Acylated calcitonin mimetics

Abstract

The invention relates to acylated calcitonin mimetics. The present invention relates to calcitonin mimetics, and discloses calcitonin mimetics acylated at lysine residues at positions (11) and (19) of the calcitonin mimetics and their use as medicaments in the treatment of various diseases and conditions, in the modulation of blood glucose levels, in the modulation of response to glucose tolerance tests, in the modulation of food intake, in the treatment of osteoporosis, and in the treatment of osteoarthritis, the various diseases and conditions include diabetes, overweight, overeating and metabolic syndrome, NASH, alcoholic and non-alcoholic fatty liver diseases.

Description

[0001] The applicant filed PCT application PCT / EP2019 / 072533 on August 22, 2019, entitled "Acylated Calcitonin Mimicry". This PCT application entered the Chinese national phase on February 22, 2021, with application number 201980055426.8. This application is a divisional application of that Chinese application. Technical Field

[0002] This invention relates to acylated calcitonin mimics and extends to their use as medicines in treating various diseases and conditions, regulating blood glucose levels, regulating responses to glucose tolerance tests, regulating food intake, treating osteoporosis, and treating osteoarthritis, including but not limited to diabetes (type I and II), overweight, overeating and metabolic syndrome, non-alcoholic steatohepatitis (NASH), and alcoholic and non-alcoholic fatty liver disease. Background Technology

[0003] Globally, there are approximately 250 million people with diabetes, and this number is projected to double in the next two decades. More than 90% of this population has type 2 diabetes (T2DM). It is estimated that currently only 50 to 60% of people are diagnosed with T2DM or are in the pre-diagnostic stage.

[0004] Type 2 diabetes mellitus (T2DM) is a heterogeneous disease characterized by abnormal carbohydrate and lipid metabolism. The etiology of T2DM is multifaceted, encompassing both genetic and environmental factors affecting β-cell function and insulin sensitivity in tissues such as muscle, liver, pancreas, and adipose tissue. As a result, impaired insulin secretion is observed, and this impairment is synchronized with a progressive decline in β-cell function and chronic insulin resistance. The endocrine pancreas fails to compensate for peripheral insulin resistance, leading to hyperglycemia and the onset of clinical diabetes. Currently, tissue resistance to insulin-mediated glucose uptake is considered a major pathophysiological determinant of T2DM.

[0005] The optimal criteria for successful intervention in type 2 diabetes mellitus (T2DM) is a reduction in blood glucose levels, which can be either a long-term reduction in blood glucose levels or an enhanced ability to tolerate high glucose levels after food intake. This can be described by a decrease in peak glucose levels and faster clearance. Both of these conditions place less stress on β-cell insulin output and function.

[0006] Type 1 diabetes is characterized by a loss of the ability to produce insulin in response to food intake, thus failing to regulate blood sugar to normal physiological levels.

[0007] The physical structure of bones can be affected by a variety of factors, including disease and injury. One of the most common bone diseases is osteoporosis, characterized by low bone mass and deterioration of bone tissue structure, leading to bone fragility, particularly increased susceptibility to fractures of the hip, spine, and wrist. Osteoporosis develops when there is an imbalance where bone resorption exceeds bone formation. It has been shown that administering effective amounts of anti-resorption agents such as calcitonin can prevent bone resorption.

[0008] Inflammatory or degenerative diseases, including joint diseases such as osteoarthritis (OA), rheumatoid arthritis (RA), or juvenile rheumatoid arthritis (JRA) and inflammation caused by autoimmune responses such as lupus, ankylosing spondylitis (AS), or multiple sclerosis (MS), can lead to substantial loss of mobility due to pain and joint destruction. The cartilage that covers and cushions the bones within an joint can degenerate over time, undesirably bringing two bones into direct contact. This can restrict the movement of one bone relative to the other during joint movement and / or cause damage from one bone to the other. The subchondral bone beneath the cartilage may also degenerate. Administration of effective amounts of anti-resorption agents such as calcitonin can prevent bone resorption.

[0009] Calcitonin is highly conserved across a wide range of species. The full-length natural calcitonin is 32 amino acids long. Sequences of examples of natural calcitonin are shown below:

[0010] Salmon CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP

[0011] Eel CSNLSTCVLGKLSQELHKLQTYPRTDVGAGTP

[0012] Chicken CASLSTCVLGKLSQELHKLQTYPRTDVGAGTP

[0013] Mouse CGNLSTCMLGTYTQDLNKFHTFPQTSIGVEAP

[0014] Rat CGNLSTCMLGTYTQDLNKFHTFPQTSIGVGAP

[0015] Horse CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP

[0016] Dog-1 CSNLSTCVLGTYSKDLNNFHTFSGIGFGAETP

[0017] Dog-2 CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP

[0018] pigCSNLSTCVLSAYWRNLNNFHRFSGMGFGPETP

[0019] PeopleCGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP

[0020] Synthetic variants of natural calcitonin are disclosed in WO2013 / 067357 and WO 2015 / 071229, which have modified amino acid sequences designed to provide improved properties.

[0021] However, peptides such as calcitonin and calcitonin mimics typically have poor absorption, distribution, metabolism, and excretion properties, as well as rapid clearance and short half-lives. Therefore, peptide drugs usually require daily parenteral administration. Currently, daily subcutaneous (SC) administration is not the optimal method of administration because it is inconvenient for individual patients and may lead to non-compliance with the treatment plan to avoid the discomfort associated with daily injections. Therefore, weekly subcutaneous administration would improve the quality of life for relevant patients and further help with adherence to the treatment plan.

[0022] Numerous methods are known in the art to attempt to improve the in vivo half-life of peptide drugs. Such methods include improving proteolytic stability (e.g., by protecting the N- and C-termini, replacing amino acids with D-amino acids or non-natural amino acids, cyclizing peptides, etc.) and reducing renal clearance (e.g., by conjugating the peptide to macromolecules such as macropolymers, albumins, immunoglobulins, etc.). However, it is also known in the art that such modifications to pharmaceutical peptides can be detrimental in terms of, for example, reduced drug efficacy and unpredictable adverse side effects such as drug sensitization. Therefore, it is impossible to predict whether such modifications will necessarily improve the therapeutic spectrum of peptide drugs.

[0023] Therefore, developing peptide drugs that only require weekly administration is a challenging prospect.

[0024] One approach to improving the pharmacokinetic and pharmacodynamic properties of peptide drugs is peptide acylation. Trier et al. (PhD dissertation, 2016, "Acylation of Therapeutic Peptides", DTU; available for download at: http: / / orbit.dtu.dk / files / 127682557 / PhD_thesis_Sofie_Trier.pdf) investigated the effects of acylation on two therapeutic peptides (glucagon-like peptide-2 (GLP-2) and salmon calcitonin (sCT)) with acyl groups of different lengths (C8-C9). 16While previous observations of similar peptides have shown that the effects of acylated GLP2 are largely predictable, the effects observed with acylated sCT are unpredictable. For example, Trier et al. found that acylation of sCT (at different positions on the peptide backbone) consistently resulted in a substantial loss of receptor potency (60 to 80%), whereas GLP-2 retained its receptor potency after acylation. Therefore, while Trier et al. did discover some useful properties associated with acylated sCT (particularly with short-chain (C8) acylation), it is also clear that acylated sCT has many unpredictable and significant adverse effects, most notably the significant decrease in receptor potency. Another noteworthy point is that Trier et al.'s study focused on acylation of position 18 (Lys18) of salmon calcitonin. This is because previous studies aimed at improving the efficacy of salmon calcitonin identified position 18 as a preferred site for modification (in this case, modified via PEGylation rather than acylation). In those studies, it was found that PEGylation of sCT at the Lys18 position resulted in better efficacy than similar peptides modified at the Cys1 or Lys11 positions (Youn et al., J. Control. Release, 2006, 334-342). Summary of the Invention

[0025] The inventors have discovered that acylation of calcitonin mimics at the 11th or 19th lysine residue, particularly by certain specific acyl moieties, results in a surprisingly improved peptide efficacy compared to equivalent unacylated peptides, and also increases the duration of peptide action. Similarly, the greatest improvement in the efficacy of calcitonin mimics was found to correspond to acylation at the 11th or 19th residue, while acylation at the 18th residue produced poorer results, contrary to the findings of Youn et al. Therefore, the inventors have developed a novel, effective acylated calcitonin mimic that can be administered only once a week instead of daily.

[0026] Therefore, in one aspect, the present invention provides a calcitonin mimic that is acylated at a lysine residue at position 11 and / or at a lysine residue at position 19. The ε-amino group of the side chain of the lysine residue is acylated with an acyl group selected from any of the following: C with an optional linker. 16 Or longer fatty acids; or C with optional connectors 16 Or longer fatty acids.

[0027] As used herein, “calcitonin mimic” means a peptide that activates the calcitonin receptor (i.e., a calcitonin receptor agonist) and preferably also activates the amylin receptor (i.e., a dual agonist of amylin and calcitonin receptors).

[0028] In some preferred embodiments, the calcitonin mimic is 32 to 37 amino acids in length. Most preferably, the calcitonin mimic is 32 amino acids in length.

[0029] In a preferred aspect, wherein the calcitonin mimic is acylated at the lysine residue at position 11, the present invention relates to calcitonin mimics of formula (I)(a):

[0030] CX2X3LSTCX8LGK Ac ...

[0031] in

[0032] X2 = A, G, or S

[0033] X3 = N or S

[0034] X8 = M, V, or α-aminoisobutyric acid (AiB)

[0035] And among them, K Ac It is a lysine residue in which the ε-amino group of the side chain is acylated by an acyl group selected from any of the following:

[0036] C with optional connector 16 Or longer fatty acids; or

[0037] C with optional connector 16 Or longer fatty acids.

[0038] In another preferred aspect, wherein the calcitonin mimic is acylated at the lysine residue at position 19, the present invention relates to calcitonin mimics of formula (I)(b):

[0039] CX2X3LSTCX8LGX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 K Ac ...

[0040] in

[0041] X2 = A, G, or S

[0042] X3 = N or S

[0043] X8 = M, V, or α-aminoisobutyric acid (AiB)

[0044] X 11 =R, K, T, A or K Ac (Preferred R, K or K) Ac(Option R or K)

[0045] X 12 =L or Y (L is the optimal choice)

[0046] X 13 =S, T, W or Y (preferably T, S or Y)X 14 =Q, K, R, or A (Q or A is preferred, Q is the best choice)

[0047] X 15 =D, E, or N (Optimal choice: D or E)

[0048] X 16 =L or F (optimal choice L)

[0049] X 17 =H or N

[0050] X 18 =R, K, or N (R or K are preferred)

[0051] And among them, K Ac It is a lysine residue in which the ε-amino group of the side chain is acylated by an acyl group selected from any of the following:

[0052] C with optional connector 16 Or longer fatty acids; or

[0053] C with optional connector 16 Or longer fatty acids.

[0054] Preferably, the calcitonin mimic of formula (I)(a) or (I)(b) is 32 to 37 amino acids in length, more preferably 32, 33, 35, 36 or 37 amino acids in length. Most preferably, the calcitonin mimic of formula (I)(a) or (I)(b) is 32 amino acids in length.

[0055] In a preferred aspect of the invention, the calcitonin mimic is a 32-mer calcitonin mimic of formula (II):

[0056] CX2X3LSTCX8LGX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27GX 29 X 30 X 31 P

[0057] in

[0058] X2 = A, G, or S

[0059] X3 = N or S

[0060] X8 = M, V, or α-aminoisobutyric acid (AiB)

[0061] X 11 =K Ac R, K, T or A (K is the most preferred) Ac (R or K)

[0062] X 12 =L or Y

[0063] X 13 =S, T, W or Y

[0064] X 14 =Q, K, R or A

[0065] X 15 =D, E or N

[0066] X 16 =L or F

[0067] X 17 =H or N

[0068] X 18 =R, K or N

[0069] X 19 =K Ac L, F, or K (K is the most preferred) Ac (L or F)

[0070] X 20 =Q, H or A

[0071] X 21 =T or R

[0072] X 22 =Y or F

[0073] X 23 =S or P

[0074] X 24 =G, K, Q or R

[0075] X 25 =T, I or M

[0076] X 26 =S, N, D, G or A

[0077] X 27 =T, V, F or I

[0078] X 29 =S, A, P or V

[0079] X 30 =N, G or E

[0080] X 31 =A, T, or S (A or T is the most preferred)

[0081] Where X 11 For K Ac and / or X 19 For K Ac (make X) 11 For K Ac And X 19 It can be L, F, or K, preferably L or F; or X 11 The value is R, K, T, or A, preferably R or K, and X 19 For K Ac ; or X 11 For K Ac And X 19 For K Ac ),

[0082] And among them, K Ac It is a lysine residue in which the ε-amino group of the side chain is acylated by an acyl group selected from any of the following:

[0083] C 16 Or longer fatty acids,

[0084] C 16 Or longer fatty acids,

[0085] Connector-C 16 Or longer fatty acids, or

[0086] Connector-C 16 Or longer fatty acids.

[0087] Preferably, the 32-mer calcitonin mimic of formula (II) is:

[0088] CX2X3LSTCX8LGX 11 LX 13 X 14 X 15 LX 17 X 18 X 19 X 20 TX 22 PX 24 TDVGANAP

[0089] in

[0090] X2 = A, G, or S

[0091] X3 = N or S

[0092] X8 = M, V, or AiB

[0093] X 11 =K Ac R, K, T or A (K is the most preferred) Ac (R or K)

[0094] X 13 =T, S or Y

[0095] X 14 =Q or A (Q is the optimal choice)

[0096] X 15 =D or E

[0097] X 17 =H or N

[0098] X 18 =R or K

[0099] X 19 =K Ac L, F, or K (K is the most preferred) Ac (L or F)

[0100] X 20 =Q, H or A

[0101] X 22 =Y or F

[0102] X 24 =K, Q or R

[0103] Among them, X 11 For K Ac and / or X 19 For K Ac ,

[0104] And among them, K Ac It is a lysine residue in which the ε-amino group of the side chain is acylated by an acyl group selected from any of the following:

[0105] C 16 Or longer fatty acids,

[0106] C 16 Or longer fatty acids,

[0107] Connector-C 16 Or longer fatty acids, or

[0108] Connector-C16 Or longer fatty acids.

[0109] Preferably, X2 is S and X3 is N; or X2 is G and X3 is N; or X2 is A and X3 is S.

[0110] Preferably, X 13 It can be S or T, with S being the most preferred. Preferably, X... 24 It can be R or K.

[0111] In a preferred embodiment,

[0112] -X 11 For K Ac X 17 For H, X 18 Let K, X 19 Let L be the integer part of the integer part, and X be the integer part of the integer part. 20 For Q or A; or

[0113] -X 11 For K Ac X 17 For H, X 18 Let R, X 19 Let L be the integer part of the integer part, and X be the integer part of the integer part. 20 For Q or A; or

[0114] -X 11 For K Ac X 17 Let N, X 18 Let K, X 19 Let F be the integer part of the integer part, and X be the integer part of the integer part. 20 For H or A; or

[0115] -X 11 For K Ac X 17 Let N, X 18 Let R, X 19 Let F be the integer part of the integer part, and X be the integer part of the integer part. 20 For H or A; or

[0116] -X 11 For R or K, X 17 For H, X 18 Let K, X 19 For K Ac And X 20 For Q or A; or

[0117] -X 11 For R or K, X 17 For H, X 18 Let R, X 19 For K Ac And X 20 For Q or A; or

[0118] -X 11 For R or K, X 17 Let N, X 18 Let K, X 19 For K Ac And X 20 For H or A; or

[0119] -X 11 For R or K, X 17 Let N, X 18 Let R, X 19 For K Ac And X 20 It can be H or A.

[0120] In a preferred embodiment, X2 is S, X3 is N, and X... 11 For K Ac X 13 Let S and X be the values ​​of S and X. 17 For H, X 18 For K or R, X 19 Let L and X be the numbers. 20 It is either Q or A, and X 22 For Y; or X2 is S, X3 is N, X 11 For R or K, X 13 Let S and X be the values ​​of S and X. 17 For H, X 18 For K or R, X 19 For K Ac X 20 It is either Q or A, and X 22 Let Y be X2. In a preferred embodiment, X2 is A, X3 is S, and X... 11 For K Ac X 13 Let S and X be the values ​​of S and X. 17 For H, X 18 For K or R, X 19 Let L and X be the numbers. 20 It is either Q or A, and X 22 For F; or X2 is A, X3 is S, X 11 For R or K, X 13 Let S and X be the values ​​of S and X. 17 For H, X 18 For K or R, X 19 For K Ac X 20 It is either Q or A, and X 22 Let F be X2. In a preferred embodiment, X2 is G, X3 is N, and X... 11 For K Ac X 13 Let T, X 17 Let N, X 18 For K or R, X 19 Let F, X20 It is H or A, and X 22 For F; or X2 is G, X3 is N, X 11 For R or K, X 13 Let T, X 17 Let N, X 18 For K or R, X 19 For K Ac X 20 It is H or A, and X 22 It is F.

[0121] In another preferred aspect, the present invention relates to calcitonin mimics, wherein the calcitonin mimics are 33-meric peptides according to formula (III):

[0122] CSNLSTCX6LGX7LSQDLHRX8QTYPKX1TX5VGANAP(III)

[0123] Or the calcitonin mimicry described therein is a 35-meric peptide according to formula (IV):

[0124] CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3TX5VGANAP(IV)

[0125] Or the calcitonin mimicry described therein is a 36-mer peptide according to formula (V):

[0126] CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3X4TX5VGANAP(V)

[0127] Or the calcitonin mimicry described therein is a 37-mer peptide according to formula (VI):

[0128] CSNLSTCX6LGK Ac LZX1X2X3X4TX5VGANAP(VI)

[0129] Each of X1 to X4 is any amino acid, provided that at least one of X1 to X4 is a basic amino acid residue, and / or at least two of X1 to X4 are independently polar amino acid residues or basic amino acid residues, at least one of X1 to X4 is a Gly residue, and none of X1 to X4 are acidic residues.

[0130] Where X5 is either D or N;

[0131] Where X6 is AiB or M;

[0132] Where X7 is K Ac And X8 is L, or X7 is R, or X8 is K. Ac ,

[0133] Where Z is selected from SQDLHRLSNNFGA, SQDLHRLQTYGAI, or ANFLVHSSNNFGA; and

[0134] Among them, K Ac It is a lysine residue, wherein the ε-amino group of the side chain is acylated by an acyl group selected from any of the following:

[0135] C 16 Or longer fatty acids,

[0136] C 16 Or longer fatty acids,

[0137] Connector-C 16 Or longer fatty acids, or

[0138] Connector-C 16 Or longer fatty acids.

[0139] Preferably, at least one of X1 or X4 in formulas (III) to (VI) is a basic amino acid residue. More preferably, at least one of X1 or X4 is a basic amino acid residue, and at least one or more of X1 to X4 are independently polar or basic amino acid residues, and none of X1 to X4 are acidic residues. Even more preferably, at least three of X1 to X4 are independently polar or basic amino acid residues, and none of X1 to X4 are acidic residues. More preferably, all of X1 to X4 are independently polar or basic amino acid residues, and none of X1 to X4 are acidic residues. Most preferably, all of X1 to X4 are independently polar or basic amino acid residues, at least three of X1 to X4 are basic amino acid residues, and none of X1 to X4 are acidic residues.

[0140] Basic amino acid residues can be any natural or non-natural amino acid residues with a basic side chain, selected from, but not limited to, Arg, His, or Lys. Polar amino acid residues can be any natural or non-natural amino acid residues with a polar, uncharged side chain, selected from, but not limited to, Ser, Thr, Asn, Gln, or Cys. As used herein, the term "acidic residue" refers to any natural or non-natural amino acid residue with an acidic side chain, such as Glu or Asp.

[0141] In a preferred embodiment, X1 is selected from Asn, Phe, Val, Gly, Ile, Leu, Lys, His, or Arg;

[0142] X2 is selected from Ala, Asn, His, Leu, Ser, Thr, Gly, or Lys;

[0143] X3 is selected from Ala, Phe, Ile, Ser, Pro, Thr, Gly, or Lys; and / or

[0144] X4 is selected from Ile, Leu, Gly, His, Arg, Asn, Ser, Lys, Thr, or Gln;

[0145] The condition is that at least one of X1 or X4 is a basic amino acid residue, and / or at least two of X1 to X4 are independently polar amino acid residues and / or basic amino acid residues, and / or at least one of X1 to X4 is a Gly residue.

[0146] In a preferred embodiment, X1 is selected from Asn, Gly, Ile, His, or Arg;

[0147] X2 is selected from Asn, Leu, Thr, Gly, or Lys;

[0148] X3 is selected from Phe, Pro, Ile, Ser, Thr, Gly, or Lys; and / or

[0149] X4 is selected from Gly, His, Asn, Ser, Lys, Thr, or Gln;

[0150] The condition is that at least one of X1 or X4 is a basic amino acid residue, and / or at least two of X1 to X4 are independently polar amino acid residues and / or basic amino acid residues, and / or at least one of X1 to X4 is a Gly residue.

[0151] The peptides of the present invention according to formulas (III) to (V) above may contain one or more of the following conservative substitutions:

[0152] -The Asp residue at position 15 of the peptide is replaced by Glu;

[0153] - The Arg residue at position 18 of the peptide is replaced by Lys; and / or

[0154] - The Lys residue at position 24 of the peptide is replaced by Arg.

[0155] The peptides of the present invention according to formula (VI) above may contain one or more of the following conservative substitutions:

[0156] - The Asp residue at position 15 of the peptide is replaced by Glu; and / or

[0157] - The Arg residue at position 18 of the peptide is replaced by Lys.

[0158] The Z component of the peptide in formula (VI) is either SQDLHRLSNNFGA or SQDLHRLQTYGAI.

[0159] In all aspects of the invention, the linker preferably comprises a glutamic acid residue and / or an oligoethylene glycol (OEG) amino acid linker, the OEG amino acid linker comprising one OEG amino acid or two or more OEG amino acids linked together, wherein the OEG amino acid is:

[0160]

[0161] And n is 1 to 10, preferably 1 to 5, preferably 1 to 3, preferably 1 or 2, and most preferably 1.

[0162] The OEG amino acid linker preferably comprises 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 or carboxyl terminus of the OEG amino acid linker. Preferably, the OEG amino acid linker is selected from any of the following:

[0163]

[0164] Preferably, the OEG amino acid linker is:

[0165] In a preferred embodiment, the acyl group is selected from C. 18 Or longer fatty acids, C 18 Or longer fatty acids, connector-C 18 Or longer fatty acids, or junction-C 18 Or a longer fatty acid diacid. Preferably, the acyl group is selected from any of the following:

[0166] C 18 To C 30 Fatty acids, preferably C 18 To C 22 fatty acid,

[0167] C 18 To C 30 Fatty acid, preferably C 18 To C 22 Fatty diacids,

[0168] Connector-C 18 To C 30 Fatty acids, preferred linker-C 18 To C 22 fatty acid,

[0169] Connector-C 18 To C30 Fatty acid, preferred connector-C 18 To C 22 Fatty diacids.

[0170] Preferably, C 18 Fatty acid diacid is octadecanoic acid (CAS No. 871-70-5).

[0171] In a preferred embodiment, K Ac Acrylation with a fatty diacid by a connector, wherein the fatty diacid is C 18 To C 22 Fatty acid, and the connector is Preferably, C 18 Fatty acid diacid is octadecanoic acid.

[0172] Preferably, the calcitonin mimic of the present invention is selected from any one of the following:

[0173] CSNLSTCMLGK Ac LSQDLHRLQTYPKTDVGANAP

[0174] CSNLSTCMLGK Ac LSQELHRLQTYPKTDVGANAP

[0175] CSNLSTCVLGK Ac LSQELHKLQTYPRTDVGANAP

[0176] CASLSTCVLGK Ac LSQDLHKLQTFPKTDVGANAP

[0177] CGNLSTCMLGK Ac LSQDLNKFHTFPQTDVGANAP

[0178] CSNLSTC(AiB)LGK Ac LSQDLHRLQTYPKTDVGANAP

[0179] CGNLSTC(AiB)LGK Ac LTQDLNKFHTFPKTDVGANAP

[0180] CSNLSTC(AiB)LGK Ac LANFLVHSSNNFGAILPKTDVGANAP

[0181] CSNLSTCMLGK Ac LSQDLHRLQTYPKHTDVGANAP

[0182] CSNLSTCMLGK Ac LSQDLHRLQTYPKHSSTDVGANAP

[0183] CSNLSTCMLGK Ac LSQDLHRLQTYPKHSSNTDVGANAP

[0184] CSNLSTCMLGK Ac LSQDLHRLSNNFGAILSSTNVGANAP

[0185] CSNLSTCMLGK Ac LSQDLHRLQTYGAILSPKTDVGANAP

[0186] CSNLSTCMLGK Ac LANFLVHSSNNFGAILPKTDVGANAP

[0187] CSNLSTCMLGK Ac LSQDLHRLQTYPKILSSTDVGANAP

[0188] CSNLSTCMLGK Ac LSQDLHRLQTYPKGLITTDVGANAP

[0189] CSNLSTCMLGK Ac LSQDLHRLQTYPKNNFGTDVGANAP

[0190] CSNLSTCMLGK Ac LSQDLHRLQTYPKRTTQTDVGANAP

[0191] CSNLSTCMLGK Ac LSQDLHRLQTYPKHTTNTDVGANAP

[0192] CSNLSTCMLGK Ac LSQDLHRLQTYPKHGGQTDVGANAP

[0193] CSNLSTCMLGK Ac LSQDLHRLQTYPKHKKNTDVGANAP

[0194] CSNLSTCMLGK Ac LSQDLHRLQTYPKHKKHTDVGANAP

[0195] CSNLSTC(AiB)LGRLSQDLHRK Ac QTYPKTDVGANAP

[0196] CSNLSTCMLGRLSQELHRK Ac QTYPKTDVGANAP

[0197] Where K Ac As defined above. The amino acid residue at position 8 of the aforementioned peptide, if not otherwise specified, may optionally be substituted with AiB.

[0198] Preferably, the calcitonin mimic of the present invention is selected from any one of the following:

[0199] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKTDVGANAP-NH2

[0200] AcCSNLSTC(AiB)LGK Ac LSQDLHRLQTYPKTDVGANAP-NH2

[0201] AcCGNLSTC(AiB)LGK Ac LTQDLNKFHTFPKTDVGANAP-NH2

[0202] AcCSNLSTCVLGK Ac LSQELHKLQTYPRTDVGANAP-NH2

[0203] AcCSNLSTCMLGK Ac LSQELHRLQTYPKTDVGANAP-NH2

[0204] AcCASLSTCVLGK Ac LSQDLHKLQTFPKTDVGANAP-NH2

[0205] AcCGNLSTCMLGK Ac LSQDLNKFHTFPQTDVGANAP-NH2

[0206] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHTDVGANAP-NH2

[0207] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHSSTDVGANAP-NH2

[0208] AcCSNLSTCMLGKAc LSQDLHRLQTYPKHSSNTDVGANAP-NH2

[0209] AcCSNLSTCMLGK Ac LSQDLHRLSNNFGAILSSTNVGANAP-NH2

[0210] AcCSNLSTCMLGK Ac LSQDLHRLQTYGAILSPKTDVGANAP-NH2

[0211] AcCSNLSTCMLGK Ac LANFLVHSSNNFGAILPKTDVGANAP-NH2

[0212] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKILSSTDVGANAP-NH2

[0213] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKGLITTDVGANAP-NH2

[0214] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKNNFGTDVGANAP-NH2

[0215] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKRTTQTDVGANAP-NH2

[0216] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHTTNTDVGANAP-NH2

[0217] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHGGQTDVGANAP-NH2

[0218] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHKKNTDVGANAP-NH2

[0219] AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHKKHTDVGANAP-NH2

[0220] AcCSNLSTC(AiB)LGK AcLANFLVHSSNNFGAILPKTDVGANAP-NH2

[0221] AcCSNLSTC(AiB)LGRLSQDLHRK Ac QTYPKTDVGANAP-NH2

[0222] AcCSNLSTCMLGRLSQELHRK Ac QTYPKTDVGANAP-NH2

[0223] Where K Ac Acrylation with a fatty diacid by a connector, wherein the fatty diacid is C 18 To C 22 Fatty acid, and the connector is

[0224]

[0225] Preferably, C 18 The fatty acid diacid is octadecanoic acid. The amino acid residue at position 8 of the above peptide is optionally substituted with AiB, unless otherwise specified. 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.

[0226] The calcitonin mimics of the present invention can be formulated for enteral administration. For example, the calcitonin mimics can be formulated in a pharmaceutical composition for oral administration, the pharmaceutical composition comprising coated citric acid particles, wherein the coated citric acid particles increase the oral bioavailability of the peptide. Alternatively or otherwise, the calcitonin mimics can be formulated together with a carrier for oral administration. Exemplary carriers may include 5-CNAC, SNAD, or SNAC. The calcitonin mimics of the present invention can also be formulated for parenteral administration. For example, the calcitonin mimics can be formulated for injection.

[0227] The present invention also relates to pharmaceutical compositions comprising calcitonin mimics as described above.

[0228] The present invention also relates to the use of calcitonin mimics as described above as pharmaceuticals. In this regard, calcitonin mimics can be used to treat diabetes (type I and / or type II), overweight, overeating, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, poor regulation of blood glucose levels, poor regulation of response to glucose tolerance tests, or poor regulation of food intake. Calcitonin mimics can also be administered in combination with metformin or another insulin sensitizer.

[0229] The peptides of the present invention may be acylated or otherwise modified at their N-terminus to reduce the positive charge of the first amino acid, and may independently be amidated at their C-terminus.

[0230] Peptides can be formulated for drug administration, and can be formulated for enteral or parenteral administration. Preferred formulations are injectable, preferably for subcutaneous injection; however, peptides can be formulated with a carrier for oral administration, and optionally the carrier increases the oral bioavailability of the peptide. Suitable carriers include those containing 5-CNAC, SNAD, or SNAC.

[0231] Optionally, the peptide is formulated into a pharmaceutical composition for oral administration, the pharmaceutical composition comprising coated citric acid particles, wherein the coated citric acid particles increase the oral bioavailability of the peptide.

[0232] This invention includes the use of the peptides of this invention as pharmaceuticals. The peptides can be used to treat diabetes (type I and / or type II), overweight, overeating, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, poor regulation of blood glucose levels, poor regulation of response to glucose tolerance tests, or poor regulation of food intake. In particular, the peptides can be used to reduce undesirably high fasting blood glucose levels or undesirably high HbA1c or undesirably high responses to glucose tolerance tests. The peptides of this invention can also be used to reduce triglycerides and / or reduce fat accumulation in the liver in subjects.

[0233] The peptides of the present invention can be produced using any suitable method known in the art for generating peptides, such as synthetic (chemical) techniques and recombinant techniques. Preferably, synthetic methods are used to produce the peptides. The synthesis of synthetic peptides 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.).

[0234] In some embodiments, the N-terminal side of the calcitonin mimics discussed above is modified to reduce the positive charge of the first amino acid. For example, an acetyl, propionyl, or succinyl group may be substituted at cysteine-1. Alternatives to reduce the 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 to the N-terminus. Alternatively, other amino acids may be added to the N-terminus of the peptides discussed above, including, but not limited to, lysine, glycine, formylglycine, leucine, alanine, acetylanine, and diallanyl. As those skilled in the art will understand, peptides having multiple cysteine ​​residues typically form disulfide bridges between two such cysteine ​​residues. All such peptides described herein are defined as optionally including one or more such disulfide bridges, particularly at the Cys1-Cys7 positions. Mimicking this, the cysteines at positions 1 and 7 may be jointly substituted with an α-aminooctanoic acid bond. While the calcitonin mimics of this disclosure may be present in free acid form, it is preferred that the C-terminal amino acid be amidated. The applicant anticipates that such amidation may contribute to the peptide's effectiveness and / or bioavailability. Synthetic chemical methods can be used to amidate the C-terminal amino acid. Another technique for preparing the amidated form of the calcitonin mimic of this disclosure is to react a precursor (with a glycine-substituted C-terminal amino group of the desired amidation product) according to known techniques in the presence of peptidylglycine α-amidoxylase, wherein the precursor is converted to the amidation product in the reaction, as described in, for example, US4708934, EP0308067, and EP0382403.

[0235] The production of amidation products can also be accomplished using the methods and amidases described in the following literature: Consalvo et al., 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.

[0236] The preferred amidated peptide can be prepared by, for example, by generating a glycine-extended precursor in *E. coli* as a soluble fusion protein with glutathione S-transferase, or by directly expressing the precursor according to the technique described in US6103495. Such a glycine-extended precursor has the same molecular structure as the desired amidated product, except at the C-terminus (where the product terminates with --X--NH2, while the precursor terminates with --X-gly, where X is the C-terminal amino acid residue of the product). The α-amidase described in the aforementioned publication catalyzes the conversion of the precursor to the product. This enzyme is preferably recombinantly generated, for example, in Chinese hamster ovary (CHO) cells, as described in the aforementioned article, *Biotechnology and Biopharm*.

[0237] The free acid form of the peptide activator disclosed herein can be generated in a similar manner, except that it does not include a C-terminal glycine in the "precursor," which is the final peptide product and does not require an amidation step.

[0238] Unless otherwise stated, the preferred dosages of the calcitonin mimics disclosed herein are the same for both therapeutic and prophylactic purposes. The required dosages will be discussed in more detail below and vary depending on the method of administration.

[0239] Unless otherwise stated or apparent from the context, dosage as used herein refers to the weight of the active compound (i.e., calcitonin analogue) unaffected by or without regard to pharmaceutical excipients, diluents, carriers, or other components, although it is desirable to include such additional components. Any dosage form (capsules, tablets, injections, etc.) commonly used in the pharmaceutical industry for delivering peptide active agents is suitable for use herein, and the terms “excipient,” “diluent,” or “carrier” include both the inactive components commonly included and the active components of such dosage forms in the industry. Preferred oral dosage forms are discussed in more detail below, but should not be considered as the exclusive means of administering the active agents of this disclosure.

[0240] The calcitonin mimics of this disclosure can be administered to patients to treat a variety of diseases or conditions. As used herein, the term "patient" refers to any organism belonging to the animal kingdom. In one embodiment, the term "patient" refers to a vertebrate, more preferably a mammal including humans.

[0241] Therefore, this disclosure includes the use of peptides in treating type 1 diabetes, type 2 diabetes or metabolic syndrome, obesity or appetite suppression, or for reducing insulin resistance, or for reducing undesirable high fasting blood glucose levels, or for reducing undesirable peak serum glucose levels, or for reducing undesirable peak serum insulin levels, or for reducing undesirable large responses to glucose tolerance tests, or for treating osteoporosis, or for treating osteoarthritis, or for treating non-alcoholic steatohepatitis (NASH), or for treating alcoholic fatty liver disease, or for reducing liver triglycerides, or for methods of reducing fat accumulation in the liver of a subject.

[0242] There are several field-recognized measurements of normal weight that take into account various factors such as sex, age, and height. Patients requiring treatment or prevention programs as described herein include those who are overweight or have a higher risk of becoming overweight or obese than the general population due to genetic, environmental, or other recognized risk factors. According to this disclosure, calcitonin mimics are intended for use in the treatment of diabetes, where weight control is an aspect of the treatment.

[0243] In one embodiment, the method includes administering an enteral amount of any of the pharmaceutically effective peptides described herein to a patient who requires treatment of the condition.

[0244] In one embodiment, the method comprises administering a pharmaceutically effective amount of any of the peptides described herein parenterally to a patient requiring treatment of the condition. For parenterial administration (including intraperitoneal, subcutaneous, intravenous, intradermal, or intramuscular injection), solutions of the peptides of the present invention in sesame oil or peanut oil, or in aqueous propylene glycol solution, can be used. If necessary, the aqueous solution should be appropriately buffered, and the liquid diluent should first be isotonic. These aqueous solutions are suitable for intravenous injection purposes. Oily solutions are suitable for intra-articular, intramuscular, and subcutaneous injection purposes. The preparation of all these solutions under aseptic conditions can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. Examples of suitable formulations for parenterial application include solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants including suppositories. The peptides can be formulated in a sterile form, such as dispersed in a fluid carrier, for example, sterile saline or 5% saline glucose solution commonly used for injectable formulations, in multiple-dose or single-dose formulations.

[0245] The method may include a preparatory step to determine whether a patient has the condition, and / or a subsequent step to determine the extent to which the treatment effectively alleviates the patient's condition, for example, in each case, performing an oral glucose tolerance test or resting blood glucose level.

[0246] Oral enteric preparations are intended to be ingested by swallowing so that they are subsequently released in the intestine below the stomach and thus delivered to the liver via the portal vein, rather than keeping the preparation in the mouth so that it can be transferred to the bloodstream via the sublingual or oral route.

[0247] Suitable dosage forms for use in this disclosure include tablets, microtablets, capsules, granules, pills, powders, effervescent solids, and chewable solid dosage forms. These formulations may include gelatin, preferably hydrolyzed gelatin or low molecular weight gelatin. Such formulations can be obtained by freeze-drying a homogeneous aqueous solution containing a calcitonin analogue and hydrolyzed gelatin or low molecular weight gelatin and further processing the resulting solid material into the oral pharmaceutical formulation, wherein the gelatin may have an average molecular weight of 1,000 to 15,000 Daltons. Such formulations may include protective carrier compounds, such as 5-CNAC or other compounds disclosed herein.

[0248] While oral formulations such as tablets and capsules are preferred, the compositions used in this disclosure may be in the form of syrups, elixirs, and suppositories. Oral delivery is generally the route of choice because it is convenient, relatively easy, and usually painless, resulting in greater patient compliance compared to other delivery methods. However, biological, chemical, and physical barriers (such as varying pH in the gastrointestinal tract, potent digestive enzymes, and gastrointestinal membranes impermeable to active agents) make oral delivery of calcitonin-like peptides to mammals problematic. For example, oral delivery of calcitonin, a long-chain polypeptide hormone secreted by the parafollicular cells of the mammalian thyroid gland and by the suprabronchial glands of birds and fish, initially proved difficult, at least in part due to the insufficient stability of calcitonin in the gastrointestinal tract and its inability to be easily transported across the intestinal wall into the bloodstream.

[0249] However, suitable oral formulations are described below.

[0250] Patient treatment

[0251] In one embodiment, the calcitonin mimic of this disclosure is administered at an adequate dose to maintain the patient's serum mimic serum level at 5 picograms to 1000 nanograms per milliliter, preferably 50 picograms to 500 nanograms per milliliter, for example 1 nanogram to 300 nanograms per milliliter. Serum levels can be measured by any suitable technique known in the art, such as radioimmunoassay or mass spectrometry. The attending physician can monitor the patient's response and then slightly adjust the dose to account for the individual patient's metabolism and response. Near-simultaneous release is best achieved by administering all components of this disclosure as a single pill or capsule. However, this disclosure also includes, for example, dividing the required amount of calcitonin mimic into two or more tablets or capsules, which can be administered together such that they together provide the necessary amount of all components. As used herein, "pharmaceutical composition" includes, but is not limited to, the complete dose suitable for specific administration to a patient, whether or not one or more tablets or capsules (or other dosage forms) are recommended in a given administration.

[0252] Unigene can be used The methods used in the product are employed to formulate the calcitonin mimics of this disclosure for oral administration. These may include the methods described in U.S. Patent Nos. 5,912,014, 6,086,918, 6,673,574, 7,316,819, 8,093,207, and U.S. Publication No. 2009 / 0317462. In particular, it may include co-formulating the compound with a protein transduction domain of a membrane transport protein such as HIV TAT protein, with one or more protease inhibitors and / or a pH-lowering agent and / or an acid-resistant protective solvent that may be coated, and / or an absorption enhancer that may be a surfactant.

[0253] In one embodiment, the calcitonin mimic of the present disclosure is preferably formulated for oral delivery in a manner known in U.S. Patent Publication No. 2009 / 0317462.

[0254] In one embodiment, the calcitonin mimic of the present invention can be formulated for enteral administration, particularly for oral administration, by mixing with a suitable carrier compound. Suitable carrier compounds include those described in U.S. Patent Nos. 5,773,647 and 5,866,536, of which 5-CNAC (N-(5-chlorosalicyloyl)-8-aminooctanoic acid, typically its disodium salt) is particularly effective. Other preferred carriers or delivery agents are SNAD (sodium salt of 10-(2-hydroxybenzoyl)decanoic acid) and SNAC (sodium salt of N-(8-[2-hydroxybenzoyl]amino)octanoic acid). In one embodiment, the pharmaceutical composition of the present disclosure comprises a delivery-effective amount of a carrier such as 5-CNAC, i.e., an amount sufficient to deliver the compound to achieve the desired effect. Typically, the carrier such as 5-CNAC is present in an amount from 2.5% to 99.4% by weight of the total composition, more preferably from 25% to 50% of the total composition.

[0255] In addition, WO 00 / 059863 discloses the disodium salt of Formula I.

[0256]

[0257] in

[0258] R 1 R 2 R 3 and R 4 Independently hydrogen, -OH, -NR 6 R 7 Halogens, C1-C4 alkyl or C1-C4 alkoxy groups;

[0259] R 5 C2-C, whether substituted or not 16 Alkylene, substituted or unsubstituted C2-C 16 alkenyl, substituted or unsubstituted C1-C 12 Alkyl (arylene), or substituted or unsubstituted aryl (C1-C2) 12 (alkylene); R 6 and R 7 Independently hydrogen, oxygen, or C1-C4 alkyl; its hydrates and solvates are particularly effective for oral delivery of active agents such as calcitonin (e.g., salmon calcitonin), which may be used in this disclosure.

[0260] Preferred enteric formulations of 5-CNAC, which may be optionally micronized, are generally as described in WO2005 / 014031.

[0261] The compound can be formulated for oral administration using methods used in Bone Medical Limited's Capsitonin products. These include methods incorporated in Axcess formulations. More specifically, the active ingredient can be encapsulated in enteric-coated capsules capable of withstanding gastric transport. This may contain the active compound along with a hydrophilic aromatic alcohol absorption enhancer, such as that described in WO02 / 028436. In a known manner, enteric coatings can become permeable in a pH-sensitive manner, for example, at pH values ​​between 3 and 7. WO2004 / 091584 also describes suitable formulation methods using aromatic alcohol absorption enhancers.

[0262] Compounds can be formulated using methods seen in Oramed products, which may contain formulations containing ω-3 fatty acids, as seen in WO2007 / 029238 or as described in US5,102,666.

[0263] Typically, pharmaceutically acceptable salts (especially monosodium or disodium salts), solvates (e.g., alcohol solvates), and hydrates of these carriers or delivery agents can be used.

[0264] Oral administration of the pharmaceutical compositions according to this disclosure can be achieved as follows: periodically, such as once or more daily or weekly; intermittently, such as irregularly within a day or week; or cyclically, such as periodically for several days or weeks, followed by a period without administration. The dosage form of the pharmaceutical compositions of the embodiments disclosed in this invention can be any known form, such as liquid or solid dosage forms. Liquid dosage forms include solution emulsions, suspensions, syrups, and elixirs. In addition to the active compound and carrier such as 5-CNAC, liquid formulations may also contain inert excipients commonly used in the art, such as solubilizers, such as ethanol; oils, such as cottonseed, castor oil, and sesame oil; wetting agents; emulsifiers; suspending agents; sweeteners; flavoring agents; and solvents, such as water. Solid dosage forms include capsules, soft gel capsules, tablets, sac-tablets, powders, granules, or other solid oral dosage forms, all of which can be prepared by methods well known in the art. The pharmaceutical composition may additionally contain additives in commonly used amounts, including but not limited to pH adjusters, preservatives, flavorings, masking agents, fragrances, humectants, tonicators, colorants, surfactants, plasticizers, lubricants such as magnesium stearate, flow aids, compression aids, solubilizers, excipients, diluents such as microcrystalline cellulose (e.g., Avicel PH 102 from FMC), or any combination thereof. Other additives may include phosphate buffer salts, citric acid, glycols, and other dispersants. The composition may also include one or more enzyme inhibitors, such as actinamide or epiactinonin and their derivatives; aprotinin, Trasylol, and Bowman-Birk inhibitors. Furthermore, transport inhibitors, i.e., [rho]-glycoproteins such as ketoprofin, may be present in the compositions disclosed herein. The solid pharmaceutical compositions of this disclosure can be prepared by conventional methods, for example, by blending, kneading, and filling a mixture of the active compound, a carrier such as 5-CNAC, and any other components into capsules, or, if not filled into capsules, by subsequently molding, followed by further compression or molding to obtain tablets. Alternatively, solid dispersions can be formed by known methods and then further processed to form tablets or capsules. Preferably, the components of the pharmaceutical compositions of this disclosure are homogenized or uniformly mixed throughout the solid dosage form.

[0265] Alternatively, the active compound can be formulated as a conjugate with the support, which may be, for example, an oligomer described in US2003 / 0069170.

[0266] compound

[0267] Such conjugates can be applied together with fatty acids and bile salts, as described herein.

[0268] Conjugates with polyethylene glycol (PEG), as described by Mansoor et al., can be used.

[0269] Alternatively, the active compound can be mixed with nitroso-N-acetyl-D,L-penicillamine (SNAP) and Carbopol solution or with taurocholate and Carbopol solution to form a mucosal adhesion emulsion.

[0270] Active compounds can be formulated by loading them into chitosan nanocapsules disclosed by Prego et al. (optionally modified with PEG such as Prego Prego C, Torres D, Fernandez-Megia E, Novova-Carballal R, Quihoa E, Alonso MJ.) or into chitosan or PEG-coated lipid nanoparticles disclosed by Garcia-Fuentes et al. As described by Guggi et al., chitosan nanoparticles for this purpose can be modified with iminotetrahydrothiophene. As described by Dogru et al., they can be formulated into water / oil / water emulsions. As described by Sinko et al. or Song et al., the bioavailability of active compounds can be increased by using taurine deoxycholate or lauroylcarnitine. Generally, nanoparticles suitable as carriers are discussed in de laFuente et al., and said nanoparticles can be used in this disclosure.

[0271] Other suitable strategies for oral formulations include the use of Chiasma's Transient Permeability Enhancer (TPE) system, as described in WO2005 / 094785. TPEs utilize an oily suspension of solid hydrophilic particles in a hydrophobic medium to protect drug molecules from inactivation due to the harsh gastrointestinal (GI) environment, while simultaneously acting on the GI wall to induce the permeation of its cargo drug molecules.

[0272] Further methods include using glutathione or compounds containing numerous thiol groups, as described in US2008 / 0200563, to inhibit the action of the efflux pump on the mucosa. Practical examples of these techniques are also described in the following literature: Caliceti, P., Salmaso, S., Walker, G., and Bernkop-Schnurch, A. (2004) 'Development and in vivo evaluation of an oral insulin-PEG delivery system'. Eur. J. Pharm. Sci., 22, 315-323; Guggi, D., Krauland, AH, and Bernkop-Schnurch, A. (2003) 'Systemic peptide delivery via the stomach: in vivo evaluation of an oral dosage form for salmon'. 'calcitonin'. J. Control. Rel. 92, 125-135, and Bernkop-Schnurch, A., Pinter, Y., Guggi, D., Kahlbacher, H., Schoffmann, G., Schuh, M., Schmerold, I., Del Curto, MD, 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, 26-33.

[0273] Active compounds can be formulated in seamless microspheres, as described in WO2004 / 084870, wherein the active pharmaceutical ingredient is dissolved in an emulsion, microemulsion, or suspension formulated into microspheres; and variable coating is performed using conventional or novel coating techniques. The result is an encapsulated drug in a “pre-dissolved” form, which, upon oral administration, provides predetermined immediate or sustained release of the active pharmaceutical ingredient to a specific location and at a specific rate along the gastrointestinal tract. Essentially, pre-dissolving the drug enhances the predictability of the pharmacokinetic profile while simultaneously improving permeability and drug stability.

[0274] Chitosan-coated nanocapsules, as described in US2009 / 0074824, can be used. Active molecules applied using this technique are protected within the nanocapsules because they are stable against the action of gastric juices. Furthermore, the mucosal adhesive properties of this system enhance adhesion time to the intestinal wall (a delay in gastrointestinal transit has been demonstrated in these systems), thereby facilitating more efficient absorption of the active molecules.

[0275] Methods developed by TSR1 can be used. These include hydrophilic solubilization technology (HST), in which gelatin (a naturally derived collagen extract with both positive and negative charges) coats particles containing the active ingredient in lecithin micelles and prevents them from agglomerating or clumping. This results in improved wettability of the hydrophobic drug particles through polar interactions. Furthermore, the amphiphilic lecithin reduces the surface tension between the dissolving fluid and the particle surface.

[0276] The active ingredient can be formulated together with cucurbituril as an excipient.

[0277] Alternatively, Merrion Pharmaceuticals’ GIPET technology can be used to produce enteric-coated tablets containing an active ingredient and an absorption enhancer, which may be a medium-chain fatty acid or a medium-chain fatty acid derivative as described in US2007 / 0238707 or a membrane transpeptide as described in US7268214.

[0278] GIRES can be used TM The technology comprises a controlled-release dosage form contained within an inflatable sachet placed within a drug capsule for oral administration. After the capsule dissolves, a gas-generating system inflates the sachet in the stomach. Clinical trials have shown that the sachet remains in the stomach for 16–24 hours.

[0279] Alternatively, the active ingredient can be conjugated with a protective modifier to enable it to withstand enzymatic degradation in the stomach and facilitate its absorption. The active ingredient can also be covalently conjugated with a monodisperse short-chain methoxy polyethylene glycol glycol lipolipide derivative, which, after purification, is crystallized and lyophilized into a dry active pharmaceutical ingredient. These methods are described in US5438040 and www.biocon.com.

[0280] Hepatic-directed vesicles (HDVs) can also be used for active delivery. HDVs can consist of liposomes (≤150 nm in diameter) encapsulating an active substance, the liposomes also containing hepatocyte-targeting molecules within their lipid bilayer. The targeting molecules guide the delivery of the encapsulated active substance to hepatocytes, thus requiring relatively small amounts of the active substance to be effective. Such a technique is further described in US2009 / 0087479 and at www.diasome.com.

[0281] The active substance can be incorporated into a composition that further comprises a substantially non-aqueous hydrophilic medium comprising an alcohol and a co-solvent, the hydrophilic medium being combined with a medium-chain moiety of glycerides, optionally mixed with a long-chain PEG substance, as described in US2002 / 0115592 concerning insulin.

[0282] Alternatively, the intestinal patch described in the following literature may be used: Shen Z, Mitragotri S, PharmRes. 2002 Apr; 19(4):391-5 'Intestinal patches for oral drug delivery'.

[0283] As described in U.S. Patent No. 7,189,414, active materials can be incorporated into an easily eroded matrix formed from a hydrogel blended with a hydrophobic polymer.

[0284] The appropriate oral dose level for adults to be treated can be 0.05 to 5 mg, preferably about 0.1 to 2.5 mg.

[0285] The patient's dosage and treatment frequency can be 1 to 4 times per week, preferably 1 to 2 times per week, and most preferably once per week. It is desired that the treatment will be maintained for an extended period of at least 6 weeks, preferably at least 6 months, preferably at least 1 year, and optionally for life.

[0286] The compositions according to this disclosure can be used in combination with one or more other therapeutic agents for the treatment of related conditions. Alternatively, the compositions according to this disclosure can be combined with one or more other therapeutic agents for combined administration.

[0287] Combination therapies according to this disclosure include combinations of the active compounds as described with insulin, GLP-2, GLP-1, GIP, or amylin, or generally combinations with other antidiabetic drugs. Therefore, combination therapies including co-formulations can be prepared with: insulin sensitizers, including biguanides such as metformin, buformin, and phenformin; TZDs (PPARs) such as balagliflozin, pioglitazone, linaglitazone, rosiglitazone, and troglitazone; dual PPAR agonists such as agliflozin, mogtazol, and ticaglitazone; or secretagogues, including sulfonylureas such as sulfamethoxazole, chlorpropamide, gliclazide, toluenesulfonamide, tolazoline, glipizide, glibenclamide, glibenclamide, glimepiride, glipizide, and glimepiride; megliflozin / glinides (K+) such as nateglinide, repaglinide, and mitiglinide; G… LP-1 analogs such as exenatide, lixilatide, liraglutide, semaglutide, dulaglutide, and abiglutide; DPP-4 inhibitors such as alogliptin, linagliptin, saxagliptin, sitagliptin, and vidagliptin; insulin analogs or special formulations such as (rapid-acting) lispro insulin, aspart insulin, glutalis insulin, (long-acting) insulin glargine insulin, detemir insulin, inhalable insulin-Exubra, and NPH insulin; and others, including α-glucosidase inhibitors such as acarbose, miglitol, and voglibose; amylin analogs such as pramlintide; SGLT2 inhibitors such as dapagliflozin, empagliflozin, repagliptin, and seragliflozin; and miscellaneous classes including benflurexate and toresistat.

[0288] Further combinations include co-administration or co-formulation with leptin. Leptin resistance is a well-established component of type 2 diabetes; however, to date, injectable leptin has failed to improve this condition. Conversely, there is evidence supporting amylin and therefore molecules with amylin-like capabilities, such as salmon calcitonin mimics, to improve leptin sensitivity. Amylin / leptin combinations have been shown to have synergistic effects on body weight and food intake, as well as insulin resistance [Kusakabe T et al.].

[0289] Further preferred combination therapies include co-formulating or co-administering the peptides of the present 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 amphetamine derivatives (e.g., phenbutylide), topiramate, etc. (Phenylbutazone / Topiramate combination), 5-HT 2C Receptor agonists (e.g., Locaserin), (naltrexone / bupropion combination), glucagon-like peptide-1 [GLP-1] analogs and derivatives (e.g., liraglutide, semaglutide), sarcoplasmic / endoplasmic reticulum (SR) Ca 2+ATPase (SERCA) inhibitors (e.g., myolipin), fibroblast growth factor 21 (FGF-21) receptor agonists (e.g., FGF-21 analogs), and β3-adrenergic receptor agonists (e.g., Mirabegron). Such combinations can be used to treat overweight conditions, such as obesity. Attached Figure Description

[0290] Figure 1 Comparison of food intake and body weight using KBP346, KBP347, KBP349, KBP351, KBP352, KBP353, and KBP089. A) Food intake, 0 to 4 hours. B) Body weight change, 4 hours. C) Food intake, 4 to 24 hours. D) Body weight change, 24 hours. E) Food intake, 24 to 49 hours. F) Body weight change, 48 hours.

[0291] Figure 2 Single-dose testing of KBP375, KBP376, and KBP377. A single dose was administered at t=0, and the effects of each molecule at a single dose of 36 nmol / kg on food intake and body weight were monitored for 168 hours, with head-to-head comparisons to an unacylated baseline. A) Food intake. B) Body weight change.

[0292] Figure 3 Dose-response testing of KBP356, KB358, KBP362, KBP364, KB368, and KBP370. A single dose was administered at t=0, and the effect of a single dose of 36 nmol / kg on food intake and body weight was monitored for 168 hours. AB) Food intake and body weight of the acylated KBP-066 variant. CD) Food intake and body weight of the acylated KBP-062 variant. EF) Food intake and body weight of the acylated KBP-110 variant.

[0293] Figure 4 Effects of single high doses of KBP372 and KBP356 on food intake and body weight. A) Effect of KBP-042A11.03 (KBP372) on food intake. B) Effect of KBP-042A11.03 (KBP372) on body weight. C) Effect of KBP-066A11.03 (KBP356) on food intake. D) Effect of KBP-066A11.03 (KBP356) on body weight.

[0294] Figure 5 : A 4-hour food intake study of KBP350.

[0295] Figure 6Cumulative food intake. A) Cumulative food intake over time. Food intake was monitored daily for the first 21 days of the study. n = 3 to 4 cages + / - SEM. B) In Figure 9 The total area under the curve shown in A. n = 9-10. + / -SEM.

[0296] Figure 7 ZDF body weight during the study period. A) Individual rat body weight (g), B) Body weight percentage normalized to solvent. Body weight was recorded daily for the first 21 days, then twice weekly until one week before the end of the study (day 62). Body weight in the KBP-066A11.03 group was monitored daily until one week before the end of the study (day 62). n = 9–10 rats. + / - SEM.

[0297] Figure 8 Fasting blood glucose (ZDF). Fasting blood glucose was measured on days 0, 14, 28, 42, and 62 after a 6-hour fast. n = 9–10. + / - SEM.

[0298] Figure 9 ZDF HbA1c values: A) Baseline HbA1c. B) HbA1c at the end of the study. HbA1c was measured from day -3 at the start of the study. HbA1c was measured at the end of the study, day 62. n = 9-10. + / - SEM.

[0299] Figure 10 Oral glucose tolerance test (OGTT). A) OGTT over 180 minutes in male ZDF rats. B) Total area under the curve during the OGTT shown in A). OGTT was performed after 8 weeks of treatment. Rats were fasted for 11 hours prior to the -30 minute time point. Blood glucose levels were measured at time points -30, 0, 15, 30, 60, 120, and 180 minutes. Oral glucose was administered at the 0 minute time point. A result above 33.3 mmol / L was considered acceptable. -1 The blood glucose level was designated as the upper limit for detection; 33.3 mmol / L. -1 Rats were not pre-administered saline, KBP-066, or KBP-066A on the same day. n = 9-10. + / - SEM.

[0300] Figure 11Single-dose testing of KBP-305, KBP-306, KBP-307, KBP-356, KBP-381, KBP-382, and KBP-383. A single dose was administered at t=0, and the effects of a single dose of 3 nmol / kg of each molecule on food intake and body weight were monitored for 96 hours, with head-to-head comparisons with each other and with the solvent to determine the optimal acylation length. A) Short-term food intake (g). B) Body weight change (g). n=4 rats per group. Data are presented as + / - SEM.

[0301] Figure 12 A six-week weight loss study in HFD SD rats using KBP-066A11 compounds with different acylation lengths, .03, .04, and .05. Rats were treated with KBP-066A11.03, KBP-066A11.04, KBP-066A11.05, or a solvent, and administered to rats via a single subcutaneous injection of 4 nmol / kg of the compound every 3 days. Body weight was recorded daily throughout the study. A) Daily food intake (g); B) Individual rat weight loss (g). n = 4 rats per group. Data are presented as + / - SEM.

[0302] Figure 13 Other parameters in a weight loss study in HFD SD rats using KBP-066A11 compounds with different acylation lengths, .03, .04, or .05. Rats were treated with KBP-066A11.03, KBP-066A11.04, KBP-066A11.05, or a solvent. Rats were administered a single subcutaneous injection of 4 nmol / kg of the compound every 3 days. A) Oral glucose tolerance test. B) Area under the curve of OGTT. C) Weight of epididymal WAT at the end of the study (g). D) Weight of inguinal WAT at the end of the study (g). E) Weight of perirenal WAT at the end of the study (g). F) Change in body weight relative to baseline at the end of the study (g). Body weight was recorded daily throughout the study. n = 4 rats per group. Data are presented as + / - SEM.

[0303] Figure 14 : Radiolabeled salmon calcitonin ( 125 Competitive ligand binding assays were performed using I-sCT as a tracer, and 2% serum albumin was measured in two different species: rats (Rattus norvegicus) and humans (Homo sapiens). 0.25 nM sCT was used. 125I-sCT was used as a tracer. A) Competitive binding assay performed in 2% RSA. B) Competitive binding assay performed in 2% HSA. Data are expressed as + / - SEM.

[0304] Figure 15 Single-dose assays of KBP-356, KBP-386, KBP-387, KBP-388, KBP-389, and KBP-390 were used to investigate the acylation sites of the KBP-066 backbone. A single dose was administered at t=0, and the effects of a single dose of 3 nmol / kg of each molecule on food intake and body weight were monitored for 96 hours, with head-to-head comparisons with each other and with the solvent to determine the optimal acylation site. A) Short-term food intake (g). B) Body weight change (g). n=4 rats per group. Data are presented as + / - SEM.

[0305] Figure 16 Single-dose assays of KBP-391, KBP-312, KBP-313, KBP-314, KBP-315, KBP-316, KBP-317, and KBP-318 were used to investigate the acylation sites of the KBP-021 backbone. A single dose was administered at t=0, and the effects of a single dose of 3 nmol / kg of each molecule on food intake and body weight were monitored for 96 hours, with head-to-head comparisons with each other or with the solvent to determine the optimal acylation site. A) Short-term food intake (g). B) Body weight change (g). n=4 rats per group. Data are presented as + / - SEM.

[0306] Figure 17 A six-week weight loss study in HFD SD rats using KBP-066 compounds with the same acylation lengths of .03, .04, and .05 but different positions (A11 and A19). Rats were administered KBP-066A11.03 (KBP-356), KBP-066A19.03 (KBP-389), or a solvent via subcutaneous injection every 3 days. Body weight and food intake were recorded daily throughout the study. A) Daily food intake (g) during the study period. B) Individual rat weight loss (g). n = 4 rats per group. Data are presented as + / - SEM.

[0307] Figure 18Other parameters in a weight loss study in HFD SD rats using KBP-066A11 compounds with different acylation lengths, .03, .04, and .05 acylation. Rats were treated with KBP-066A11.03 or KBP-066A19.03 or the solvent. Rats were administered a single subcutaneous injection of 4 nmol / kg of the compound every 3 days. A) Oral glucose tolerance test. B) Area under the curve of OGTT. C) Weight of epididymal WAT at the end of the study (g). D) Weight of inguinal WAT at the end of the study (g). E) Weight of perirenal WAT at the end of the study (g). F) Change in body weight relative to baseline at the end of the study (g). Body weight was recorded daily throughout the study. n = 4 rats per group. Data are presented as + / - SEM.

[0308] Figure 19 The acylation linker of the KBP-066 scaffold was investigated using single doses of KBP-356, KBP-384, and KBP-385. Single doses were administered at t=0, and the effect of each molecule at a single dose of 4 nmol / kg on body weight was monitored for 96 hours and compared with each other to determine the optimal acylation linker. A) Short-term food intake (g). B) Body weight change (g). n=4 rats per group. Data are presented as + / - SEM. Detailed Implementation

[0309] Example

[0310] The embodiments of this disclosure described in the following examples are provided to aid in understanding this disclosure and should not be construed as limiting the scope of this disclosure in any way, as defined in the following claims. The following examples are presented to provide a complete disclosure and description of how to make and use the described embodiments to those skilled in the art. These examples are not intended to limit the scope of this disclosure, nor are they intended to represent all or only the experiments conducted. Every effort has been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise stated, parts are parts by weight, molecular weights are weight-average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or near atmospheric pressure. The following materials and methods are used in the following examples.

[0311] Cells and cell lines

[0312] The following cell lines expressing calcitonin, amylin, and CGRP receptors were purchased and cultured according to the manufacturer's instructions.

[0313] 1. Calcitonin receptor (CTR): U2OS-CALCR from DiscoveRx (catalog number: 93-0566C3).

[0314] 2. Amylin receptor (AMY-R): CHO-K1 CALCR+RAMP3 from DiscoveRx (catalog number: 930268C2).

[0315] chemicals

[0316] Thioflavin T (T3516, Sigma). Prepare a 10 mM solution of the stock solution ThT in 5 mM sodium phosphate at pH 7.2. Store aliquots at -20°C protected from light. Thaw and dilute the stock solution ThT just before use.

[0317] For the calcitonin mimic tested (hereinafter referred to as "acylated KBP" or simply "KBP"), the final buffer conditions were 10 mM Tris-HCl pH 7.5.

[0318] The final peptide concentration in the well should be 100 to 200 μM, and the final ThT concentration should be 4 μM. Finally, add 10 μL of ThT.

[0319] animal models

[0320] In animal model studies, the potency of acylated KBP was evaluated using 12-week-old healthy Sprague Dawley (SD) rats. In some instances, they were fed a normal diet before and during the test, while in others, the 12-week-old healthy SD rats were fed a high-fat diet (HFD) for 8 weeks before and during the test.

[0321] Acylated calcitonin mimics

[0322] Tables 1a and 1b below list the amino acid sequences of the tested acylated calcitonin mimics. As used therein:

[0323] 1. Acylation refers to K Ac -(glutamate linker)-(C16 fatty acid [palmitate]);

[0324] 2-acylation refers to K Ac -(glutamate linker)-(C18 diacid [octadecanoic acid]);

[0325] 3-acylation refers to K Ac -(2xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C18 diacid [octadecanoic acid]).

[0326] 4-acylation refers to K Ac -(2xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C20 diacid [eicosanoic acid]).

[0327] 5-acylation refers to K Ac -(2xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C22 diacid [docosahexaic acid]).

[0328] 6-acylation refers to K Ac -(2xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C16 diacid [hexadecanoic acid]).

[0329] 7-acylation refers to K Ac -(3xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C18 diacid [octadecanoic acid]).

[0330] 8-acylation refers to K Ac -(3xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C18 diacid [octadecanoic acid]).

[0331] 9-acylation refers to K Ac -(2xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C24 diacid [tetracodic acid]).

[0332] 10-acylation refers to K Ac -(2xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C26 diacid [hexadecanoic acid]).

[0333] 11-acylation refers to K Ac -(2xOEG amino acids linked together with glutamic acid residues attached to the N-terminus)-(C14 diacid [tetradecanoic acid]).

[0334] The tested calcitonin mimicry was based on the following unmodified core peptide sequence:

[0335] CSNLSTCMLGRLSQDLHRLQTYPKTDVGANAP(KBP089)

[0336] CSNLSTC(AiB)LGRLSQDLHRLQTYPKTDVGANAP(KBP066)

[0337] CGNLSTC(AiB)LGRLTQDLNKFHTFPKTDVGANAP(KBP062)

[0338] CSNLSTCVLGKLSQELHKLQTYPRTDVGANAP(KBP042)

[0339] CSNLSTC(AiB)LGRLANFLVHSSNNFGAILPKTDVGANAP(KBP110)

[0340] CSNLSTCMLGRLSQELHRLQTYPKTDVGANAP(KBP021)

[0341] The following additional naming rules were also used in Table 1b:

[0342]

[0343]

[0344] Therefore, for example, naming the peptide KBP-066A11.03 indicates that the peptide is composed of the KBP-066 core sequence, which is modified by the lysine residue at position 11 being replaced by C18 diacid 2*OEG acylation.

[0345]

[0346]

[0347]

[0348] Various acylations have the following chemical structures:

[0349] OEG-OEG-yGlu-C14 diacid (i.e. 11 acylation)

[0350]

[0351] OEG-OEG-yGlu-C16 diacid (i.e. 6-acylated)

[0352]

[0353] OEG-yGlu-C18 diacid (i.e. 8-acylated)

[0354]

[0355] OEG-OEG-yGlu-C18 diacid (i.e. 3-acylated)

[0356]

[0357] OEG-OEG-OEG-yGlu-C18 diacid (i.e. 7-acylation)

[0358]

[0359] OEG-OEG-yGlu-C20 diacid (i.e. 4-acylated)

[0360]

[0361] OEG-OEG-yGlu-C22 diacid (i.e. 5-acylated)

[0362]

[0363] OEG-OEG-yGlu-C24 diacid (i.e. 9-acylated)

[0364]

[0365] OEG-OEG-yGlu-C26 diacid (i.e. 10-acylated)

[0366]

[0367] Initial acylation studies (Examples 1 to 5)

[0368] Example 1 ( Figure 1 and Figure 5 )

[0369] Single-dose comparison of the effects of 1-acylated variants at different positions (position 9 "A09", position 11 "A11", position 16 "A16", position 18 "A18" and position 32 "A32") on food intake and body weight in 12-week-old lean SD rats with the non-acylated baseline peptide (KBP-089).

[0370] KBP core Position / Acylation KBP-346 KBP-042 A11 / 1 acylation KBP-347 KBP-089 A18 / 1 acylation KBP-349 KBP-089 A11 / 1 acylation KBP-350 KBP-089 A12 / 1 acylation KBP-351 KBP-089 A16 / 1 acylation KBP-352 KBP-089 A9 / 1 acylation KBP-353 KBP-089 A32 / 1 acylation

[0371] Rats were housed individually for four days prior to the test. Rats were randomly assigned to six groups based on body weight (solvent (0.9% NaCl), KBP (dose: 25 nmol / kg (^100 μg / kg)). They were fasted overnight and then treated with a single subcutaneous dose of either the peptide or the solvent in the morning. Food intake was monitored at the following intervals (0 to 4 hours, 4 to 24 hours, 24 to 48 hours). Body weight was measured at baseline and 24 and 48 hours after subcutaneous injection.

[0372] Acylation at positions “A09”, “A11”, and “A32” produced a durable in vivo response. Figure 1 Further testing is warranted (see below). The 12th position ( Figure 5 The 16th and 18th bits returned unacceptable results, so no further experiments were conducted.

[0373] Example 2: β-Arrestin assay

[0374] The PathHunter β-repressor GPCR assay is a whole-cell functional assay that directly measures the ability of ligands to activate GPCRs by detecting the interaction between β-repressor protein and activated GPCRs. Because the recruitment of β-repressor protein is independent of G protein signaling, these assays provide a robust and versatile screening and analysis platform applicable to virtually any Gi-coupled, Gs-coupled, or Gq-coupled receptor.

[0375] In this system, GPCR is combined with the small enzyme fragment ProLink. TM The GPCR is fused within the GPCR and co-expressed in cells that stably express a fusion protein of β-repressor protein and a larger β-gal N-terminal deletion mutant (called the enzyme receptor or EA). Activation of the GPCR stimulates the binding of the β-repressor protein to the ProLink-tagged GPCR and forces the two enzyme fragments to complement each other, thus forming an active β-gal enzyme. This interaction leads to an increase in enzyme activity, which can be utilized using chemiluminescence... The test reagents are used for measurement.

[0376] In independent bioassays, CTR and AMY-R cells were treated at specified time points with increasing KBP doses (100, 20, 4, 0.8, 0.16, 0.032 nM and solvents) as shown in Tables 2 and 3. The assays were performed in white 384-well plates (Greiner Bio-One, 784080). Cells were seeded at 2500 cells per well in 10 μL of cell type-specific medium the day before the experiment. Pathhunter was used to quantify GPCR-mediated β-repressor protein recruitment. TM The test kit (93-0001, DiscoverX) was used, and the corresponding measurements were performed according to the manufacturer's instructions.

[0377] Sustained / durable responses were assessed using calcitonin receptor (CTR): U2OS-CALCR from the DiscoveRx (catalog number: 93-0566C3) cell line. Contrary to the conventional three-hour output, β-repressor protein accumulation was measured and analyzed over 3, 6, 24, 48, or 72 hours. Tables 2 (2-acylation) and 3 (3-acylation) list the results of the β-repressor protein studies.

[0378] Table 2. p-2-acylation (K Ac Study of β-repressor protein of β-(glutamate linker)-(C18 diacid)

[0379]

[0380] Table 3. P-3 acylation (K) AcStudy of β-repressor protein of -(2xOEG amino acids linked to a glutamate residue at the N-terminus)-(C18 diacid [octadecanoic acid]))

[0381]

[0382]

[0383] The β-repressor protein study showed the following:

[0384] 1) In terms of the acylation position on the peptide, the acylation efficacy is as follows: A11>A32>A09.

[0385] 2) In terms of activating calcitonin receptor (CTR), amylin receptor (AMY-R), prolonging CTR response, and inhibiting food intake, 2-acylation or 3-acylation at position 11 is generally a far superior combination of acylation / position for each peptide core.

[0386] 3) When the same acylation modification was used, acylated KBPs with different cores demonstrated similar potency and patterns in vitro.

[0387] Example 3 ( Figure 2 )

[0388] Single-dose comparison of the effects of the 3-acylated variants of KBP089 (A09 (KBP375), A11 (KBP376), and A32 (KBP377) on food intake and body weight in 20-week-old HFD SD rats compared with the non-acylated baseline (KBP-089).

[0389] KBP core Notes Position / Acylation KBP-375 KBP-089 KBP-089A09.03 A9 / 3 acylation KBP-376 KBP-089 KBP-089A11.03 A11 / 3 acylation KBP-377 KBP-089 KBP-089A32.03 A32 / 3 acylation

[0390] Rats were housed individually for four days prior to the test. Rats were randomly assigned to 11 groups based on body weight (solvent (0.9% NaCl), KBP (dose: 36 nmol / kg (150–157 μg / kg)). They were fasted overnight and then treated with a single subcutaneous dose of either the peptide or the solvent in the morning. Food intake was monitored at the following intervals (0–4 hours, 4–24 hours, 24–48 hours…144–168 hours). Body weight was measured at baseline and every 24 hours after subcutaneous injection.

[0391] Animal model studies confirmed the results of the β-inhibitory protein study and demonstrated its improved efficacy compared to naked peptides:

[0392] 1) Using KBP-089 as the core peptide, in terms of the benefit of acylation position, A11>A32>A09.

[0393] 2) In terms of sustained in vivo activity and efficacy, 2-acylated and 3-acylated KBP-089 are far superior to unacylated KBP-089 at a given dose.

[0394] Animal model studies also showed that acylation at position 9 reduced the peptide's potency compared to naked peptides, thus ruling out position 9 as a potential site for further research.

[0395] Example 4 ( Figure 3 )

[0396] Single-dose comparison of the effects of 3-acylated variants of A11 and A32 with different peptide cores on food intake and body weight in 20-week-old HFD SD rats with their respective non-acylated baseline KBPs (KBP-066, KBP-062 and KBP-110).

[0397] KBP core Notes Position / Acylation KBP-356 KBP-066 KBP-066A11.03 A11 / 3 acylation KBP-358 KBP-066 KBP-066A32.03 A32 / 3 acylation KBP-362 KBP-062 KBP-062A11.03 A11 / 3 acylation KBP-364 KBP-062 KBP-062A32.03 A32 / 3 acylation KBP-368 KBP-110 KBP-110A11.03 A11 / 3 acylation KBP-370 KBP-110 KBP-110A32.03 A32 / 3 acylation

[0398] Rats were housed individually for four days prior to the test. Rats were randomly assigned to 11 groups based on body weight (solvent (0.9% NaCl), KBP (dose: 4 nmol / kg (^17 μg / kg), 12 nmol / kg (^50 μg / kg), or 36 nmol / kg (^150 μg / kg)). They were fasted overnight and then treated with a single subcutaneous dose of either the peptide or the solvent in the morning. Food intake was monitored at the following intervals (0 to 4 hours, 4 to 24 hours, 24 to 48 hours... 144 to 168 hours). Body weight was measured at baseline and every 24 hours after subcutaneous injection.

[0399] The results are as follows:

[0400] 1) The peptide core does not affect the improvement observed by acylation at position 11 or 32.

[0401] 2) A11 is a better acylation site than A32.

[0402] Example 5 ( Figure 4 )

[0403] Effects of single high-dose KBP-042 and KBP-066 A11 / 3 acylated variants on food intake and body weight in 20-week-old HFD SD rats. Rats were housed individually for four days prior to the test. Rats were randomly assigned to 11 groups based on body weight (solvent (0.9% NaCl), KBP (dose: 300 nmol / kg (^1000 μg / kg)).

[0404] KBP core Notes Position / Acylation KBP-372 KBP-042 KBP-042A11.03 A11 / 3 acylation KBP-356 KBP-066 KBP-066A11.03 A11 / 3 acylation

[0405] Rats were fasted overnight and then treated with a single subcutaneous dose of either the peptide or the solvent in the morning. Food intake was monitored at the following intervals (0 to 4 hours, 4 to 24 hours, 24 to 48 hours...188 to 312 hours). Body weight was measured at baseline and every 24 hours after subcutaneous injection.

[0406] High-dose testing using KBP356 and KBP372 demonstrated superior, sustained in vivo efficacy lasting for several days. Therefore, these acylated peptides are clear candidates for the development of once-weekly peptide therapeutics.

[0407] Example 6 ( Figures 6 to 10 )

[0408] Further work was conducted on compound KBP-356 (KBP-066A11.03), which contains an AiB residue at position 8 of the peptide and a preferred acylation at position 11 of the peptide.

[0409] Long-term studies were conducted in male ZDF rats. (Obese homozygous recessive (fa / fa) strain: 370) (Charles River, USA). Rats were transported at 5 weeks of age. 2-3 rats were housed in each cage.

[0410] Long-term treatment of male ZDF rats:

[0411] At 5 weeks of age (day 6), rats were transported to the Nordic Bioscience animal facility. The rats were allowed to acclimatize for three days. HbA1c and body weight (BW) were recorded (day 3). On day 4, the rats were randomly assigned to groups based on HbA1c (major) and BW (minor). The study began on day 1.

[0412] Dosage concentration and frequency

[0413] Administer KBP-066 or saline (solvent) to animals once daily. Administer KBP-066A11.03 every three days. Administer subcutaneously (SC) around midday.

[0414] Saline solution: dosage volume is 1 mL / kg.

[0415] KBP-066: The dosage volume is 1 mL / kg, and the dosage concentration is 5, 50, or 500 μg / kg, with a compound concentration of 5, 50, or 500 mg / L. The dose equivalents (in nmol / kg) are 1.43, 14.3, and 143 nmol / kg, respectively.

[0416] KBP-066A11.03: The dosage volume is 1 mL / kg, the dosage concentration is 25 μg / kg, and the compound concentration is 25 mg / L. The dose equivalent (in μg / kg) is 104 μg / kg.

[0417] Treatment group (nmol / kg)

[0418]

[0419] Treatment group (μg / kg)

[0420]

[0421] Total weekly dose for each treatment group:

[0422] 5 μg / kg KBP-066 is equivalent to 35 μg / kg / week or 10 nmol / kg / week.

[0423] 50 μg / kg KBP-066 is equivalent to 350 μg / kg / week or 100.4 nmol / kg / week.

[0424] 500 μg / kg KBP-066 is equivalent to 3500 μg / kg / week or 1004 nmol / kg / week.

[0425] 25 nmol / kg KBP-066 is equivalent to 243.4 μg / kg / week or 58.3 nmol / kg / week.

[0426] The compound was dissolved in brine and stored at -20°C. Aliquots were thawed just before application.

[0427] Collection of test results

[0428] Day -3: HbA1c measurement

[0429] Day 1: (First day of the study) Rats were fasted for 6 hours and blood and bilirubin levels were collected. Drug administration was then administered.

[0430] Day 14: Fasting blood glucose (FBG) + blood sample (fasting for 6 hours)

[0431] Day 28: FBG + blood sample (fasting for 6 hours)

[0432] Day 42: FBG + blood sample (fasting for 6 hours)

[0433] Day 57: (gr. 1+2) / Day 58 (gr. 3+4) OGTT, without prior administration of KBP-066 or KBP-066A11.03 (11-hour fast). Hb1Ac was measured at t=120 or t=180 during OGTT.

[0434] Day 62: FBG + blood sample (fasting for 6 hours)

[0435] Food intake

[0436] Monitor your food intake daily. Monitor your weight daily for the first three weeks, then twice a week thereafter.

[0437] fasting blood glucose

[0438] use The monitoring system (Roche Diagnostics, Rotkreuz, Switzerland) monitors fasting blood glucose every two weeks: the measurement is taken from the tail vein (25G needle).

[0439] HbA1c

[0440] For the first (randomized) and second (after the second OGTT) HbA1c measurements, rats were not fasted. A drop of blood was added to the HbA1c cassette, and HbA1c was measured using a DCA Vantage analyzer. Compound or saline administration was subsequently administered during the first and second HbA1c measurements.

[0441] Oral glucose tolerance test

[0442] An oral glucose tolerance test (OGTT) was performed after eight weeks of treatment. The glucose dose was calculated using the animal's body weight the previous day. Animals were fasted for 11 hours. Calories were administered approximately 45 minutes before the time point -30 minutes (see figure below). Animals were pre-administered KBP-066, KBP-066A11.03, or saline during the first OGTT, but not during the second OGTT, hence (C) in the figure below.

[0443] OGTT Table

[0444]

[0445] B = Blood sample (EDTA), approximately 200-300 μL

[0446] BG = Blood Glucose

[0447] G = Glucose (oral administration of 1g glucose / kg BW, 2mL / kg)

[0448] C = compound (or salt water) (SC.)

[0449] result

[0450] Figure 6 A+B: Cumulative food intake

[0451] Figure 6A shows the cumulative food intake during the study period. All treatment groups consumed less food than the solvent. Furthermore, higher doses resulted in greater reductions in food intake. The acylated KBP-066A11.03 treatment group showed the greatest reduction in food intake compared to all doses of KBP-066. At the end of the study, all treatment groups consumed significantly less food during the study compared to the solvent, with the acylated KBP-066A11.03 treatment showing the greatest reduction in food intake; approximately 35% less food intake compared to the solvent. Figure 6 B).

[0452] Figure 7 A+B, weight

[0453] During the first three weeks of the study, all treatment groups experienced weight loss. As the ZDF solvent rats became increasingly ill, they were unable to maintain their weight gain / weight loss rate. Figure 7 A) The treated group caught up with the solvent group in terms of weight gain. Compared to the solvent group, the rate of weight gain depended on the dosage and also on the type of compound used in the treated group. Figure 7 B). The recovery rate was slowest in the acylated KBP-066A11.03 treatment group, followed by the 14.3 and 143 nmol / kg groups, while the fastest weight recovery was observed at 1.43 nmol / kg.

[0454] This demonstrates that administering acylated KBP-66A11.03 subcutaneously every three days provides additional pharmacological benefits compared to administering unacylated KBP-066 subcutaneously once daily.

[0455] Figure 8 fasting blood glucose

[0456] As the ZDF solvent rats' condition progressively worsened, fasting blood glucose (FBG) could not be maintained. Therefore, all treatment groups effectively reduced FBG during the study period compared to the solvent group. Acylated KBP-066A11.03 treatment was the most effective, resulting in only a modest increase of 5 mM in FBG during the 62-day study in this hyperaggressive animal model of type 2 diabetes. Unacylated KBP-066 reduced FBG in a dose-dependent manner but was less effective than the acylated treatment group in reducing FBG. Similarly, this demonstrates that acylated KBP-066A11.03 provides additional pharmacological benefits compared to unacylated KBP-066.

[0457] Figure 9 HbA1c at baseline and at the end of the study

[0458] As expected, baseline HbA1c values ​​in male ZDF rats were almost identical before the onset of diabetes and the treatment program. Figure 9A). At the end of the study (day 62), HbA1c levels were significantly lower in all treatment groups compared to the solvent. Interestingly, the acylated KBP-066A11.03 treatment group had the lowest HbA1c value. Furthermore, it was significantly lower than all the unacylated KBP-066 treatment groups. Figure 9 B) demonstrates the further advantages of acylation compared to the unacylated equivalent.

[0459] Figure 10 Oral glucose tolerance test (OGTT)

[0460] An oral glucose tolerance test was performed eight weeks after treatment, and the results were as follows: Figure 10 As shown in Figure A, individual OGTT curves differ significantly due to treatment-induced differences in FBG. This difference is highlighted in the calculated tAUC values. Figure 10 B). Compared with the solvent, the tAUC of all treatment groups was significantly lower. The acylated KBP-066A11.03 treatment group had the lowest tAUC value, which was also significantly lower than two of the three unacylated KBP-066 treatment groups, with a p-value of 0.06 compared to the last group (143 nmol / kg KBP-066). Figure 10 B).

[0461] In summary, the collective data showed that in obese and diabetic ZDF rats, administration of acylated KBP-66A11.03 every three days via a subcutaneous regimen provided additional pharmacological benefits compared to once-daily subcutaneous administration of unacylated KBP-066.

[0462] Summary of Results of Examples 1 to 6

[0463] Results of acylation studies based on acylation sites

[0464] Position A09 (acylated at position 9 of the peptide)

[0465] The acylation at position "A09" with 1 acylation produced sustained, long-lasting in vivo activity, warranting further testing. Figure 1 (A to F).

[0466] Furthermore, acylation at position A09 with 2-acylation and 3-acylation weakens the EC50 of the CTR and AMYR receptors and does not produce a long-term response to CTR (Tables 3-4).

[0467] However, the acylation of A09 with both 2- and 3-acylation disrupts the previously observed long-term in vivo efficacy of the core peptide, thus rendering the acylated KBP as potent as the solvent. Therefore, following a single subcutaneous injection of 36 nmol / kg of the compound, they were superior to the unacylated core peptide in both reducing food intake and weight loss. Figure 2 AB) Low effectiveness.

[0468] Therefore, position A09 is no longer considered.

[0469] Position A11 (acylation at position 11 of the peptide)

[0470] The acylation at position A11 with 1-acylation produced sustained, long-lasting in vivo activity, warranting further testing. Figure 1 (A to F).

[0471] The acylation of position A11 with 2-acylation and 3-acylation yielded the best EC50 for the determination of CTR and AMYR receptors and produced the highest long-term response value (tAUC) among all tested core peptides (Table 3-4).

[0472] Furthermore, after a single subcutaneous injection of 36 nmol / kg of the compound, with reduced food intake ( Figure 2 A) and weight loss ( Figure 2 In aspect B), A11 / 3 acylation significantly improved the in vivo activity of the core peptide compared to non-acylated core peptides. When KBP-089 was used as the core peptide, A11 / 3 acylation was also better than any other acylation site in reducing food intake and weight loss. Figure 2 (A to B).

[0473] In dose response test ( Figure 3 This difference is further emphasized in (A to F), where all three doses (4 nmol / kg, 12 nmol / kg, and 36 nmol / kg) are superior to the corresponding A32 / 3 acylated peptides. In terms of potency, the lowest dose of A11 / 3 at 4 nmol / kg has a similar spectrum to the A32 / 3 acylated peptide at 36 nmol / kg. This provides consistent evidence for the core peptides tested. Figure 3 (A to F).

[0474] To further investigate the potency of the 3-acylated A11 position, acylated KBP-042 and KBP-066 with 3-acylation at position A11 were tested at high doses (300 nmol / kg) and compared with the non-acylated version to demonstrate the potential maximum effect of sustained in vivo efficacy and sustained bioavailability. Figure 4 (A to D).

[0475] For KBP-042 ( Figure 4 A) and KBP-066 ( Figure 4 C), 3-acylation at position A11 reduced food intake for more than 120 hours, and after 144 hours, it returned to the level of food consumption in the solvent. For KBP-042 ( Figure 4 B) and KBP-066 ( Figure 4D), treatment-mediated weight loss peaked after 96 hours and returned to baseline levels after 240 hours.

[0476] In summary, A11 is the optimal testing site in terms of preserving ligand potency and maximizing sustained in vivo efficacy.

[0477] Position A12 (acylation at position 12 of the peptide)

[0478] Compared to solvents, the A12 site with 1-acylation produced worse results in vivo in a 4-hour food intake study. Figure 5 ).

[0479] Therefore, position A12 was not a good candidate for acylation and was not further tested.

[0480] Position A16 (acylation at position 16 of the peptide)

[0481] The presence of a 1-acylated A16 position demonstrates the absence of long-term in vivo activity. Figure 1 (A to F).

[0482] Therefore, position A16 was not a good candidate for 1-acylation and was not further tested.

[0483] Position A18 (acylation at position 18 of the peptide)

[0484] The A18 position with 1-acylation was effective during the 4- to 24-hour testing period, but the observed efficacy was not maintained in extended activity studies (KBP-347, 48h, Figure 1 F).

[0485] Therefore, position A18 was not a good candidate for 1-acylation and was not further tested.

[0486] Position A32 (acylation at position 32 of the peptide)

[0487] The presence of an acylated A32 position demonstrates a long-term in vivo effect on food intake and body weight, and it is one of the best compounds tested. Figure 1 (A to F).

[0488] Compared to position A11, position A32, with both 2-acylation and 3-acylation, resulted in poorer EC50 values ​​for both CTR and AMYR receptors. Acylation at position A32 slightly attenuated the long-duration CTR-mediated response compared to position A11, but still maintained the long-duration response (Tables 3-4).

[0489] For all tested core peptides, acylation at position A32 improved the in vivo efficacy of a single-dose subcutaneous treatment compared to the non-acylated counterpart.

[0490] However, during in vivo studies, at equivalent doses, this position was worse than A11 in both 2-acylation and 3-acylation in all tests. Figure 2 From A to B, Figure 3 (A to F).

[0491] In summary, A32 is the most common position compared to position A11 in terms of maintaining ligand potency and improving in vivo efficacy using 1-acylation, 2-acylation, and 3-acylation.

[0492] Further acylation studies (Examples 7 to 12)

[0493] Example 7: Measurement of β-inhibitory protein and thioflavin T

[0494] Additional PathHunterβ-inhibitor GPCR assays were performed using the same protocol described above in conjunction with Example 2. In separate bioassays, CTR and AMY-R cells were treated at specified time points with increased KBP doses (range 1 μM to 0.1 nM and solvent) as shown in Tables 4.1 to 4.4.

[0495] Thioflavin T assays were also performed. Thioflavin T (ThT) is a dye widely used for the detection of amyloid fibrils. In the presence of fibrils, ThT exhibits maximum excitation at 450 nm and enhanced emission at 480 nm, while ThT is essentially non-fluorescent at these wavelengths when not bound to amyloid fibrils.

[0496] Therefore, the combination of ThT and a fluorescence microplate reader is an ideal tool for screening a large number of in vitro samples for the presence of amyloid fibrils.

[0497] The ThT measurement used for KBPS was performed by Nielsen et al. (Nielsen L, Khurana R, Coats A, S, Brange J, Vyas S, et al. Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry. 2001; 40(20): 6036-46.1) describes an improved method for measuring the process of insulin fibrilization.

[0498] The samples were subjected to fibrillation screening in triplicate in 384-well plates (Greiner Bio-One, 784080), with a final volume of 20 μL. The plates were sealed with an optical adhesive film to prevent sample evaporation during the assay.

[0499] Insert the plate into a fluorescence microplate reader, such as SpectraMax with SoftMax Pro 7.0.2 software, and set the template to 37°C, excitation wavelength to 450nm, and emission wavelength to 480nm.

[0500] The ELISA reader should measure fluorescence every 10 minutes for 24 hours. Shake the plate for 5 seconds before the first reading, and then for 3 seconds before all other readings. Alternatively, read the plate after the following incubation times: 0, 1, 2, 4, and 24 hours.

[0501] Relative fluorescence units (RFU) were plotted as a function of time. As described by Nielsen et al., fibrillation was identified as an increase in RFU beyond baseline.

[0502] In this submission, four fibrillation levels were defined based on 18h fluorescence signals to obtain a single output reflecting the peptide fibrillation potential: None = <1000 RFU, Low = 1000 to 3000 RFU, Medium = 3000 to 10000, High = >10000.

[0503] The results of the thioflavone T assay are also shown in Tables 4.1 to 4.4.

[0504] Table 4.1. For different acylation lengths (K) Ac Study of β-repressor protein of β-(glutamate linker)-(C14 to C26 diacid)

[0505]

[0506] Table 4.2. Use of the backbone (KBP-066) and 3-acylation (K Ac -(glutamate linker)-(C18 diacid)) for the study of β-repressor proteins at different acylation sites

[0507]

[0508]

[0509] Table 4.3 shows the use of the backbone (KBP-021) and 3-acylation (K Ac -(glutamate linker)-(C18 diacid)) for the study of β-repressor proteins at different acylation sites

[0510]

[0511] Table 4.4 Study of β-repressor proteins with different acylated linkers using the backbone (KBP-066) and the same acylation (C18 diacid).

[0512]

[0513] Results - Acylation Length

[0514] Regarding in vitro efficacy based on acylation length, there is a clear correlation between acylation length and in vitro efficacy. The shortest acylations (11 (C14 diacid) and 6 (C16 diacid)) yielded the lowest EC50 values ​​for CTR and AMYR on both receptors (Table 4.1), while the longest acylations (9 (C24 diacid) and 10 (C26 diacid)) yielded the highest recorded EC50 values ​​on both receptors.

[0515] The acylated peptides tested in this series using the KBP-066 backbone showed no fibrillation issues.

[0516] Results - Acylation sites on the KBP-066 framework

[0517] Table 4.2 lists the EC50 values ​​for CTR and AMYR for this series. In terms of in vitro potency based on the acylation sites on the KBP-066 backbone, three sites stand out as potent dual agonists of both calcitonin and amylin receptors. A11, A19, and A24 each exhibit unique 5 × 10⁻⁵ EC50 values ​​for both receptors. -9 The EC50 value in the M range was excellent, while all other test sites were defective. The improved potency of A11, A19, and A24 appears to translate into improved in vivo efficacy of the KBP-066 framework (see description below). Figure 15 , 17 Interestingly, A09 is one of the best CTR agonists but has poor AMYR activity, suggesting that acylation near the N-terminus can disrupt AMYR activity and produce biased ligands.

[0518] At most locations, fibrillation does not appear to be a problem for the KBP-066 backbone, as only one peptide (KBP-066A16.03(387)) produced a “low” score in the ThT assay.

[0519] Results - Acylation sites on the KBP-021 framework

[0520] Table 4.3 lists the EC50 values ​​for CTR and AMYR for this series. In terms of in vitro potency based on the acylation sites on the KBP-021 backbone, both sites stand out as potent dual agonists. A11 and A19 each have a unique 5 × 10⁻⁶ EC50 values ​​for both receptors. -9The EC50 value in the M range was excellent, while all other test sites were defective. The improved potency of A11 and A19 also appears to translate into improved in vivo efficacy of the KBP-021 framework (see description below). Figure 16 ).

[0521] Interestingly, fibrillation appears to be a problem with the KBP-021 backbone. As the only peptide tested, position A19, despite good potency both in vitro and in vivo, scored "high" in the ThT assay. In the ThT assay, the adjacent position "A18" also scored high with a "medium" score, suggesting that fibrillation is prone to occur when the KBP-021 backbone is acylated in this region.

[0522] Furthermore, longer acylation also appears to increase the fibrillation of the skeleton KBP-021, as the 4-acylation and 5-acylation at the A11 position scored "low" in the ThT assay; however, this issue did not affect the favorable 3-acylation position at A11.

[0523] Results - Acylated Connectors

[0524] Table 4.4 lists the EC50 values ​​for CTR and AMYR for this series. In terms of in vitro efficacy based on the acylation sites on the KBP-066 backbone, the OEG-OEG-γGLU connector (356) has an EC50 value for CTR that is almost 10 times better than OEG-OEG-OEG-γGLU (385) and OEG-γGLU (384). However, all connectors have an EC50 value of 5 × 10⁻⁶ for AMYR. -9 A very similar EC50 within the M range.

[0525] Regarding fibrillation, the shortest joint, OEG-γGLU (384), produced a “low” score in the ThT assay, while the other two joints produced a “none” score.

[0526] Example 8 ( Figure 11 )

[0527] Single-dose comparison of the effects of several acylation variants (3, 4, 5, 6, 9, 10, 11) at the same location and skeleton (A11 and KBP-066, respectively) on food intake and body weight in 20-week-old SD rats fed with HFD for 8 weeks prior to the experiment under short-term conditions.

[0528] KBP core acylation length Notes Position / Acylation KBP-356 KBP-066 C18 diacid KBP-066A11.03 A11 / 3 acylation KBP-383 KBP-066 C20 diacid KBP-066A11.04 A11 / 4 acylation KBP-382 KBP-066 C22 diacid KBP-066A11.05 A11 / 5 acylation KBP-381 KBP-066 C16 diacid KBP-066A11.06 A11 / 6 acylation KBP-307 KBP-066 C24 diacid KBP-066A11.09 A11 / 9 acylation KBP-306 KBP-066 C26 diacid KBP-066A11.10 A11 / 10 acylation KBP-305 KBP-066 C14 diacid KBP-066A11.11 A11 / 11 acylation

[0529] Rats were housed individually for four days prior to the test. Rats were then randomly divided into 8 groups according to body weight (solvent: 0.9% NaCl, KBP dosage: 3 nmol / kg (^10)). -11(μg / kg)). Fast them overnight, then administer a single dose of peptide or solvent subcutaneously in the morning. Monitor food intake at the following intervals (0 to 4 hours, 4 to 24 hours, 24 to 48 hours, 48 ​​to 72 hours, and 72 to 96 hours). Measure body weight at baseline and at 4, 24, 48, 72, and 96 hours after subcutaneous injection.

[0530] Figure 11 Results - Food intake and weight

[0531] 6-acylation, 10-acylation, and 11-acylation reduced food intake and body weight, reaching peak inhibition at 24 hours before rebounding to solvent levels. 9-acylation reduced food intake and body weight, reaching peak inhibition at 48 hours before rebounding to solvent levels. 3-acylation reduced food intake and body weight, reaching peak inhibition at 72 hours before rebounding to solvent levels. 4-acylation and 5-acylation reduced food intake and body weight, reaching peak inhibition at 96 hours before rebounding.

[0532] Therefore, 3-acylation, 4-acylation, and 5-acylation are the leading candidates for acylation length, because the initial goal was to suppress food intake and body weight for at least 72 hours, since administering the drug once every 3 days in rodents is equivalent to administering it once a week in humans.

[0533] Example 9 ( Figure 12 , 13 and 14)

[0534] Further investigations were conducted on the best-performing acylation variants (3, 4, and 5) in the short-term trials, using repeated dosing to compare the effects of acylations with the same location and skeleton (A11 and KBP-066, respectively). Prior to the study, food intake and body weight were investigated in a long-term environment (five-week study) in 20-week-old SD rats fed with HFD for 8 weeks.

[0535] KBP core acylation length Notes Position / Acylation KBP-356 KBP-066 C18 diacid KBP-066A11.03 A11 / 3 acylation KBP-383 KBP-066 C20 diacid KBP-066A11.04 A11 / 4 acylation KBP-382 KBP-066 C22 diacid KBP-066A11.05 A11 / 5 acylation

[0536] Rats were placed in pairs in cages and randomly divided into treatment groups (solvent (0.9% NaCl) and KBP (dose: 3 nmol / kg (^14 μg / kg)) according to body weight). Food intake and body weight were monitored daily for 35 days. At the end of the study, an oral gastrointestinal test (OGTT) was performed, followed by animal sacrifice, and adipose tissue was removed and weighed.

[0537] Long-term treatment of male HFD SD rats

[0538] Rats were transported to Nordic Bioscience's animal facility at 12 weeks of age and immediately fed HFD for another eight weeks. Rats were randomly assigned to groups based on body weight prior to the start of the study. The study began on day 1.

[0539] Dosage concentration and frequency

[0540] KBP was administered to animals once every three days. The administration was carried out subcutaneously (SC) around midday each day. The compound was dissolved in saline and stored at -20°C. Aliquots were thawed just before administration.

[0541] Saline solution: dosage volume is 1 mL / kg.

[0542] KBP: The dose volume is 1 mL / kg, and the dose concentration is 4 nmol / kg.

[0543] The dose equivalent (μg / kg) was ^14 μg / kg. The total weekly dose for each treatment group was 4 nmol / kg KBP, equivalent to 28 nmol / kg / week or ^100 μg / kg / week.

[0544] Collection of test results

[0545] Day 1: (The first day of the study, administration of medication)

[0546] Days 1 to 35: Monitor food intake and weight daily.

[0547] Day 35: Weight at the end of the study

[0548] Day 35: Oral glucose tolerance test.

[0549] Day 35: Eradication + Weighing of Fat Tissue

[0550] Oral glucose tolerance test.

[0551] An oral glucose tolerance test (OGTT) was performed five weeks after treatment.

[0552] Calculate the glucose dose using the animal's body weight from the previous day. Fast the animal for 11 hours. Apply calories approximately 45 minutes before the time point -30 minutes (see diagram below). Administer KBP or the solvent to the animal the day before the OGTT.

[0553] OGTT Table

[0554]

[0555] BG = Blood Glucose

[0556] G = Glucose (oral administration of 1g glucose / kg BW, 2mL / kg)

[0557] Weighing white adipose tissue (WAT)

[0558] Dissect the entire epididymis and perirenal WAT depot and weigh it. For the inguinal WAT, cut off the fixed anatomically restricted area and weigh it.

[0559] Figure 12 As a result, food intake and weight

[0560] Figure 12 The changes in food intake and body weight as a function of treatment time are shown during the long-term study period. Figure 12 A shows the dynamics of food intake between 3-acylation, 4-acylation, and 5-acylation, while Figure 12 B showed weight loss mediated by 3-acylation, 4-acylation, and 5-acylation. Clearly, after 5 weeks of treatment, all three acylations resulted in significant weight reduction; however, there was no difference in the efficacy of 3-acylation, 4-acylation, and 5-acylation in terms of weight loss.

[0561] Figure 13 As a result, OGTT and adipose tissue

[0562] Figure 13 The results of the OGTT with corresponding iAUC (OGTT) are shown (Figures A and B), as well as the weights of three different adipose tissues: epididymis, groin, and perirenal. Figure 13 (C to E), and weight at the study endpoint ( Figure 13 F). 3-acylation treatment resulted in significant reductions in iAUC (OGTT), epididymal WAT size, peripheral WAT size, and body weight as a study endpoint. 4-acylation and 5-acylation treatments resulted in significant reductions in iAUC (OGTT), epididymal WAT size, and body weight as a study endpoint, but not in peripheral WAT size. None of the treatments significantly reduced the size of the inguinal WAT. 3-acylation performed slightly better than the solvent compared to 4 / 5, but there was no significant difference between the treatment groups.

[0563] Figure 14 As a result, competitive I-125sCT ligand binding

[0564] To investigate whether the improved efficacy of 4-acylation and 5-acylation under short-term conditions could be transferred to humans, competitive ligand binding assays were performed to study the binding of acylation to serum albumin in rodents and humans. Figure 14 The results show that KBP-066A11.03 and KBP-066A11.05, along with radiolabeled I-125 salmon calcitonin (NEX423, Perkin Elmer), were effective in 2% rodent serum albumin (RSA). Figure 4 A) or 2% human serum albumin (HSA) Figure 4Competitive binding in B). No difference in EC50 was observed between 3-acylation and 5-acylation when measured in 2% HSA. However, when measured in RSA, 5-acylation shifted the IC50 further to the right, suggesting a stronger affinity for RSA in the assay. Therefore, the improved efficacy observed in short-term rodent environments is most likely a rodent-specific phenomenon and cannot be transferred to humans.

[0565] Example 10 ( Figure 15 )

[0566] Single-dose comparison of the effects of 3-acylated variants at different positions (position 9 "A09", position 11 "A11", position 12 "A12", position 16 "A16", position 18 "A18", position 19 "A19" and position 32 "A32") on food intake and body weight in 20-week-old HFD SD rats.

[0567] KBP core Notes Position / Acylation KBP-354 KBP-066 KBP-066A09.03 A9 / 3 acylation KBP-356 KBP-066 KBP-066A11.03 A11 / 3 acylation KBP-386 KBP-066 KBP-066A12.03 A12 / 3 acylation KBP-387 KBP-066 KBP-066A16.03 A16 / 3 acylation KBP-388 KBP-066 KBP-066A16.03 A18 / 3 acylation KBP-389 KBP-066 KBP-066A18.03 A19 / 3 acylation KBP-390 KBP-066 KBP-066A19.03 A24 / 3 acylation KBP-358 KBP-066 KBP-066A24.03 A32 / 3 acylation

[0568] Rats were housed individually for four days prior to testing. Rats were randomly assigned to eight groups based on body weight (solvent (0.9% NaCl), KBP (dose: 4 nmol / kg (^10⁻¹¹ μg / kg)). They were fasted overnight and then treated with a single subcutaneous dose of either the peptide or the solvent in the morning. Food intake was monitored at the following intervals (0–4 h, 4–24 h, 24–48 h, 48–72 h, and 72–96 h). Body weight was measured at baseline and at 4, 24, 48, 72, and 96 h after subcutaneous injection. Two skeletons, KBP-066 and KBP-021, were tested.

[0569] Figure 15 Results – Food Intake and Weight

[0570] Regarding its location on the skeletal structure, the results for KBP-066 are as follows. In a short-term environment, at a dose of 4 nmol / kg ( Figure 5 Positions A11 and A19 are the two best positions in terms of inhibiting food intake for 72 hours and inhibiting weight gain for 96 hours. The third best position is A24, followed by A18 and A16. The least effective acylation position is A12, which appears to be an unfavorable acylation position because it seems to interfere to some extent with the DACRA-mediated efficacy on food intake and weight gain.

[0571] Based on these data, A11 and A19 are the preferred sites for acylation of the KBP-066 backbone.

[0572] Figure 16 Results – Food Intake and Weight

[0573] When using a different skeleton KBP-021 in the same experimental environment as KBP-066, the positional patterns are slightly different.

[0574] KBP core Notes Position / Acylation KBP-312 KBP-021 KBP-021A09.03 A9 / 3 acylation KBP-391 KBP-021 KBP-021A11.03 A11 / 3 acylation KBP-313 KBP-021 KBP-021A12.03 A12 / 3 acylation KBP-314 KBP-021 KBP-021A16.03 A16 / 3 acylation KBP-315 KBP-021 KBP-021A16.03 A18 / 3 acylation KBP-316 KBP-021 KBP-021A18.03 A19 / 3 acylation KBP-317 KBP-021 KBP-021A19.03 A24 / 3 acylation KBP-318 KBP-021 KBP-021A24.03 A32 / 3 acylation

[0575] In a short-term environment, at a dose of 3 nmol / kg ( Figure 6 Positions A11 and A19 are two far optimal sites in terms of inhibiting food intake for 72 hours and body weight for 96 hours, as observed in the KBP-066 backbone. The third best site is A18, but it is significantly worse than A11 and A19. Position A24 is better than the solvent, but still far from what was observed in the KBP-066 backbone. Positions A16, A12, and A09 are comparable to the solvent in terms of both food intake and body weight, indicating that their acylation sites are unfavorable, as inferred from peptide activity.

[0576] However, as shown in Table 4.3 of the in vitro properties, A19 and A18 have significant problems with fibrillation potential when used in combination with KBP-021, which makes A11 the preferred acylation site for the KBP-021 skeleton.

[0577] Example 11 ( Figure 17 and 18 )

[0578] Further investigation was conducted on the acylation sites (A11 and A19) that performed best in the short-term trials, and repeated doses were used to compare the effects of acylations with the same acylation and backbone (i.e., 3-acylation and KBP-066, respectively).

[0579] Prior to the start of the study, food intake and body weight were investigated in a long-term environment (five-week study) in 20-week-old SD rats fed with HFD for 8 weeks.

[0580] KBP core Acylation site Notes Position / Acylation KBP-356 KBP-066 A11 KBP-066A11.03 A11 / 3 acylation KBP-389 KBP-066 A19 KBP-066A19.03 A19 / 3 acylation

[0581] The experimental protocol described in Example 9 above was followed. In short, rats were placed in pairs in cages and randomly assigned to treatment groups (solvent (0.9% NaCl), KBP (dose: 4 nmol / kg (^14 μg / kg)) based on body weight). Food intake and body weight were monitored daily for 35 days. At the end of the study, an oral glucose tolerance test (OGTT) was performed, followed by animal sacrifice, and adipose tissue was removed and weighed.

[0582] Figure 17 Results - Food intake and body weight of A11 and A19 under long-term conditions

[0583] Figure 17 This shows food intake over time as a function of treatment during the long-term study period. Figure 17 A) and weight ( Figure 17 Changes in B). Clearly, both A11 and A19 inhibited food intake in a similar manner and resulted in significant weight loss after 5 weeks of treatment; however, there was no difference in the effectiveness of A11 and A19 in terms of weight loss.

[0584] Figure 18 As a result, OGTT and adipose tissue

[0585] Figure 18 The results of the OGTT with corresponding iAUC (OGTT) are shown (Figures A and B), as well as the weights of three different adipose tissues: epididymis, groin, and perirenal. Figure 18 (C to E), and weight at the study endpoint ( Figure 18 F). Treatment at position A11 resulted in significant reductions in iAUC (OGTT), epididymal WAT size, perirenal WAT size, and body weight as studied. Treatment at position A19 resulted in significant reductions in iAUC (OGTT), epididymal WAT size, and body weight as studied, but not in perirenal WAT size. None of the treatments significantly reduced inguinal WAT size. Compared to position A19, position A11 showed slightly better solvent performance, but there was no significant difference between the treatment groups.

[0586] Example 12 ( Figure 19 )

[0587] Single-dose comparison of the effects of three acylated linker variants (3, 7, and 8) at the same location and skeleton (A11 and KBP-066, respectively) on food intake and body weight in 20-week-old SD rats fed with HFD for 8 weeks prior to the experiment in a short-term environment.

[0588] KBP core acylation length Acylated connector Notes Position / Acylation KBP-356 KBP-066 C18 diacid OEG-OEG-γGLU KBP-066A11.03 A11 / 3 acylation KBP-385 KBP-066 C18 diacid OEG-OEG-OEG-γGLU KBP-066A11.07 A11 / 7 acylation KBP-384 KBP-066 C18 diacid OEG-γGLU KBP-066A11.08 A11 / 8 acylation

[0589] Rats were housed individually for four days prior to the test. Rats were randomly assigned to eight groups based on body weight (solvent (0.9% NaCl), KBP (dose: 4 nmol / kg (^13-14 μg / kg)). They were fasted overnight and then treated with a single subcutaneous dose of either the peptide or the solvent in the morning. Food intake was monitored at the following intervals (0 to 4 hours, 4 to 24 hours, 24 to 48 hours, 48 ​​to 72 hours, and 72 to 96 hours). Body weight was measured at baseline and at 4, 24, 48, 72, and 96 hours after subcutaneous injection.

[0590] Figure 19 Results - Food intake and weight

[0591] All three types of connectors tested functioned well under short-term conditions and all reduced food intake for up to 72 hours before rebound. Figure 19A). After 96 hours, 3-acylation was slightly superior to 7-acylation and 8-acylation. This was evident in the corresponding weight loss. Figure 19 B), in which 3-acylation separates earlier from 7-acylation and 8-acylation within 24 hours, and continues to separate at two subsequent time points, 72 hours and 96 hours. Furthermore, 8-acylation appears to have a slightly lower tendency in terms of fibrillation potential (Table 4.4), which may complicate further development of compounds using this type of acylation.

[0592] Summary of results from Examples 7 to 12

[0593] acylation length

[0594] from Figures 11 to 14 The data collected in Table 4.1 indicate that 3-acylation, 4-acylation, and 5-acylation are interchangeable in the range of C18 diacids to C22 diacids when developing acylated peptides for once-weekly dosing regimens in humans.

[0595] Therefore, for the present invention, C18, C20 and C22 diacids are preferred acylation lengths.

[0596] Acylation site

[0597] from Figure 15 , 17 Data collected from 18 and 18 indicate that A19 may have a slight advantage in the short term when using the KBP-066 backbone and 3-acylation, while A11 may have a slight advantage in the long term.

[0598] When combined with the KBP-066 skeleton, neither A11 nor A19 exhibits any fibrillation issues (Table 4.2).

[0599] Overall, these data suggest that, together with the acylation sites A11 and A19 of KBP-066, they represent two of the most comprehensive acylation sites for developing once-weekly dosing regimens for humans.

[0600] Therefore, A11 and A19 are the preferred sites for acylation of KBP-066.

[0601] Similarly, based on Table 4.3 and Figure 16 Clearly, when combined with KBP-021, A11 and A19 are the two optimal acylation sites.

[0602] However, since A19 is very easy to fibrillate in this case, based on overall performance, A11 with 3 acylation is the preferred acylation site and length for the KBP-021 skeleton.

[0603] Acylated connector

[0604] Based on the test and Table 4.4, the OEG-OEG-γGLU connector appears to be the optimal connector, as shortening it introduces potential fibrillation problems, while lengthening it at best offers no benefit. Furthermore, as... Figure 19 As shown, the OEG-OEG-γGLU connector also performs best in the short-term in vivo environment, making it the preferred acylation connector overall.

[0605] In this specification, unless otherwise expressly stated, the word "or" means an operator that returns a true value when either or both of the stated conditions are met, not an operator that requires only one of the conditions to be met, i.e., "exclusively OR". The word "contains" means "including", not "consisting of". All previously accepted teachings above are incorporated herein by reference.

Claims

1. A calcitonin mimetic acylated at a lysine residue located at position 11 of the calcitonin mimetic and / or acylated at a lysine residue located at position 19 of the calcitonin mimetic, wherein, the side chain epsilon-amino group of the lysine residue is acylated with an acyl group selected from any one of the following: C with optional linker 16 or longer aliphatic acid; or C with optional linker 16 or longer aliphatic diacids.

2. The calcitonin mimetic of claim 1, wherein, the calcitonin mimetic is a calcitonin mimetic of formula (I)(a): CX2X3LSTCX8LGK Ac ... wherein X2 = A, G or S X3 = N or S X8 = M, V or alpha-amino isobutyric 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: C with optional linker 16 or longer aliphatic acids; or C with optional linker 16 or longer aliphatic diacids.

3. The calcitonin mimetic of claim 1, wherein, the calcitonin mimetic is a calcitonin mimetic of formula (I)(b): CX2X3LSTCX8LGX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 K Ac ... wherein X2 = A, G or S X3 = N or S X8 = M, V or alpha-amino isobutyric acid (AiB) X 11 = R, K, T, A or K Ac X 12 = L or Y X 13 = S, T, W or Y X 14 = Q, K, R or A X 15 = D, E or N X 16 = L or F X 17 = H or N X 18 = R, K or N 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: C with optional linker 16 or longer aliphatic acids; or C with optional linker 16 or longer aliphatic diacids.

4. The calcitonin mimetic of any one of the preceding claims, wherein, the calcitonin mimetic is a calcitonin mimetic of formula (II): CX2X3LSTCX8LGX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 G X 29 X 30 X 31 P wherein X2 = A, G or S X3 = N or S X8 = M, V or alpha-amino isobutyric acid (AiB) X 11 = K Ac , R, K, T or A X 12 = L or Y X 13 = S, T, W or Y X 14 = Q, K, R or A X 15 = D, E or N X 16 = L or F X 17 = H or N X 18 = R, K or N X 19 = K Ac , L, F or K X 20 = Q, H or A X 21 = T or R X 22 = Y or F X 23 = S or P X 24 = G, K, Q or R X 25 = T, I or M X 26 = S, N, D, G or A X 27 = T, V, F or I X 29 = S, A, P or V X 30 = N, G or E X 31 = A, T or S wherein X 11 is K Ac and / or X 19 is K Ac , 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: C 16 or longer fatty acids, C 16 or longer aliphatic diacids, Linker-C 16 or longer fatty acids, or Linker-C 16 or longer aliphatic diacids.

5. The calcitonin mimetic of claim 4, wherein, the calcitonin mimetic of formula (II) is: CX2X3LSTCX8LGX 11 LX 13 X 14 X 15 LX 17 X 18 X 19 X 20 TX 22 PX 24 TDVGANAP wherein X2 = A, G or S X3 = N or S X8 = M, V or AiB X 11 = K Ac , R, K, T or A X 13 = T, S or Y X 14 = Q or A X 15 = D or E X 17 = H or N X 18 = R or K X 19 = K Ac , L, F or K X 20 = Q, H or A X 22 = Y or F X 24 = K, Q or R wherein X 11 = K Ac and / or X 19 = K Ac , 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: C 16 or longer fatty acids, C 16 or longer aliphatic diacids, Linker-C 16 or longer fatty acids, or Linker-C 16 or longer aliphatic diacids.

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.

7. The calcitonin mimetic of any one of claims 4 to 6, wherein - X 11 is K Ac , X 17 is H, X 18 is K, X 19 is L, and X 20 is Q or A; or - X 11 is K Ac , X 17 is H, X 18 is R, X 19 is L, and X 20 is Q or A; or - X 11 is K Ac , X 17 is N, X 18 is K, X 19 is F, and X 20 is H or A; or - X 11 is K Ac , X 17 is N, X 18 is R, X 19 is F, and X 20 is H or A; or - X 11 is R or K, X 17 is H, X 18 is K, X 19 is K Ac , and X 20 is Q or A; or - X 11 is R or K, X 17 is H, X 18 is R, X 19 is K Ac , and X 20 is Q or A; or - X 11 is R or K, X 17 is N, X 18 is K, X 19 is K Ac , and X 20 is H or A; or - X 11 is R or K, X 17 is N, X 18 is R, X 19 is K Ac , and X 20 is H or A.

8. The calcitonin mimetic of any one of claims 4 to 7, wherein, X2is S, X3is N, X 11 is K Ac , X 13 is S, X 17 is H, X 18 is K or R, X 19 is L, X 20 is Q or A, and X 22 is Y; or wherein X2is S, X3is N, X 11 is R or K, X 13 is S, X 17 is H, X 18 is K or R, X 19 is K Ac , X 20 is Q or A, and X 22 is Y.

9. The calcitonin mimetic of any one of claims 4 to 7, wherein, X2is A, X3is S, X 11 is K Ac , X 13 is S, X 17 is H, X 18 is K or R, X 19 is L, X 20 is Q or A, and X 22 is F; or wherein X2is A, X3is S, X 11 is R or K, X 13 is S, X 17 is H, X 18 is K or R, X 19 is K Ac , X 20 is Q or A, and X 22 is F.

10. The calcitonin mimetic of any one of claims 4 to 7, wherein, X2is G, X3is N, X 11 is K Ac , X 13 is T, X 17 is N, X 18 is K or R, X 19 is F, X 20 is H or A, and X 22 is F; or wherein X2is G, X3is N, X 11 is R or K, X 13 is T, X 17 is N, X 18 is K or R, X 19 is K Ac , X 20 is H or A, and X 22 is F.

11. The calcitonin mimetic of claim 1, wherein, the calcitonin mimetic is a 33-mer peptide according to formula (III): CSNLSTCX6LGX7LSQDLHRX8QTYPKX1TX5VGANAP or wherein the calcitonin mimetic is a 35-mer peptide according to formula (IV): CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3TX5VGANAP or wherein the calcitonin mimetic is a 36-mer peptide according to formula (V): CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3X4TX5VGANAP or wherein the calcitonin mimetic is a 37-mer peptide according to formula (VI): CSNLSTCX6LGK Ac LZX1X2X3X4TX5VGANAP wherein each of X1to X4is any amino acid, with the proviso that at least one of X1to X4is a basic amino acid residue, and / or at least two of X1to X4are independently a polar amino acid residue or a basic amino acid residue, and / or at least one of X1to X4is a Gly residue, and wherein none of X1to X4is an acidic residue; wherein X5is D or N; wherein X6is AiB or M; wherein X7 is K Ac and X8 is L, or X7 is R or K and X8 is K Ac ; 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: C 16 or longer fatty acids, C 16 or longer aliphatic diacids, Linker-C 16 or longer aliphatic acids, or Linker-C 16 or longer aliphatic diacids.

12. The calcitonin mimetic of claim 11, wherein, at least one of X1or X4is a basic amino acid residue.

13. The calcitonin mimetic of claim 11 or 12, wherein, at least one of X1or X4is a basic amino acid residue, and at least two of X1to X4are independently a polar amino acid residue or a basic amino acid residue, and none of X1to X4is an acidic residue.

14. The calcitonin mimetic of claims 11 to 13, wherein, at least three of X1to X4are independently a polar amino acid residue or a basic amino acid residue, and none of X1to X4is an acidic residue.

15. The calcitonin mimetic of claims 11 to 14, wherein, all of X1to X4are independently a polar amino acid residue or a basic amino acid residue, and none of X1to X4is an acidic residue.

16. The calcitonin mimetic of claims 11 to 15, wherein, all of X1to X4are independently a polar amino acid residue or a basic amino acid residue, at least three of X1to X4are basic amino acid residues, and none of X1to X4is an acidic residue.

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

18. The calcitonin mimetic of claims 11 to 17, wherein, X1is selected from Asn, Phe, Val, Gly, lie, Leu, Lys, His or Arg; X2is selected from Ala, Asn, His, Leu, Ser, Thr, Gly or Lys; X3is selected from Ala, Phe, lie, Ser, Pro, Thr, Gly or Lys; and / or X4is selected from lie, Leu, Gly, His, Arg, Asn, Ser, Lys, Thr or Gin; with the proviso that at least one of X1or X4is a basic amino acid residue, and / or at least two of X1to X4are independently a polar amino acid residue and / or a basic amino acid residue, and / or at least one of X1to X4is a Gly residue.

19. The calcitonin mimetic of claims 11 to 18, wherein X1is selected from Asn, Gly, lie, His or Arg; X2is selected from Asn, Leu, Thr, Gly or Lys; X3is selected from Phe, Pro, lie, Ser, Thr, Gly or Lys; and / or X4is selected from Gly, His, Asn, Ser, Lys, Thr or Gin; with the proviso that at least one of X1or X4is a basic amino acid residue, and / or at least two of X1to X4are independently a polar amino acid residue and / or a basic amino acid residue, and / or at least one of X1to X4is a Gly residue.

20. The calcitonin mimetic of claims 11 to 19, wherein the calcitonin mimetic according to formulae (III) to (V) comprises one or more of the following conservative substitutions: - the Asp residue at position 15 of the peptide is substituted by Glu; - the Arg residue at position 18 of the peptide is substituted by Lys; and / or - the Lys residue at position 24 of the peptide is substituted by Arg.

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

22. The calcitonin mimetic of claims 1 to 21, wherein, said linker comprises a glutamic acid residue and / or an oligoethylene glycol (OEG) amino acid linker comprising one OEG amino acid or two or more OEG amino acids linked together, wherein the OEG amino acid is: wherein n is 1 to 10.

23. The calcitonin mimetic of claim 22, wherein, said 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 terminal end or the carboxyl terminal end 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, said OEG amino acid linker is selected from any one of the following:

27. The calcitonin mimetic of claim 26, wherein, said linker is 28. The calcitonin mimetic of any one of claims 1 to 27, wherein, said acyl is selected from C 18 or longer aliphatic diacid, linker-C 18 or longer aliphatic diacid, linker-C 18 or longer aliphatic diacid, linker-C 18 or longer aliphatic diacid.

29. The calcitonin mimetic of any one of claims 1 to 28, wherein, said acyl group is selected from any one of the following: C 18 to C 30 fatty acids, C 18 to C 30 fatty diacids, Linker-C 16 to C 30 fatty acid, or Linker-C 16 to C 30 Fatty diacids.

30. The calcitonin mimetic of any one of claims 1 to 29, wherein, said acyl group is selected from any one of the following: C 18 to C 22 fatty acids, C 18 to C 22 fatty diacids, Linker-C 18 to C 22 fatty acid, or Linker-C 18 to C 22 Fatty diacids.

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 C 18 to C 22 fatty diacid, and the linker is 32. The calcitonin mimetic of any one of claims 1 to 31, wherein, The peptide is selected from any one of the following: CSNLSTCMLGK Ac LSQDLHRLQTYPKTDVGANAP CSNLSTC(AiB)LGK Ac LSQDLHRLQTYPKTDVGANAP CGNLSTC(AiB)LGK Ac LTQDLNKFHTFPKTDVGANAP CSNLSTCVLGK Ac LSQELHKLQTYPRTDVGANAP CSNLSTCMLGK Ac LSQELHRLQTYPKTDVGANAP CASLSTCVLGK Ac LSQDLHKLQTFPKTDVGANAP CASLSTCMLGK Ac LSQDLHKLQTFPKTDVGANAP CGNLSTCMLGK Ac LSQDLNKFHTFPQTDVGANAP CSNLSTC(AiB)LGK Ac LANFLVHSSNNFGAILPKTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKHTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKHSSTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKHSSNTDVGANAP CSNLSTCMLGK Ac LSQDLHRLSNNFGAILSSTNVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYGAILSPKTDVGANAP CSNLSTCMLGK Ac LANFLVHSSNNFGAILPKTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKILSSTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKGLITTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKNNFGTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKRTTQTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKHTTNTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKHGGQTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKHKKNTDVGANAP CSNLSTCMLGK Ac LSQDLHRLQTYPKHKKHTDVGANAP CSNLSTC(AiB)LGRLSQDLHRK Ac QTYPKTDVGANAP CSNLSTCMLGRLSQELHRK Ac QTYPKTDVGANAP and wherein K Ac 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: AcCSNLSTCMLGK Ac LSQDLHRLQTYPKTDVGANAP-NH2 AcCSNLSTC(AiB)LGK Ac LSQDLHRLQTYPKTDVGANAP-NH2 AcCGNLSTC(AiB)LGK Ac LTQDLNKFHTFPKTDVGANAP-NH2 AcCSNLSTCVLGK Ac LSQELHKLQTYPRTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQELHRLQTYPKTDVGANAP-NH2 AcCASLSTCVLGK Ac LSQDLHKLQTFPKTDVGANAP-NH2 AcCASLSTCMLGK Ac LSQDLHKLQTFPKTDVGANAP-NH2 AcCGNLSTCMLGK Ac LSQDLNKFHTFPQTDVGANAP-NH2 AcCSNLSTC(AiB)LGK Ac LANFLVHSSNNFGAILPKTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHSSTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHSSNTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLSNNFGAILSSTNVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYGAILSPKTDVGANAP-NH2 AcCSNLSTCMLGK Ac LANFLVHSSNNFGAILPKTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKILSSTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKGLITTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKNNFGTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKRTTQTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHTTNTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHGGQTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHKKNTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHKKHTDVGANAP-NH2 AcCSNLSTC(AiB)LGRLSQDLHRK Ac QTYPKTDVGANAP-NH2 AcCSNLSTCMLGK Ac LSQELHRLQTYPKTDVGANAP-NH2 and wherein K Ac is acylated with a linker-fatty diacid, wherein the fatty diacid is C 18 to C 22 fatty diacid, and the linker is 34. A peptide according to any one of claims 1 to 33, formulated for enteral administration.

35. A peptide according to claim 34, wherein, The peptide is formulated into a pharmaceutical composition for oral administration comprising coated citric acid particles, and wherein the coated citric acid particles increase oral bioavailability of the peptide.

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

37. A peptide according to claim 36, wherein, The carrier comprises 5-CNAC, SNAD or SNAC.

38. A peptide according to any one of claims 1 to 33, formulated for parenteral administration.

39. A peptide according to claim 38, formulated for injection.

40. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 33.

41. Use of a peptide according to any one of claims 1 to 33 as a medicament.

42. Use of a peptide according to any one of claims 1 to 33 in the treatment of diabetes (type I and / or type II), excess body weight, hyperphagia, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, poor regulation of blood glucose levels, poor regulation of response to a glucose tolerance test or poor regulation of food intake.

43. Use of a peptide according to any one of claims 1 to 33 in combination with metformin or another insulin sensitizing agent in the treatment of diabetes (type I and / or type II), excess body weight, hyperphagia, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, poor regulation of blood glucose levels, poor regulation of response to a glucose tolerance test or poor regulation of food intake.

44. Use of a peptide according to any one of claims 1 to 33 in combination with a weight loss drug in the treatment of an overweight condition.

45. A peptide according to claim 44, wherein, The overweight condition is obesity.

46. A co-formulation comprising a peptide according to any one of claims 1 to 33 and an insulin sensitizing agent.

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

48. A method of treating diabetes (type I and / or type II), excess body weight, hyperphagia, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, poor regulation of blood glucose levels, poor regulation of response to a glucose tolerance test or poor regulation of food intake, comprising administering to a patient in need of such treatment an effective amount of a peptide according to any one of claims 1 to 33.

49. A method of treating diabetes (type I and / or type II), excess body weight, hyperphagia, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, poor regulation of blood glucose levels, poor regulation of response to a glucose tolerance test, or poor regulation of food intake, comprising administering to a patient in need of such treatment an effective amount of a peptide of any one of claims 1 to 33 in combination with metformin or another insulin sensitizing agent.

50. A method of treating an overweight condition, comprising administering to a patient in need of such treatment an effective amount of a peptide of any one of claims 1 to 33 in combination with a weight loss drug.

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