arrestin	β-arrestin	Thioflavin T	ÛFOOD
Fold Recruitment	Fold Recruitment	ÛFluorescence 18h Assay	Sustained Atténuation (4 nmol/kg)
NO	Core Sequence	Acylation Type	EC50 values (ΙΟ⁹ M)	EC50 values (10 ⁹ M)	Score	Hours (h)
354	KBP-066	A09.03	4.7 ±0.6 (3) *	93.0 ±26 (3) *	None (3)	4h
356	KBP-066	Ail.03	3.0 ± 2.4 (23)	7.2 ±4.7 (26)	None (10)	72-96h
386	KBP-066	A12.03	56.5 ±21.1 (3)	98.9 ±74.8 (3)	None (3)	0-4h
387	KBP-066	A16.03	25.4 ±9.9 (3)	21.1 ±9.9 (3)	Low (3)	48h
388	KBP-066	A18.03	22.8 ± 11.6 (3)	34.7 ±33.8 (3)	None (3)	72h
389	KBP-066	A19.03	9.4 ±3.2 (3)	6.5 ±3.1 (3)	None (3)	72-96h
390	KBP-066	A24.03	11.1 ±2.8 (3)	6.0 ±3.3 (3)	None (3)	72h
358	KBP-066	A32.03	44.2 ±5.7 (3) *	98.6 ±53 (3) *	None (3)	72h
Table 4.2: In vitro peptide screening characteristics * Data from original patent filing

Table 4.3. β-arrestin study for different acylations positions using backbone (KBP-021) and 3 acylation (KAc-

10	(glutamic acid linker	-(C18 diacid)) __
	Compounds	U2OS (CTR)	CHO-K1 (AMY-R)	Peptide Fibrillation	Food Intake
Acylated KBPs	β-arrestin	β-arrestin	Thioflavin T	ÛFOOD
Fold Recruitment	Fold Recruitment	ÛFluorescence 18h Assay	Sustained Atténuation (4 nmol/kg)
NO	Core Sequence	Acylation Type	EC50 values (10 ⁹ M)	EC50 values (10 ⁹ M)	Score	Hours (h)
312	KBP-021	A09.03	16.7 ±2.9 (3)	1528±1201(3)	Low (3)	4h
391	KBP-021	Ail.03	12.5 ± 10.9 (9)	9.0 ±2.2 (5)	None (3)	72-96h
393	KBP-021	Ail.04	55.1 ±48.9 (3)	32.8 ± 14.6 (4)	Low (3)	72-96h____
394	KBP-021	Ail.05	56.9 ±33.4 (3)	53.9 ±21.2 (4)	Low (3)	24h
313	KBP-021	A12.03	314 ±116 (3)	330 ±124 (4)	None (3)	4h
314	KBP-021	A16.03	183 ±149 (3)	428 ±175 (5)	None (3)	4h
315	KBP-021	A18.03	19.2 ±6.6 (3)	44.9 ±6.6 (5)	Medium (3)	48h
316	KBP-021	A19.03	2.5 ±1.4 (3)	6.1 ± 1.8 (5)	High (3)	72-96h

395	KBP-021	A19.05	95.2 ±95.2 (3)	32.9 ± 12.7 (4)	High (3)	72-96h
317	KBP-021	A24.03	12.8 ±12.8 (3)	31.5 ±7.4 (5)	None (3)	24h
318	KBP-021	A32.03	197 ±83.6 (3)	301 ±225 (5)	Low (3)	4h
Table 4.3: In vitro peptide screening characteristics

Table 4.4. β-arrestin study for different acylations linkers using same backbone (KBP-066) and same acylation(C18 diacid))

Compounds	U2OS (CTR)	CHO-K1 (AMY-R)	Peptide Fibrillation	Food Intake
Acylated KBPs	β-arrestin	β-arrestin	Thioflavin T	AFOOD
Fold Recruitment	Fold Recruitment	AFIuorescence 18h Assay	Sustained Atténuation (4 nmol/kg)
NO	Core Sequence	Acylation Type	EC50 values (10 ⁹ M)	EC50 values (ΙΟ ⁹ M)	Score	Hours (h)
385	KBP-066	Ail.07	20.1 ±11.1 (3)	9.3 ± 1.7 (2)	None (3)	72h
384	KBP-066	Ail.08	29.7 ±29.2 (3)	6.9 ±1.1 (3)	Low (3)	72h
356	KBP-066	Ail. 03	3.0 ±2.4 (23)	7.2 ±4.7 (26)	None (10)	72-96h
Table 4.4: In vitro peptide screening characteristics

RESULTS - Acylation Length

In terms of in vitro potency as a function of acylation length there was a clear corrélation 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.

None of the tested acylated peptides in this sériés using the KBP-066 backbone had any fibrillation issues.

RESULTS - Acylation Position on the KBP-066 Backbone

EC50 values on the CTR and AMYR on this sériés 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. Ail, A19 and A24 ail hâve EC50 values on both receptors in the 5xl0~⁹M range as the only ones, whereas ail other tested positions are impaired in comparison. The increased potency of Ail, A19 and A24 appears to translate into improved in vivo efficacy for the KBP-066 backbone (see Figure 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.

Fibrillation does not appear to be an issue for the KBP066 backbone at most positions, as only one peptide (KBP066A16.03 (387)) produced a Low score in the ThT assay.

RESULTS - Acylation Position on the KBP-021 Backbone

EC50 values on the CTR and AMYR for this sériés 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. Ail and A19 both hâve EC50 values on both receptors in the 5xl0~⁹M range as the only ones, whereas ail other tested positions are impaired in comparison. The increased potency of Ail and A19 also appear to translate into improved in vivo efficacy for the KBP-021 backbone (see Figure 16 described infra).

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.

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

RESULTS - Acylation linker

EC50 values on the CTR and AMYR for this sériés 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-yGLU linker (356) hâve an almost 10-fold better EC50 10 on the CTR compared to OEG-OEG-OEG-yGLU (385) and OEG-yGLU (384), however, ail linkers hâve very similar EC50 in the 5xl0~⁹ M range on the AMYR.

In terms of fibrillation, the shortest linker, OEG-yGLU (384), produced a low score in the ThT assay, whereas the 15 two other linkers produce a None score.

Example 8: (Figure 11)

Single dose comparative effect of several acylated variants (3, 4, 5, 6, 9, 10, 11) at the same position and backbone, Ail 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.

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

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 (^Λ10-11 pg/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 (0-4hours, 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 inj ection.

Figure 11 results - food intake and body weight

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.

Hence, acylation 3, 4, and 5 are ail 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^rdday dosing in rodents appears to translate into once weekly dosing in man.

Example 9 (Figure 12, 13 and 14)

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, Ail and KBP066 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.

KBP	Core	Acylation length	Annotation	Position/Acylation
KBP-356	KBP-066	C18 diacid	KBP-066A11.03	All/3 acylation
KBP-383	KBP-066 1	C20 diacid	KBP-066A11.04	Ail / 4 acylation
KBP-382	KBP-066	C22 diacid	KBP-066A11.05	Ail / 5 acylation

Rats were caged two and two and were randomized by weight into treatment groups (Vehicle (0.9% NaCl), KBPs (doses: 3 nmol/kg (^Λ14 pg/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

Rats were delivered to the animal facility of Nordic Bioscience at twelve weeks of âge 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

Animais 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.

The dose équivalent in pg/kg was ^Λ14 pg/kg. Weekly total dose per treatment group: 4 nmol/kg KBP equals to 28 nmol/kg/week or ^Λ100 pg/kg/week

Collection of test results

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 tolérance test

DAY 35: Termination + adipose tissue weighed 5

Oral Glucose Tolérance Test

A glucose tolérance test (OGTT) was performed after five weeks of treatment. Body weight from the day prior was used to calculate glucose dose given. Animais were fasted for 11 10 h. Heat was applied app. 45 min prior to time point -30 min (see below figure). Animais were dosed with KBPs or vehicle the day before the OGTT.

OGTT chart

0 15 30 60 120 minutes

BG BG BG BG BG BG 20 G

BG = blood glucose.

G = glucose (oral. 1g glucose/kg BW, 2 mL/kg)) 25

White Adipose Tissue (WAT) Weighing

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.

Figure 12 results, food intake and body weight

Figure 12 shows the change in food intake and body weight over time during the chronic study as a function of treatment. Figure 12A shows the dynamic in food intake between acylation 35 3, 4 and 5, whereas Figure 12B shows the body weight loss mediated by acylation 3, 4 and 5. It is évident that ail three acylations give a significant réduction in body weight after 5 weeks of treatment, however, there are no différences between acylation 3, 4 and 5 in terms on efficacy on body weight. 5

Figure 13 results, OGTT and adipose tissue

Figure 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 (Figure 13C-E) and the body weight as study end (Figure 13F). Treatment with acylation 3 resulted in a significant réduction in iAUC (OGTT), epididymal WAT size, perinal WAT size, and body weight as study. Treatment with acylation 4 and 5 resulted in a significant réduction 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 différences between treatment groups.

Figure 14 results, Compétitive 1-125 sCT ligand binding

To investigate whether the improved efficacy in the acute setting of acylation 4 and 5 could be translated to man, a compétitive ligand binding assay was conducted to explore acylation binding to sérum albumin in rodent and man. Figure 14 shows the compétitive binding of KBP-066A11.03 and KBP066A11.05 with radio-labelled 1-125 salmon calcitonin (NEX423, Perkin Elmer) in 2% sérum albumin from rodents (RSA) (Figure 4A) or 2% sérum albumin from humans (HSA)(Figure 4B). When the assay is conducted 2% HSA there are no différences in EC50 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 (Figure 15)

Single dose comparative effect of 3 acylated variants at different positions (9 position A09, 11 position Ail, 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.

KBP	Core	Annotation	Position/Acylation
KBP-354	KBP-066	KBP-066A09.03	A9 / 3 acylation
KBP-356	KBP-066	KBP-066A11.03	Ail / 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

Rats were single caged four days prior to the test. Rats 15 were randomized by weight into eight groups (Vehicle (0.9% NaCl), KBPs (doses: 4 nmol/kg (^Λ10-11 pg/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 20 intervals (0-4hours, 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.

Figure 15 results - Food intake and body weight

In terms of position on backbone, the KBP-066 results are as follows. At 4 nmol/kg in an acute setting (Figure 5), position Ail 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 5 like a disadvantageous position to acylate as it appears to somewhat interfère with the DACRA mediated efficacy on both food intake and body weight.

Based on these data. Ail and A19 are the preferred positions to acylate backbone KBP-066.

Figure 16 results- Food intake and body weight

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

KBP	Core	Annotation	Position/Acylation
KBP-312	KBP-021	KBP-021A09.03	A9 / 3 acylation
KBP-391	KBP-021	KBP-021A11.03	Ail / 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

At 3 nmol/kg in an acute setting (Figure 6), position Ail 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 Ail and A19. Position A24 was better than vehicle, but nowhere near what was observed for the KBP-066 backbone. Position A16, A12 and A09 ail 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.

However, as the in vitro characteristics table 4.3 shows, A19 and A18 hâve major issues in terms of fibrillation potential in combination with KBP-021, which make Ail the preferred position to acylate when it cornes to backbone KBP021.

Example 11 (Figure 17 and 18)

Further work was conducted on the best performers from the acute testing, acylated positions (Ail 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.

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.

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

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 (^Λ14 pg/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.

Figure 17 results - Food intake and body weight for Ail vs Al9 in a chronic setting

Figure 17 shows the change in food intake (Figure 17A) and in body weight (Figure 17B) over time during the chronic study as a function of treatment. It is évident that Ail and A19 both supress food intake in a similar fashion and give a significant réduction in body weight after 5 weeks of treatment, however, there is no différence between position Ail and A19 in terms on efficacy on body weight-.

Figure 18 results, OGTT and adipose tissue

Figure 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 (Figure 18C-E) and the body weight as study end (Figure 18F). Treatment with position Ail resulted in a significant réduction in iAUC (OGTT), epididymal WAT size, perirenal WAT size, and body weight as study. Treatment with position A19 resulted in a significant réduction 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 Ail performed slightly better against vehicle compared to Position A19, but there was no significant différence between treatment groups.

Example 12 (Figure 19)

Single dose comparative effect of three acylated linker variants (3, 7 and 8) at the same position and backbone, Ail 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 25 prior to the experiment.

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

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 (^Λ13-14 pg/kg)). They were 30 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 (0-4hours, 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 inj ection.

Figure 19 results - food intake and body weight

Ail three linkers tested worked well in an acute setting and ail attenuated food intake (Figure 19A) for up to 72 hours before rebounding. Acylation 3 was slightly better than 7 and 8 after 96 hours. This was évident on the corresponding body weight loss (Figure 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. Furthermore, in terms of fibrillation potential (Table 4.4), acylation 8 appear to hâve some minor tendencies that could complicate further development of compound using that type of acylation.

SUMMARY OF RESULTS OF EXAMPLES 7-12

Acylations Length

The collected data from Figure 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.

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

Acylation position

The collected data from Figure 15, 17 and 18 demonstrated that A19 may hâve a slight edge in an acute setting, whereas Ail has a slight edge in a chronic setting when using the KBP066 backbone and 3 acylation.

Neither Ail nor A19 had any issues with fibrillation when combined with the KBP-066 backbone (Table 4.2)

Overall, these data suggest that the acylations position Ail 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.

Hence Ail and A19 are the preferred positions for acylating KBP-066.

Similarly, based on Table 4.3 and Figure 16 it is évident that Ail and A19 are the two best acylation positions when combined with KBP-021.

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

Acylation Linker

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

In this spécification, 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' 5 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'. Ail prior teachings acknowledged above are hereby incorporated by reference.

Claims

1. A calcitonin mimetic that is 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 ε-amino group of said lysine residue is acylated with an acyl group selected from any one of the following:

Ci6 or longer fatty acid with an optional linker, or Ci6 or longer fatty diacid with an optional linker.

2. The calcitonin mimetic of claim 1, wherein the calcitonin mimetic is a calcitonin mimetic of formula (I) (a) :

CX₂X3LSTCX₈LGKac. ..

wherein

X2 = A, G or S

X3 = N or S χ₈ = Μ, V or α-aminoisobutyric acid (AiB) and wherein K_Ac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following:

Ci6 or longer fatty acid with an optional linker, or C16 or longer fatty diacid with an optional linker.

3. The calcitonin mimetic of claim 1, wherein the calcitonin mimetic is a calcitonin mimetic of formula (D (b) :

CX2X3LSTCX8LGX11X12X13X14X15X16X17X18KAC... wherein

X₂ = A, G or S

X₃ = N or S

Xg = Μ, V or α-aminoisobutyric acid (AiB)

X11 = R, K, T, A or Ka<

X12 = L or Y

5 X13 = S, T, w or Y

X14 = Q, K, R or A

X15 = D, E or N

X16 = L or F

X17 = H or N

10 X18 = R, K or N and wherein Kac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following:

016 or longer fatty acid with an optional linker, or

15 C16 or longer fatty diacid with an optional linker.

4 . The calcitonin mimetic of any one of the preceding claims, wherein the calcitonin mimetic is a calcitonin mimetic of formula (II):

2 0 CX2X3LSTCX8LGX11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27GX29X30X31P wherein

X2 = A, G or S

X₃ = N or S

X₈ = Μ, V or a-aminoisobutyr ic acid (AiB)

25 Xll = Kacz Rz K, T or A

X12 = L or Y

X13 = S, T, W or Y

X14 = Qz Kz R or A

X15 = D, E or N

30 X16 = L or F

X17 = H or N

X18 = R, K or N

X19 = Kac, L, F or K X20 = Q, H or A X21 = T or R X22 = Y or F X23 = S or P X₂4 = G, K, Q or R X25 = T, I or M X26 = S, N, D, G or A X27 = T, V, F or I X29 = S, A, P or V X30 = N, G or E X31 = A, T or S wherein either Xn is Kac and/or X19 is Kac, and wherein K_Ac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following: Ci6 or longer fatty acid, Ci6 or longer fatty diacid, Iinker-Ci6 or longer fatty acid, or Iinker-Ci6 or longer fatty diacid.

5. The calcitonin mimetic of claim 4, wherein the calcitonin mimetic of formula (II) is: CX2X3LSTCX8LGX11LX13X14X15LX17X18X19X20TX22PX24TDVGANAP wherein X₂ = A, G or S X3 = N or S Xg = Μ, V or AiB Xn = Kacr R, K, T or A X13 = T, S or Y X14 = Q or A X15 = D or E

X17 = H or N

Xi₈ = R or K

X19 = Kac, L, F or K

X20 = Q, H or A

5 X22 = Y or F

X24 = K, Q or R wherein either Xn = Kac and/or X19 = Kac, and wherein K_Ac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group

10 selected from any one of the following:

Ci6or longer fatty acid,

Ci6 or longer fatty diacid,

Iinker-Ci6 or longer fatty acid, or Iinker-Ci6 or longer fatty diacid. 15

6. The calcitonin mimetic of claim 4 or 5, wherein X2 is S and X₃ is N; or X₂ is G and X₃ is N; or X₂ is A and X3 is S.

20

7 . The calcitonin mimetic of any one of claims 4 to 6 r wherein - X11 is Kac, X17 is H, X18 is K, X19 is L and X20 is Q or

A; or - X11 is Kac, X17 is H, X18 is R, X19 is L and X20 is Q or

25 A; or - X11 is Kac, X17 is N, X18 is K, X19 is F and X20 is H or

A; or - X11 is Kac, X17 is N, Xi8 is R, X19 is F and X20 is H or

30 A; or - X11 is R or K, X17 is H, Xi8 is K, Xi 9 is Ka< ₃ and X20 is

Q or A; or

- Xn is R or K, Xi₇ is H, Xi₈ is R, X19 is Kac and X20 is Q or A; or

- Xn is R or K, X17 is N, Xis is K, X19 is Kac and X20 is H or A; or

- Xn is R or K, X17 is N, Xis is R, X19 is Kac and X20 is H or A.

8. The calcitonin mimetic of any one of claims 4 to 7, wherein X2 is S, X3 is N, Xn is Kac, X13 is S, X17 is H, X18 is K or R, X19 is L, X20 is Q or A and X22 is Y; or wherein X2 is S, X3 is N, Xn is R or K, X13 is S, X17 is H, Xi8 is K or R, X19 is Kac, X20 is Q or A and X22 is Y.

9. The calcitonin mimetic of any one of claims 4 to 7, wherein X2 is A, X3 is S, Xn is Kac, X13 is S, X17 is H, Xi8 is K or R, X19 is L, X20 is Q or A and X22 is F; or wherein X2 is A, X3 is S, Xn is R or K, X13 is S, X17 is H, Xi8 is K or R, X19 is K_Ac, X20 is Q or A and X22 is F.

10. The calcitonin mimetic of any one of claims 4 to 7, wherein X2 is G, X3 is N, Xn is Kac, X13 is T, X17 is N, X18 is K or R, X19 is F, X20 is H or A and X22 is F; or wherein X2 is G, X3 is N, Xn is R or K, X13 is T, X17 is N, Xi8 is K or R, X19 is K_Ac, X20 is H or A and X22 is F.

11. The calcitonin mimetic of claim 1, wherein the calcitonin mimetic is a 33mer peptide in accordance with formula (III):

CSNLSTCX₆LGX₇LSQDLHRX₈QTYPKXiTX₅VGANAP or wherein the calcitonin mimetic is a 35mer peptide in accordance with formula (IV): CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3TX5VGANAP or wherein the calcitonin mimetic is a 36mer peptide in accordance with formula (V): CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3X4TX5VGANAP or wherein the calcitonin mimetic is a 37mer peptide in accordance with formula (VI): CSNLSTCX6LGK_AcLZXiX2X3X4TX₅VGANAP wherein each of Xi to Χ4 is any amino acid, with the proviso that at least one of Xi to X4 is a basic amino acid residue, and/or at least two of Xi to X4 are independently a polar amino acid residue or a basic amino acid residue, and/or at least one of Xi to X4 is a Gly residue, and wherein none of Xi to X4 is an acidic residue; wherein X5 is D or N; wherein Χθ is AiB or M; wherein either X7 is Kac and Xs is L, or X7 is R or K and Xs is Ka_c; wherein Z is selected from SQDLHRLSNNFGA, SQDLHRLQTYGAI or ANFLVHSSNNFGA; and wherein K_Ac is a lysine residue wherein the side chain ε-amino group is acylated with an acyl group selected from any one of the following: Ci6 or longer fatty acid, Ci6 or longer fatty diacid, linker-Ci₆ or longer fatty acid, or linker-Cis or longer fatty diacid.

12 . The calcitonin mimetic of claim 11, wherein at least one of Xi or X4 is a basic amino acid residue.

13. The calcitonin mimetic of claims 11 or 12, wherein at least one of Xi or X4 is a basic amino acid residue, and at least two of Xi to X4 are independently a polar amino acid residue or a basic amino acid residue, and none of Xi to X4 is an acidic residue.

5

14. The calcitonin mimetic of claims 11 to 13, wherein at least three of Xi to X4 are independently a polar amino acid residue or a basic amino acid residues, and none of Xi to X4 is an acidic residue.

10

15. The calcitonin mimetic of claims 11 to 14, wherein ail of Xi to X4 are independently a polar amino acid residue or a basic amino acid residue, and none of Xi to X4 is an acidic residue.

15

16. The calcitonin mimetic of claims 11 to 15, wherein ail of Xi to X4 are independently a polar amino acid residue or a basic amino acid residue, at least three of Xi to X4 are basic amino acid residues, and none of Xi to X4 is an acidic residue.

17. The calcitonin mimetic of claims 11 to 16, wherein the basic amino acid residues are selected from Arg, His or Lys, and/or the polar amino acid residues are selected from Ser, Thr, Asn, Gin or Cys.

18. The calcitonin mimetic of claims 11 to 17, wherein Xi is selected from Asn, Phe, Val, Gly, Ile, Leu, Lys, His or Arg;

X2 is selected from Ala, Asn, His, Leu, Ser, Thr, Gly

30 or Lys;

X₃ is selected from Ala, Phe, Ile, Ser, Pro, Thr, Gly or Lys; and/or

X₄ is selected from Ile, Leu, Gly, His, Arg, Asn, Ser, Lys, Thr or Gin;

with proviso that at least one of Xi or X4 is a basic amino acid residue, and/or at least two of Xi to X4 are

5 independently a polar amino acid residue and/or a basic amino acid residue, and/or at least one of Xi to X4 is a Gly residue.

19. The calcitonin mimetic of daims 11 to 18, wherein Xi

10 is selected from Asn, Gly, Ile, His or Arg;

X2 is selected from Asn, Leu, Thr, Gly or Lys;

X3 is selected from Phe, Pro, Ile, Ser, Thr, Gly or

Lys; and/or

X₄ is selected from Gly, His, Asn, Ser, Lys, Thr or

15 Gin;

with proviso that at least one of Xi or X4 is a basic amino acid residue, and/or at least twc of Xi to X4 are independently a polar amino acid residue and/or a basic amino acid residue, and/or at least one of Xi to X4 is

20 a Gly residue.

20. The calcitonin mimetic of daims 11 to 19, wherein the calcitonin mimetic in accordance with formulae (III)(V) comprises one or more of the following conservative

25 substitutions:

- Asp residue at position 15 of the peptide is substituted with Glu;

- Arg residue at position 18 of the peptide is substituted with Lys; and/or

30 - Lys residue at position 24 of the peptide is substituted with Arg.

21. The calcitonin mimetic of claims 11 to 19, wherein the calcitonin mimetic in accordance with formulae (VI), wherein the Z component of the peptide of formula (VI) is SQDLHRLQTYGAI or SQDLHRLSNNFGA, comprises one or more of the following conservative substitutions: - Asp residue at position 15 of the peptide is substituted with Glu; and/or - Arg residue at position 18 of the peptide is substituted with Lys.

22 . The calcitonin mimetic of claims 1 to 21, 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: JL ^{1 J} o wherein n is from 1 to 10.

23. The calcitonin mimetic of claim 22, wherein the OEG amino acid linker comprises two to six OEG amino acids linked together.

24 . The calcitonin mimetic of claim 22 or 23, 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. The calcitonin mimetic of any one of claims 22 to 24, wherein n is 1.

26. The calcitonin mimetic of any one of claims 22 to 24, wherein the OEG amino acid linker is selected from any one of the following:

^H O O H ^H°Y° H ο ^H%° _H 5 ° JL o H 0^0 Y H 2 >, H Ν'^Λ^^^^Ν·^^Ο'^χ^°^Αν^χ^°'^Ο'^ιΤ¹JL ο H O O^OH h ο γ JL O HO h O h 9 ψ |/V^Û^0^^N^0‘^a^⁰'^ ⁵ H 0 H 0^0 7 H O H H N^^sZNi^N'^/sO^zx'^OYn^xx-’°^sZ'O'Ni^N^>'O'^Q' X 0 H 0 o^oh _h H n^xXx^V^N'^O-^^O^^z*'O^^^O^^x'O^-'^O-^-^O'^ 10 Ua o O^o H H Ο V H O , φ ^Ν'_γζ''Ύ^Ι Ν^χίχ^ν θ'—νθχΛ Ν^^θ^-^Ο^ΥιΑ 1 η ο Η Ο Η 0^0 Ο^ΟΗ _Η 0%°^Η Η 0 0α 0 A Η ο Η Ο^ΟΗ 27. The calcitonin mimetic of claim 26, HO te Y H o H Y— 15 is JL O H O ,0^0^ O O , •θΥν^Ον^ο· «A H O 0 .^O^^A 0 0 wherein the linker

28 . The calcitonin mimetic of any one of claims 1 to 27, wherein the acyl group is selected from Cis or longer fatty acid, Cis or longer fatty diacid, linker-Cis or longer fatty acid, or linker-Cis or longer fatty diacid.

29. The calcitonin mimetic of any one of claims 1 to 28, wherein the acyl group is selected from any one of the following: C18 to C30 fatty acid, Cis to C30 fatty diacid, linker-Cis to C30 fatty acid, or Iinker-Ci6 to C30 fatty diacid.

30. The calcitonin mimetic of any one of claims 1 to 29, wherein the acyl group is selected from any one of the following: Cis to C22 fatty acid, Cis to C22 fatty diacid, linker-Cis to C22 fatty acid, or linker-Cis to C22 fatty diacid.

31. The calcitonin mimetic of any one of claims 1 to 30, wherein K_Ac is acylated with a linker-fatty diacid, wherein the fatty diacid is a Cis to C22 fatty diacid H O^O Y h 0 and the linker is JL O H O

32. The calcitonin mimetic of any one of claims 1 to 31, wherein the peptide is selected from any one of the following:

CSNLSTCMLGKacLSQDLHRLQTYPKTDVGANAP CSNLSTC (AiB) LGKacLSQDLHRLQTYPKTDVGANAP CGNLSTC (AiB) LGKacLTQDLNKFHTFPKTDVGANAP CSNLSTCVLGKacLSQELHKLQTYPRTDVGANAP CSNLSTCMLGKacLSQELHRLQTYPKTDVGANAP CASLSTCVLGKacLSQDLHKLQTFPKTDVGANAP CASLSTCMLGKacLSQDLHKLQTFPKTDVGANAP CGNLSTCMLGKacLSQDLNKFHTFPQTDVGANAP

CSNLSTC (AiB) LGKacLANFLVHSSNNFGAILPKTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKHTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKHSSTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKHSSNTDVGANAP CSNLSTCMLGKacLSQDLHRLSNNFGAILSSTNVGANAP CSNLSTCMLGKacLSQDLHRLQTYGAILSPKTDVGANAP CSNLSTCMLGKacLANFLVHSSNNFGAILPKTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKILSSTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKGLITTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKNNFGTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKRTTQTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKHTTNTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKHGGQTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKHKKNTDVGANAP CSNLSTCMLGKacLSQDLHRLQTYPKHKKHTDVGANAP CSNLSTC (AiB) LGRLSQDLHRKacQTYPKTDVGANAP CSNLSTCMLGRLSQELHRKacQTYPKTDVGANAP and wherein Ka_c is as defined in any one of claims 1 to 32.

33. The calcitonin mimetic of any one of claims 1 to 32, wherein the peptide is selected from any one of the following:

AcCSNLSTCMLGKacLSQDLHRLQTYPKTDVGANAP-NH₂

AcCSNLSTC (AIE) LGKa_cLSQDLHRLQTYPKTDVGANAP-NH₂ AcCGNLSTC (AIE) LGKacLTQDLNKFHTFPKTDVGANAP-NH₂ AcCSNLSTCVLGKacLSQELHKLQTYPRTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQELHRLQTYPKTDVGANAP-NH₂ AcCASLSTCVLGKacLSQDLHKLQTFPKTDVGANAP-NH₂ AcCASLSTCMLGKacLSQDLHKLQTFPKTDVGANAP-NH₂ AcCGNLSTCMLGKacLSQDLNKFHTFPQTDVGANAP-NH₂ AcCSNLSTC (AIE) LGKa_cLANFLVHSSNNFGAILPKTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKHTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKHSSTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKHSSNTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLSNNFGAILSSTNVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYGAILSPKTDVGANAP-NH₂ AcCSNLSTCMLGKacLANFLVHSSNNFGAILPKTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKILSSTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKGLITTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKNNFGTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKRTTQTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKHTTNTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKHGGQTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKHKKNTDVGANAP-NH₂ AcCSNLSTCMLGKacLSQDLHRLQTYPKHKKHTDVGANAP-NH₂ AcCSNLSTC (AiB) LGRLSQDLHRKa_cQTYPKTDVGANAP-NH₂AcCSNLSTCMLGKacLSQELHRLQTYPKTDVGANAP-NH₂ and wherein K_Ac is acylated with a linker-fatty diacid wherein the fatty diacid is a Cis to C₂₂ fatty diacid and the linker is HO^O Y H O H O'^rr¹ JL O H O

34. A peptide as claimed in any one of daims 1 to 33, formulated for enterai administration.

100

35. A peptide as claimed in claim 34, wherein the peptide is formulated in a pharmaceutical composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increase the oral bioavailability of the peptide

36. A peptide as claimed in any one of claims 1 to 35, formulated with a carrier for oral administration.

37. A peptide as claimed in claim 36, wherein the carrier comprises 5-CNAC, SNAD, or SNAC.

38. A peptide as claimed in any one of claims 1 to 33, formulated for parentéral administration.

39. A peptide as claimed in claim 38, formulated for inj ection.

40. A pharmaceutical composition comprising the peptide as claimed in any one of claims 1 to 33.

41. A peptide as claimed in any one of claims 1 to 33 for use as a médicament.

42. A peptide as claimed in any one of claims 1 to 33, for use in treating diabètes (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

101 levels, poorly regulated response to glucose tolérance tests, or poor régulation of food intake.

43. A peptide as claimed in any one of claims 1 to 33 in combination with metformin or another insulin sensitizer for use in treating diabètes (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 tolérance tests, or poor régulation of food intake.

44. A peptide as claimed in any one of claims 1 to 33 in combination with a weight loss drug for use in treating an overweight condition.

45. A peptide as claimed in claim 44, wherein the overweight condition is obesity.

46. A co-formulation comprising a peptide as claimed in any one of claims 1 to 33 and an insulin sensitizer.

47. A co-formulation comprising a peptide as claimed in any one of claims 1 to 33 and a weight loss drug.

48. A method of treating diabètes (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,

102 or osteoarthritis, poorly regulated blood glucose levels, poorly regulated response to glucose tolérance tests, or poor régulation of food intake, comprising administering an effective amount of peptide as claimed in any one of claims 1 to 33 to a patient in need of said treatment.

49. A method of treating diabètes (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 tolérance tests, or poor régulation of food intake, comprising administering an effective amount of peptide as claimed in any one of claims 1 to 33 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 a peptide as claimed in any one of claims 1 to 33 in combination with a weight loss drug to a patient in need of said treatment.