Acylated calcitonin mimetics

Abstract

To provide calcitonin mimetics.SOLUTION: The present invention provides: a calcitonin mimetic that is acylated at a lysine residue located at the 11 position of the calcitonin mimetic and / or that is acylated at a lysine residue located at the 19 position of the calcitonin mimetic, the calcitonin mimetic being acylated with a fatty acid and the like having a chain length of C16 or more, which may have a linking group; and uses thereof as a drug in the treatment of various diseases and disorders, including the treatment of diabetes, overweight, excessive food intake and metabolic syndrome, NASH, alcoholic and non-alcoholic fatty liver disease, regulating blood glucose levels, regulating the response to a glucose tolerance test, regulating food intake, and treating osteoporosis and osteoarthritis.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to acylated calcitonin mimetics and their use as pharmaceutical agents in the treatment of various diseases and disorders including, but not limited to, diabetes (type I and / or type II), overweight, overfeeding and metabolic syndrome, non-alcoholic steatohepatitis (NASH), alcoholic and non-alcoholic fatty liver disease, regulating blood glucose levels, regulating the response to a glucose tolerance test or regulating food intake, treatment of osteoporosis, and osteoarthritis. [Background technology]

[0002] There are approximately 250 million people with diabetes worldwide, a number that is predicted to double over the next 20 years. Over 90% of this population has type 2 diabetes mellitus (T2DM). It is estimated that currently, only 50-60% of people with T2DM or in stages preceding overt T2DM are diagnosed.

[0003] T2DM is a heterogeneous disease characterized by abnormalities in carbohydrate and fat metabolism. The causes of T2DM are multifactorial and include both genetic and environmental components that affect β-cell function and insulin sensitivity in tissues such as muscle, liver, pancreas, and adipose tissue. As a consequence, impaired insulin secretion is observed, paralleled by a progressive decline in β-cell function and chronic insulin resistance. The inability of the endocrine pancreas to compensate for peripheral insulin resistance leads to the development of hyperglycemia and clinical diabetes. Tissue resistance to insulin-mediated glucose uptake is now recognized as the major pathophysiological determinant of T2DM.

[0004] The criterion for successful optimal intervention in T2DM is lowering blood glucose levels, which can be both a chronic reduction in blood glucose levels, explained by lower peak glucose levels and more rapid clearance, and an increased ability to tolerate higher glucose levels after food ingestion, both of which place less strain on β-cell insulin production and function.

[0005] Type I diabetes is characterized by the loss of ability to produce insulin in response to food intake and, therefore, the inability to regulate blood glucose to normal physiological levels.

[0006] The physical structure of bone can be damaged by various factors, including disease and injury.One of the most common bone diseases is osteoporosis, which is characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and susceptibility to fractures, especially in the hip, spine and wrist.Osteoporosis develops when there is an imbalance in the rate of bone resorption that exceeds the rate of bone formation.It has been shown that the administration of an effective amount of anti-resorptive agent, such as calcitonin, can prevent bone resorption.

[0007] Inflammatory and degenerative diseases, including joint diseases such as osteoarthritis (OA), rheumatoid arthritis (RA) or juvenile rheumatoid arthritis (JRA), and inflammation resulting from autoimmune responses such as lupus, ankylosing spondylitis (AS) or multiple sclerosis (MS), can result in substantial loss of movement due to pain and destruction of the joint. Cartilage that covers and cushions bones in joints can deteriorate over time, thus unnecessarily allowing direct contact between the two bones, which can limit the movement of one bone relative to the other and / or cause damage to one bone by the other during joint movement. Subchondral bone immediately below the cartilage can also deteriorate. Bone resorption can be prevented by administration of an effective amount of an anti-resorptive agent, such as calcitonin.

[0008] Calcitonin is highly conserved across a wide range of species. Natural full-length calcitonin is 32 amino acids in length. An example of a natural calcitonin sequence is shown below:

[0009] Salmon CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP Eel CSNLSTCVLGKLSQELHKLQTYPRTDVGAGTP Chicken CASLSTCVLGKLSQELHKLQTYPRTDVGAGTP Mouse CGNLSTCMLGTYTQDLNKFHTFPQTSIGVEAP Rat CGNLSTCMLGTYTQDLNKFHTFPQTSIGVGAP Horse CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP Dog-1 CSNLSTCVLGTYSKDLNNFHTFSGIGFGAETP Dog-2 CSNLSTCVLGTYTQDLNKFHTFPQTAIGVGAP Pig CSNLSTCVLSAYWRNLNNFHRFSGMGFGPETP Human CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP

[0010] Synthetic variants of natural calcitonin with modified amino acid sequences intended to provide improved properties are disclosed in WO2013 / 067357 and WO2015 / 071229.

[0011] However, peptides such as calcitonin and calcitonin mimetics generally have rapid clearance and short half-lives, leading to poor absorption, distribution, metabolism and excretion characteristics. Therefore, peptide drugs generally require daily oral administration. Daily administration of treatment via subcutaneous (sc) injection is currently not the best method of administration, as it is inconvenient for individual patients and may lead to non-compliance with the treatment regimen to avoid the discomfort associated with daily injections. Therefore, weekly dosing via sc injection would improve the quality of life of patients and further aid in compliance with the treatment regimen.

[0012] There are many approaches known in the art that attempt to improve the in vivo half-life of peptide drugs. These include proteolytic stability (e.g., by N- and C-terminal protection, substitution of amino acids with D-amino acids or non-natural amino acids, ring closure of the peptide, etc.) and reducing renal clearance (e.g., by conjugation of the peptide to large polymers or large molecules such as albumin or immunoglobulins). However, such modifications of peptide drugs are It is also known in the art that such modifications may be detrimental, for example in terms of reduced drug efficacy, unexpected adverse side effects such as drug hypersensitivity, etc. Therefore, it is not possible to predict whether such modifications will necessarily improve the therapeutic properties of peptide drugs. It would be difficult to develop a peptide drug that would be administered only once a week.

[0013] One approach to improve the pharmacokinetic and pharmacodynamic properties of peptide drugs is acylation of peptides. Trier et al. acylated two therapeutic peptides, glucagon-like peptide-2 (GLP2) and salmon calcitonin (sCT), with different lengths (C8-C 16) acyl group (PhD thesis, 2016, "Acylation of Therapeutic Peptides", DTU; available at http: / / orbit.dtu.dk / files / 127682557 / PhD_thesis_Sofie.Trier.pdf). The effects of acylation of GLP2 proved to be broadly predictable based on previous observations of similar peptides, whereas the effects observed when acylating sCT proved to be unpredictable. For example, Trier et al. found that acylation of sCT (at a different position on the peptide backbone) consistently resulted in substantial loss of receptor potency (60% to 80%), whereas receptor potency was preserved for acylated GLP2. Thus, although Trier et al. did not find any beneficial properties associated with acylation of sCT, particularly with short chain (C8) acylation, they did find a number of unexpected and significant adverse effects associated with acylation of sCT, most notably a significant loss in receptor efficacy. Another noteworthy point is that Trier et al.'s study focused on acylation at position 18 (Lys18) of salmon calcitonin, because previous studies aimed at improving the efficacy of salmon calcitonin had identified position 18 as a superior position for modification (by PEGylation, but not by acylation). In those studies, PEGylation at position 18 of sCT was found to provide superior efficacy over similar peptides modified at positions Cys1 or Lys11 (Youn et al, J. Control. Release, 2006, 334-342). [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2013 / 067357 [Patent Document 2] International Publication No. WO2015 / 071229 [Non-Patent Document 1] PhD thesis, 2016, "Acylation of Therapeutic Peptides", DTU; http: / / orbit.dtu.dk / files / 127682557 / PhD_thesis_Sofie.Trier.pdf [Non-Patent Document 2] Youn et al, J. Control. Release, 2006, 334-342 Summary of the Invention [Problem to be solved by the invention]

[0015] [Means for solving the problem]

[0016] Summary of the Invention The present inventors have found that acylation of a calcitonin mimetic at the lysine residue at position 11 of the calcitonin mimetic or at position 19 of the calcitonin mimetic, particularly with certain acyl moieties, unexpectedly improves the efficacy of the peptide and increases the duration of action of the peptide compared to the equivalent non-acylated peptide. Similarly, the efficacy of the calcitonin mimetic was found to be significantly improved in response to acylation at positions 11 or 19, whereas acylation at position 18 gave inferior results, contrary to the findings of Youn et al. The present inventors have thus developed a novel acylated calcitonin mimetic with high efficacy that does not require daily administration but only requires once a week.

[0017] Thus, in one embodiment, the present invention provides a calcitonin mimetic in which the lysine residue at position 11 of the calcitonin mimetic is acylated and / or the lysine residue at position 19 of the calcitonin mimetic is acylated, the side chain ε-amino group of which is an amino group selected from the following: 16 or a fatty acid having a chain length of 1 or more; or a C 16 and acylated with dibasic fatty acids of chain length 1 or greater.

[0018] As used herein, "calcitonin mimetics" refers to peptides that activate the calcitonin receptor (i.e., calcitonin receptor agonists), and preferably peptides that also activate the amylin receptor (i.e., dual amylin and calcitonin receptor agonists).

[0019] In certain preferred embodiments, the calcitonin mimetic is 32 to 37 amino acids in length. Most preferably, the calcitonin mimetic is 32 amino acids in length.

[0020] In one preferred embodiment, the calcitonin mimetic is The lysine residue at position 11 is acylated, as defined by formula (I)(a): CX2X3LSTCX8LGK Ac … [During the ceremony, X2=A, G or S X3=N or S X8=M, V or α-aminoisobutyric acid (AiB); K Ac has a side chain ε-amino group which is any one of the following: C which may have a linking group 16 or C which may have a linking group 16 Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from or a calcitonin mimetic represented by the formula:

[0021] Or, the lysine residue at position 19 is acylated, as represented by formula (I)(b): CX2X3LSTCX8LGX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 K Ac … [During the ceremony, X2=A, G or S X3=N or S X8 = M, V or α-aminoisobutyric acid (AiB) X 11 =R, K, T, A or K Ac (Preferably R, K or K Ac and most preferably R or K. X 12 =L or Y (preferably L). X 13 =S, T, W or Y (preferably T, S or Y). X 14 =Q, K, R or A (preferably Q or A, most preferably Q). X 15 =D, E or N (preferably D or E). X 16 =L or F (most preferably L). X 17 =H or N X 18 =R, K or N (preferably R or K), K Ac has a side chain ε-amino group which is any one of the following: C which may have a linking group 16 or C which may have a linking group 16 Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from It is a calcitonin mimetic represented by the formula:

[0022] Preferably, the calcitonin mimetic of formula (I)(a) or formula (I)(b) is 32 to 37 amino acids in length, more preferably 32, 33, 36 or 37 amino acids in length. Most preferably, the calcitonin mimetic of formula (I)(a) or formula (I)(b) is 32 amino acids in length.

[0023] In one preferred embodiment of the invention, the calcitonin mimetic has the 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 GX 29 X 30 X 31 P [During the ceremony, X2=A, G or S X3=N or S X8 = M, V or α-aminoisobutyric acid (AiB) X 11 =K Ac , R, K, T or A (most preferably K Ac , R or K.) 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 X19 =K Ac , L, F or K (most preferably K Ac , L or F.) 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 (most preferably A or T), X 11 is K Ac and / or X 19 is K Ac And (X 11 is K Ac And X 19 is L, F or K, preferably L or F; X 11 is R, K, T or A, preferably R or K, and X 19 is K Ac Is it X? 11 is K Ac And X 19 is K Ac or K Ac has a side chain ε-amino group which is any one of the following: C 16 or longer chain length fatty acids C 16 Dibasic fatty acids with a chain length of Linking group -C 16 or Linking group -C 16 Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from It is a calcitonin mimetic represented by the formula:

[0024] Preferably, the 32-mer 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 [During the ceremony, X2=A, G or S X3=N or S X8=M, V or AiB X 11 =K Ac , R, K, T or A (most preferably K Ac , R or K.) X 13 =T, S or Y X 14 =Q or A (most preferably Q). X 15 =D or E X 17 =H or N X 18 =R or K X 19 =K Ac , L, F or K (most preferably K Ac , L or F.) X 20 = Q, H or A X 22 = Y or F X 24 =K, Q or R; X 11 is K Ac and / or X 19 is K Ac and K Ac has a side chain ε-amino group which is any one of the following: C 16 or longer chain length fatty acids C 16 Dibasic fatty acids with a chain length of Linking group -C 16 or Linking group -C 16 Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from It is a calcitonin mimetic represented by the formula:

[0025] Preferably, X2 is S and X3 is N; or X2 is G and X3 is N; or X2 is A and X3 is S. Preferably, X 13 is S or T, most preferably S. Preferably, X 24 is R or K.

[0026] In a preferred embodiment, X 11 K Ac and X 17 is H and X 18 is K and X 19 is L and X 20 is Q or A; or X 11 K Ac and X 17 is H and X 18 is R and X 19 is L and X 20 is Q or A; or X 11 K Ac and X 17 is N and X 18 is K and X 19 is F and X 20 is H or A; or X 11 K Ac and X 17 is N and X 18 is R and X 19 is F and X20 is H or A; or X 11 is R or K, and X 17 is H and X 18 is K and X 19 K Ac And X 20 is Q or A; or X 11 is R or K, and X 17 is H and X 18 is R and X 19 K Ac And X 20 is Q or A; or X 11 is R or K, and X 17 is N and X 18 is K and X 19 K Ac And X 20 is H or A; or X 11 is R or K, and X 17 is N and X 18 is R and X 19 K Ac And X 20 is H or A.

[0027] In a preferred embodiment, X2 is S, X3 is N, and X 11 K Ac and X 13 is S and X 17 is H and X 18 is K or R, and X 19 is L and X 20 is Q or A, and X 22 is Y; or X2 is S and X3 is N, and X 11 is R or K, and X 13 is S and X 17 is H and X 18 is K or R, and X 19 K Ac and X 20 is Q or A, and X 22 is Y. In a preferred embodiment, X2 is A, X3 is S, and X 11 K Ac and X 13 is S and X 17 is H and X 18 is K or R, and X 19 is L and X 20 is Q or A, and X 22 is F; or X2 is A, X3 is S, and X 11 is R or K, and X 13 is S and X 17 is H and X 18 is K or R, and X 19 K Ac and X 20 is Q or A, and X 22 is F. In a preferred embodiment, X2 is G, X3 is N, and X 11 K Ac and X 13 is T and X 17 is N and X 18 is K or R, and X 19 is F and X 20 is H or A, and X 22 is F; or X2 is G and X3 is N, and X 11 is R or K, and X 13 is T and X 17 is N and X 18 is K or R, and X 19 K Ac and X 20 is H or A, and X 22 is F.

[0028] In another preferred embodiment, the present invention relates to a calcitonin mimetic, the calcitonin mimetic having the formula (III): CSNLSTCX6LGX7LSQDLHRX8QTYPKX1TX5VGANAP or a 33-mer peptide represented by The calcitonin mimetic has the formula (IV): CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3TX5VGANAP or a 35-mer peptide represented by The calcitonin mimetic has the formula (V): CSNLSTCX6LGX7LSQDLHRX8QTYPKX1X2X3X4TX5VGANAP or a 36-mer peptide represented by The calcitonin mimetic has the formula (VI): CSNLSTCX6LGK Ac LZX1X2X3X4TX5VGANAP A 37-mer peptide represented by [During the ceremony, each of X1 to X4 is an amino acid, at least one of X1 to X4 is a basic amino acid residue, and / or at least two of X1 to X4 are independently a polar amino acid residue or a basic amino acid residue, and / or at least one of X1 to X4 is a Gly residue, with the proviso that none of X1 to X4 is an acidic residue; X5 is D or N; X6 is AiB or M; X7 is K Ac and X8 is L, or X7 is R or K and X8 is K Ac Either; Z is selected from SQDLHRLSNNFGA, SQDLHRLQTYGAI, or ANFLVHSSNNFGA; K Ac has a side chain ε-amino group which is any one of the following: C 16 or longer chain length fatty acids C 16 Dibasic fatty acids with a chain length of Linking group -C 16 or Linking group -C 16 Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from It is.

[0029] Preferably, in formulae (III) to (VI), at least one of X1 and X4 is a basic amino acid residue. More preferably, at least one of X1 and X4 is a basic amino acid residue, and at least one or more of X1 to X4 are independently a polar amino acid residue or a basic amino acid residue, and none of X1 to X4 is an acidic residue. More preferably, at least three of X1 to X4 are independently a polar amino acid residue or a basic amino acid residue, and none of X1 to X4 is an acidic residue. More preferably, all of X1 to X4 are independently a polar amino acid residue or a basic amino acid residue, and none of X1 to X4 is an acidic residue. Most preferably, all of X1 to X4 are independently polar amino acid residues or basic amino acid residues, at least three of X1 to X4 are basic amino acid residues, and none of X1 to X4 is an acidic residue.

[0030] The amino acid residue may be any natural or unnatural amino acid residue having a basic side chain and may be selected from, but is not limited to, Arg, His, or Lys. A polar amino acid residue may be any natural or unnatural amino acid residue having a polar, uncharged side chain and may be selected from, but is not limited to, Ser, Thr, Asn, Gln or Cys. The term "acidic residue" as used herein refers to a natural or unnatural amino acid residue having an acidic side chain, such as, for example, Glu or Asp.

[0031] In a preferred embodiment, X1 is selected from Asn, Phe, Val, Gly, Ile, Leu, Lys, His, or Arg; X2 is selected from Ala, Asn, His, Leu, Ser, Thr, Gly, or Lys; X3 is selected from Ala, Phe, Ile, Ser, Pro, Thr, Gly or Lys; and / or X4 is selected from Ile, Leu, Gly, His, Arg, Asn, Ser, Lys, Thr, or Gln; At least one of X1 or X4 is a basic amino acid residue, and / or at least two of X1 to X4 are independently a polar amino acid residue and / or a basic amino acid residue, and / or at least one of X1 to X4 is a Gly residue.

[0032] In a preferred embodiment, X1 is selected from Asn, Gly, Ile, His, or Arg; X2 is selected from Asn, Leu, Thr, Gly, or Lys; X3 is selected from Phe, Pro, Ile, Ser, Thr, Gly or Lys; and / or X4 is selected from Gly, His, Asn, Ser, Lys, Thr, or Gln; At least one of X1 or X4 is a basic amino acid residue, and / or at least two of X1 to X4 are independently a polar amino acid residue and / or a basic amino acid residue, and / or at least one of X1 to X4 is a Gly residue.

[0033] The peptides of the present invention represented by the above formulas (III) to (V) may contain the following conservative substitutions: The Asp residue located at position 15 of the peptide is replaced by Glu; The Arg residue at position 18 of the peptide is substituted with Lys; and / or The Lys residue at position 24 of the peptide is replaced with Arg. It may contain one or more conservative substitutions selected from:

[0034] The peptide of the present invention represented by the above formula (VI) is the Z moiety of the peptide of formula (VI) is SQDLHRLSNNFGA or SQDLHRLQTYGAI, Conservative substitutions include: The Asp residue at position 15 of the peptide is substituted with Glu; and / or The Arg residue at position 18 of the peptide is replaced with Lys. It may contain one or more conservative substitutions selected from:

[0035] In all embodiments of the invention, the linking group comprises a glutamic acid residue and / or an oligoethylene glycol (OEG) amino acid linking group comprising one OEG amino acid or two or more OEG amino acids linked together; The OEG amino acid has the following formula:

[0036] [ka]

[0037] (In the formula, n is a number from 1 to 10, preferably a number from 1 to 5, a number from 1 to 3, a number from 1 or 2, and most preferably a number from 1 to 10.) It is expressed as:

[0038] The OEG amino acid linking group can include a single OEG amino acid or a linkage of two to six OEG amino acids together. More preferably, the OEG amino acid linking group may comprise one OEG amino acid or a linkage of two or three OEG amino acids together. Most preferably, the OEG amino acid linking group may comprise a linkage between two OEG amino acids. The OEG amino acid linking group may further comprise one or more glutamic acid residues at the amino or carboxy terminus of the OEG amino acid linking group.

[0039] Preferably, the OEG amino acid linking group is selected from the following:

[0040] [ka]

[0041] Preferably, the OEG amino acid linking groups are as follows:

[0042] [ka]

[0043] In a preferred embodiment, the acyl group is 18 fatty acids with chain length of C or more 18 Divalent fatty acids with a chain length of 1 or more, linking group -C 18 or a fatty acid having a chain length of -C or more 18 The dibasic fatty acids are selected from those having a chain length of 1 or more. Preferably, the acyl group is selected from the following: C 18 ~C 30 fatty acids, preferably C 18 ~C 22 Fatty acids C 18 ~C 30 dibasic fatty acids, preferably C 18 ~C 22 Dibasic fatty acids Linking group -C 18 ~C 30 Fatty acids, preferably with a linking group -C 18 ~C 22 or Linking group -C 18 ~C 30 divalent fatty acid, preferably a linking group -C 18 ~C 22 Fatty acids

[0044] Preferably, C 18 The dibasic fatty acid is octadecanedioic acid (CAS No. 871-70-5).

[0045] In a preferred embodiment, K Ac is acylated with a linking group-divalent fatty acid, The linking group-divalent fatty acid is a divalent fatty acid having a C 18 ~C 22and the linking group is as follows:

[0046] [ka]

[0047] Preferably, the calcitonin mimetic of the present invention is any of the following: CSNLSTCMLGK Ac LSQDLHRLQTYPKTDVGANAP CSNLSTCMLGK Ac LSQELHRLQTYPKTDVGANAP CSNLSTCVLGK Ac LSQELHKLQTYPRTDVGANAP CASLSTCVLGK Ac LSQDLHKLQTFPKTDVGANAP CGNLSTCMLGK Ac LSQDLNKFHTFPQTDVGANAP CSNLSTC(AiB)LGK Ac LSQDLHRLQTYPKTDVGANAP CGNLSTC(AiB)LGK Ac LTQDLNKFHTFPKTDVGANAP 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 is selected from The K Ac is as defined above. The amino acid residue at position 8 of the above peptide may optionally be substituted with AiB.

[0048] Preferably, the calcitonin mimetic of the present invention is any 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 AcCGNLSTCMLGK Ac LSQDLNKFHTFPQTDVGANAP-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)LGK Ac LANFLVHSSNNFGAILPKTDVGANAP-NH2 AcCSNLSTC(AiB)LGRLSQDLHRK Ac QTYPKTDVGANAP-NH2 AcCSNLSTCMLGRLSQELHRK Ac QTYPKTDVGANAP-NH2 is selected from The K Ac is acylated with a linking group-divalent fatty acid, The divalent fatty acid is C 18 ~C 22 and the linking group is as follows:

[0049] [ka]

[0050] Preferably, C 18 The dibasic fatty acid is octadecanedioic acid. The amino acid residue at position 8 of the above peptides may be optionally substituted with AiB. In the above peptides, "Ac" indicates that the N-terminus of the peptide is acetylated, and "-NH2" indicates that the C-terminus of the peptide is amidated.

[0051] The calcitonin mimetics of the present invention may be formulated for enteral administration. For example, a calcitonin mimetic may be formulated into an oral pharmaceutical composition comprising coated citric acid particles, which may increase the oral bioavailability of the peptide. Alternatively or additionally, the composition may be formulated with a carrier for oral administration, such as a carrier containing 5-CNAC, SNAD, or SNAC. The calcitonin mimetics of the present invention may be formulated for parenteral administration, for example, the calcitonin mimetics may be formulated for injection.

[0052] The present invention also relates to pharmaceutical compositions comprising the calcitonin mimetics described above. The present invention also relates to the calcitonin mimetics described above for use as a medicament, in which the calcitonin mimetics may be for use in the treatment of 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, dysregulated blood glucose levels, dysregulated response to glucose tolerance test or dysregulated food intake. Calcitonin mimetics may also be administered conjugated to metformin or another insulin sensitizer.

[0053] The peptides of the invention may be acylated or otherwise modified at their N-terminus to reduce the positive charge of the first amino acid and, independently, may be amidated at their C-terminus.

[0054] The peptides can be formulated for pharmaceutical administration, and can be formulated for enteral or parenteral administration. The preferred formulation is injection, preferably subcutaneous injection. However, the peptides can also be formulated for oral administration with a carrier, optionally increasing oral bioavailability. Suitable carriers include those that contain 5-CNAC, SNAD, or SNAC.

[0055] Optionally, the peptide is formulated into an oral pharmaceutical composition comprising coated citric acid particles, which increase the oral bioavailability of the peptide.

[0056] The present invention encompasses the peptides of the invention for use as a medicament. The peptides can be used to treat diabetes (type I and / or type II), overweight, overeating, metabolic syndrome, rheumatoid arthritis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis, or osteoarthritis, dysregulated blood glucose levels, dysregulated response to a glucose tolerance test, or dysregulated food intake. In particular, the peptides can be used to reduce undesirably high fasting blood glucose levels, or to reduce undesirably high HbA1c, or to reduce undesirably high response to a glucose tolerance test. The peptides of the invention can also be used to reduce hepatic triglycerides and / or to reduce fat accumulation in the liver of a patient. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The peptides of the present invention can be produced using any suitable method, such as synthetic (chemical) or recombinant techniques known in the art to which the present invention pertains, to produce peptides. Preferably, the peptides are produced using synthetic methods. The synthesis of synthetic peptides is well known in the art to which the present invention pertains, and includes, but is not limited to, solid-phase peptide synthesis techniques utilizing various protecting groups (e.g., the use of Fmoc, Boc, Bzl, tBu, etc.).

[0058] In some embodiments, the N-terminal side of the calcitonin mimetics 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 onto cysteine-1. Alternative methods of reducing 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, at the N-terminus. Alternatively, other amino acids, including lysine, glycine, formylglycine, leucine, alanine, acetylalanine, and dialanyl, may be added to the N-terminus of the peptides discussed above. As will be appreciated by those skilled in the art, peptides with multiple cysteine ​​residues often form disulfide bonds between two such cysteine ​​residues. All such peptides described herein are defined as optionally including one or more such disulfide bonds, particularly at positions Cys1-Cys7. In imitation of this, the cysteines at positions 1 and 7 may be substituted together by an α-aminosuberic acid bond. Although the calcitonin mimetics of the present disclosure may exist in the form of a free acid, it is preferred that the C-terminal amino acid is amidated. The inventors anticipate that such amidation may contribute to the efficacy and / or bioavailability of the peptide. To amidate the C-terminal amino acid, synthetic chemistry methods can be applied. A preferred approach to produce the amidated forms of the calcitonin mimetics of the present disclosure is to react a precursor (having a glycine in place of the C-terminal amino group of the desired amidated product) in the presence of a peptidylglycine α-amidating monooxygenase according to known techniques, which is converted to the amidated product in reactions described, for example, in US4708934 and EP0308067 and EP0382403.

[0059] Production of amidated products can also be achieved using the processes and amidating enzymes described by Consalvo et al. in US7445911, Miller et al. in US2006 / 0292672, Ray et al. in 2002, Protein Expression and Purification, 26:249-259, and Mehta in 2004, Biopharm International, July, pp. 44-46.

[0060] The production of the preferred amidated peptides may proceed by generating glycine-extended precursors in E. coli, for example as soluble fusion proteins with glutathione-S-transferase, or by directly expressing the precursors according to the techniques described in US6103495. Such glycine-extended precursors have the same molecular structure as the desired amidated product, except for the C-terminus (the product terminates with --X--NH2, whereas the precursor terminates with --X-gly, where X is the C-terminal amino acid residue of the product). The α-amidating enzymes described in the above publications catalyze the conversion of the precursor to the product. The enzyme is preferably recombinantly produced, for example in Chinese Hamster Ovary (CHO) cells, as described in the Biotechnology and Biopharm article cited above.

[0061] The free acid forms of the peptide active agents of the present disclosure can be produced in a similar manner, except that they do not include a C-terminal glycine on the "precursor," which is instead the final peptide product, and do not require an amidation step.

[0062] Except as otherwise noted, the preferred dosages of the calcitonin mimetics of the present disclosure are the same for both therapeutic and prophylactic purposes. The desired dosages are discussed in detail below and will vary depending on the mode of administration.

[0063] Unless otherwise stated and clear from the context, dosage amounts herein refer to the weight of the active compound (i.e., calcitonin mimetic) unaffected by or not taking into account pharmaceutical excipients, diluents, carriers, or other ingredients, although such additional ingredients are preferably included. Any dosage form (capsule, tablet, injection, etc.) commonly used in the pharmaceutical industry for delivery of peptide active agents is suitable for use herein, and the terms "excipient," "diluent," or "carrier" include non-active ingredients that are typically included in such dosage forms together with active ingredients in the industry. A preferred oral dosage form is discussed in detail below, but should not be considered the limiting mode of administering the active agents of the present disclosure.

[0064] The calcitonin mimetics of the present disclosure can be administered to a patient to treat a number of diseases or disorders. As used herein, the term "patient" refers to any organism belonging to the animal kingdom. In certain embodiments, the term "patient" refers to a vertebrate, more preferably a mammal, including a human.

[0065] Thus, the present disclosure includes the use of peptides in a method for treating type I diabetes, type II diabetes or metabolic syndrome, obesity, or suppressing appetite, or reducing insulin resistance, or for lowering undesirably high fasting serum glucose levels, or for lowering undesirably high peak serum glucose levels, or for lowering undesirably high peak serum insulin levels, or for reducing an undesirably large response to a glucose tolerance test, or for treating osteoporosis, or for treating osteoarthritis, or for treating non-alcoholic steatohepatitis (NASH), or for treating alcoholic fatty liver disease, or for reducing hepatic triglycerides, or for reducing fat accumulation in the liver of a subject.

[0066] There are numerous art-recognized scales for normal body weight ranges that take into account numerous factors such as sex, age, and height. Patients in need of the treatment or prevention regimens described herein include those whose weight exceeds recognized standards or who are at higher risk of becoming overweight or obese than the general population due to genetic, environmental, or other recognized risk factors. In accordance with the present disclosure, it is envisioned that calcitonin mimetics may be used to treat diabetes, where weight management is an aspect of treatment.

[0067] In certain embodiments, the method comprises enterally administering to a patient in need thereof a pharma- ceutical effective amount of any one of the peptides described herein for treatment of said condition.

[0068] In some embodiments, the method includes parenterally administering to a patient in need thereof a pharma- ceutical effective amount of any one of the peptides described herein for the treatment of said condition. For parenteral administration (including intraperitoneal, subcutaneous, intravenous, intradermal, or intramuscular injection), for example, a solution of the peptide of the present disclosure, either in sesame or peanut oil or in aqueous propylene glycol, may be used. The aqueous solutions should be suitably buffered, if necessary, and the liquid diluent should first be rendered isotonic. These aqueous solutions are suitable for intravenous injection purposes. The oily solutions are suitable for intra-arterial, intramuscular, and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. For parenteral use, examples of suitable preparations include solutions, preferably oily or aqueous solutions, and suspensions, emulsions, or implants, including suppositories. The peptides can be formulated in sterile form for multi- or single-dose administration, such as by dispersion in a liquid carrier, such as sterile saline or 5% saline dextrose solution, commonly used in conjunction with injectables.

[0069] The method may include a preliminary step of determining whether the patient is suffering from the condition, and / or a subsequent step of determining how effective the treatment is in alleviating the condition in the patient, for example by performing an oral glucose tolerance test or measuring resting blood glucose levels in each case.

[0070] Oral enteric formulations are ingested by swallowing for later release in the intestine below the stomach and therefore delivery to the liver via the portal vein, as opposed to formulations that are held in the mouth to allow passage into the bloodstream via the sublingual or buccal routes.

[0071] Suitable dosage forms for use in the present disclosure include tablets, mini-tablets, capsules, granules, pellets, powders, effervescent solids, and chewable solid formulations. Such formulations may include gelatin, preferably hydrolyzed gelatin or low molecular weight gelatin. Such formulations may be obtained by freeze-drying a homogeneous aqueous solution containing calcitonin mimics and hydrolyzed gelatin or low molecular weight gelatin, and further processing the resulting solid material into said oral pharmaceutical formulation, where the gelatin may have an average molecular weight of 1000 Daltons to 15000 Daltons. Such formulations may include a protective carrier compound, such as 5-CNAC or other compounds disclosed herein.

[0072] Although oral formulations such as tablets and capsules are preferred, the compositions used in the present disclosure can take the form of syrups, elixirs, etc., and suppositories, etc. Oral delivery is generally the delivery route of choice, since it is convenient, relatively easy, and generally painless, and leads to increased patient compliance compared to other delivery modes.However, biological, chemical, and physical barriers such as fluctuating pH in the digestive tract, strong digestive enzymes, and active drug-impermeable digestive membranes make oral delivery of calcitonin-like peptides to mammals problematic; for example, oral delivery of calcitonin, a long-chain polypeptide hormone secreted by parafollicular cells of the thyroid gland in mammals and by the ultimobranchial gland in birds and fish, has proven inherently difficult, at least in part, due to the poor stability of calcitonin in the digestive tract and the lack of ability to easily transport calcitonin through the intestinal wall into the bloodstream. However, preferred oral formulations are described below.

[0073] Patient treatment In some embodiments, the calcitonin mimetics of the present disclosure are administered in a dosage sufficient to maintain serum levels of the mimetics in the patient 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 suitable techniques, such as radioimmunoassay and mass spectrometry, among others known in the art. The attending physician can monitor the patient's response, and then modify the dosage to some extent to take into account the metabolism and response of the individual patient. Near-simultaneous release is best achieved by administering all components of the present disclosure as a single pill or capsule. However, the present disclosure also includes, for example, splitting the required amount of the calcitonin mimetics into two or more tablets or capsules, which may be administered together to provide the required amount of all components together. As used herein, a "pharmaceutical composition" includes, but is not limited to, a complete dosage appropriate for a particular administration to a patient, regardless of whether one or more tablets or capsules (or other dosage forms) are recommended for a given administration.

[0074] The calcitonin mimetics of the present disclosure can be formulated for oral administration using methods used in the Unigene Enteripep® product. These may include methods such as those described in U.S. Patent No. 5,912,014, U.S. Patent No. 6,086,918, U.S. Patent No. 6,673,574, U.S. Patent No. 7,316,819, U.S. Patent No. 8,093,207, and U.S. Publication No. 2009 / 0317462. Specifically, these may include the use of conjugation of the compound to a membrane transporter, such as the protein transduction domain of the HIV TAT protein, the use of one or more protease inhibitors, and / or pH-reducing agents, which may be coated, and / or acid-resistant protective vehicles, and / or absorption enhancers, which may be surfactants.

[0075] In certain embodiments, the calcitonin mimetics of the present disclosure are preferably formulated for oral administration using methods known in US Patent Publication No. 2009 / 0317462.

[0076] In some embodiments, the calcitonin mimetics of the present disclosure can be formulated for enteral administration, particularly oral administration, by mixing with a suitable carrier compound. Suitable carrier compounds include those described in U.S. Pat. No. 5,773,647 and U.S. Pat. No. 5,866,536, among which 5-CNAC (N-(5-chlorosalicyloyl)-8-aminocaprylic acid, generally as its disodium salt) is particularly effective. Other preferred carriers or delivery agents are SNAD (10-(2-hydroxybenzamido)decanoic acid, sodium salt) and SNAC (N-(8-[2-hydroxybenzoyl]amino)caprylic acid, sodium salt). In some embodiments, the pharmaceutical compositions of the present disclosure include a delivery-effective amount of a carrier, such as 5-CNAC, i.e., an amount sufficient to deliver the compound for the desired effect. Generally, the carrier, such as 5-CNAC, is present in an amount of 2.5% to 99.4% by weight of the total composition, more preferably 25% to 50% by weight.

[0077] In addition, WO00 / 059863 discloses a compound of formula I

[0078] [ka]

[0079] [In the formula, R 1 , R 2 , R 3 , and R 4 are independently hydrogen, -OH, -NR 6 R 7 , halogen, C1-C4 alkyl, or C1-C4 alkoxy; R 5 is a substituted or unsubstituted C2-C 16 Alkylene, substituted or unsubstituted C2-C 16 Alkenylene, substituted or unsubstituted C2-C 12Alkyl (arylene), or substituted or unsubstituted aryl (C1-C 12 alkylene); R 6 and R 7 are independently hydrogen, oxygen, or C1-C4 alkyl.

[0080] and their hydrates and solvates that are particularly effective for oral delivery of active agents such as calcitonin, e.g., salmon calcitonin, and can be used in the present disclosure.

[0081] A preferred enteric formulation, using optionally micronized 5-CNAC, may be generally as described in WO2005 / 014031.

[0082] The compound can be formulated for oral administration using the methods used in Bone Medical Limited's Capsitoni n products. They can include the methods incorporated in Axcess formulations. More specifically, the active ingredient can be encapsulated in an enteric capsule that can withstand passing through the stomach. The enteric capsule can contain the active compound together with a hydrophilic aromatic alcohol absorption enhancer, for example, as described in WO02 / 028436. In a known manner, the enteric coating can be permeable in a pH-sensitive manner, for example, at a pH of 3 to 7. WO2004 / 091584 also describes a suitable formulation method using aromatic alcohol absorption enhancers.

[0083] The compounds can be formulated using methods found in Oramed products, which can include formulation with omega-3 fatty acids as found in WO2007 / 029238 or as described in US 5,102,666.

[0084] In general, pharma- ceutically acceptable salts (particularly monosodium or disodium salts), solvates (eg, alcohol solvates), and hydrates of these carriers or delivery agents may be used.

[0085] Oral administration of pharmaceutical compositions according to the present disclosure can be achieved by administering the compositions periodically, e.g., once or more on a daily or weekly basis; intermittently, e.g., at random intervals during a day or week; or periodically, e.g., by administering the compositions periodically for a period of several days or weeks followed by a period of no administration. The dosage form of the pharmaceutical compositions of the embodiments disclosed herein can be in any known form, e.g., liquid or solid dosage forms. Liquid dosage forms include solutions, emulsions, suspensions, syrups, and elixirs. In addition to the active compound and a carrier such as 5-CNAC, liquid formulations can also include inert excipients commonly used in the art, e.g., solubilizing agents, e.g., ethanol; oils such as cottonseed oil, castor oil, and sesame oil; wetting agents; emulsifying agents; suspending agents; sweeteners; flavoring agents; and solvents such as water. The solid dosage forms include capsules, soft gel capsules, tablets, caplets, powders, granules, or other solid oral dosage forms, all of which may be prepared by methods well known in the art. The pharmaceutical compositions may additionally include additives in amounts conventionally used, including, but not limited to, pH adjusting agents, preservatives, flavoring agents, flavoring agents, fragrances, humectants, isotonicity agents, coloring agents, surfactants, plasticizers, lubricants such as magnesium stearate, flow aids, compression aids, solubilizers, excipients, diluents such as microcrystalline cellulose, Avicel PH 102, supplied by FMC, or any combination thereof. Other additives may include phosphate buffer salts, citric acid, glycols, and other dispersing agents. The compositions may also include one or more enzyme inhibitors, such as actinonin or epiactinonin and their derivatives; aprotinin, trasylol, and Bowman-Birk inhibitors. Additionally, transport inhibitors, i.e., [rho]-glycoproteins, such as Ketoprofin, may be present in the compositions of the present disclosure. The pharmaceutical compositions of the present disclosure may 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 ingredients into capsules, or, instead of filling into capsules, molding and then tableting or compression molding to obtain tablets. Additionally, solid dispersions may be formed by known methods and then further processed to form tablets or capsules.Preferably, the ingredients in a pharmaceutical composition of the present disclosure are mixed homogeneously or uniformly throughout the solid dosage form.

[0086] Alternatively, the active compound can be formulated as a conjugate with a carrier, which can be an oligomer as described in US Publication No. 2003 / 0069170, such as the oligomer described below.

[0087] [ka]

[0088] Such conjugates can be administered in combination with fatty acids and bile salts as described therein. For example, conjugates with polyethylene glycol (PEG) may be used, as described in Mansoor et al.

[0089] 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 mucoadhesive emulsion.

[0090] The active compound can be formulated by loading into chitosan nanocapsules as disclosed in Prego et al. (optionally PEG-modified as described in Prego Prego C, Torres D, Fernandez-Megia E, Novoa-Carballal R, Quinoa E, Alonso MJ.) or by loading into lipid nanoparticles coated with chitosan or PEG as disclosed in Garcia-Fuentes et al. Chitosan nanoparticles for this purpose may be iminothiolane-modified as described in Guggi et al. They can be formulated in water / oil / water emulsions as described in Dogru et al. The bioavailability of the active compound can be increased by using taurodeoxycholate or lauroylcarnitine as described in Sinko et al. or Song et al. In general, nanoparticles suitable as carriers are discussed in de la Fuente et al. and can be used in the present disclosure.

[0091] Another strategy suitable for oral formulations involves the use of transient permeation enhancer (TPE) systems, such as those described in WO 2005 / 094785 by Chiasma Ltd. TPEs use an oil suspension of solid hydrophilic particles in a hydrophobic solvent to protect drug molecules from inactivation by the hostile gastrointestinal (GI) environment, while at the same time acting on the GI wall to induce permeation of the cargo drug molecules.

[0092] Further included is the use of glutathione or compounds containing multiple thiol groups, as described in US 2008 / 0200563, to inhibit the action of efflux pumps on mucous membranes. An example of such an approach is 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 Also described in Huck, Ch. (2005) 'The use of thiolated polymers as carrier matrix in oral peptide delivery' -Proof of concept. J. Control. Release, 106, 26-33.

[0093] The active compound can be formulated into seamless microspheres as described in WO2004 / 084870, where the active pharmaceutical ingredient is solubilized as an emulsion, microemulsion, or suspension formulated into minispheres and variously coated by either conventional or novel coating techniques. The result is a drug encapsulated in a "pre-solubilized" form that, when administered orally, provides a predetermined immediate or sustained release of the active drug at a specific rate to a specific location along the gastrointestinal tract. Essentially, pre-solubilizing the drug increases the predictability of its kinetic profile while simultaneously enhancing permeability and drug stability.

[0094] Nanocapsules coated with chitosan may be used, as described in US2009 / 0074824. Active molecules administered using this technique are protected within the nanocapsules, as they are stable against the action of gastric juices. In addition, the mucoadhesive properties of this system increase the adhesion time to the intestinal wall (a delayed gastrointestinal transit of these systems has been observed), facilitating more efficient absorption of the active molecules.

[0095] Methods developed by TSRLl Inc. may be used. These include the hydrophilic solubilization technique (HST), in which gelatin, a naturally occurring collagen extract carrying both positive and negative charges, coats the particles of active ingredients contained in lecithin micelles, preventing their aggregation or clumping. This results in improved hydration of the hydrophobic drug particles through polar interactions. In addition, the amphiphilic lecithin reduces the surface tension between the dissolution fluid and the particle surface.

[0096] The active ingredient may be formulated with cucurbituril as an excipient.

[0097] Alternatively, Merrion Pharmaceuticals' GIPET technology can be used to produce enteric coated tablets containing the active ingredient together with an absorption enhancer which can be a medium chain fatty acid or medium chain fatty acid derivative as described in US2007 / 0238707, or a membrane translocating peptide as described in US7268214.

[0098] GIRES™ technology can be used, which consists of a controlled release dosage form in a capsule for oral administration housed in an expandable pouch. As the capsule dissolves, a gas generating system expands the pouch in the stomach. Clinical trials have shown that the pouch is retained in the stomach for 16 to 24 hours.

[0099] Alternatively, the active ingredient may be conjugated to a protective modifier that allows it to withstand enzymatic degradation in the stomach and enhances its absorption. The active ingredient may be covalently bound to monodisperse short chain methoxypolyethylene glycol glycolipid derivatives, which are purified, crystallized and lyophilized to give the dry active pharmaceutical ingredient. Such methods are described in US5438040 and www.biocon.com.

[0100] Hepatotropic vesicles (HDV) may also be used to deliver active ingredients. HDV may consist of liposomes (diameter ≦150 nm) that encapsulate the active ingredient and also contain hepatocyte targeting molecules in their lipid bilayer. The targeting molecules guide the delivery of the encapsulated active ingredient to hepatocytes, and thus a relatively small amount of active ingredient is required to produce an effect. Such technology is described in US2009 / 0087479 and further described at www.diasome.com.

[0101] The active ingredient may be incorporated in association with medium chain partial glycerides, optionally mixed with long chain PEG species, as described in US 2002 / 0115592 for insulin, into a composition additionally containing a substantially non-aqueous hydrophilic vehicle comprising alcohol and a co-solvent.

[0102] Alternatively, intestinal patches may be used as described in Shen Z, Mitragotri S, Pharm Res. 2002 Apr;19(4):391-5 'Intestinal patches for oral drug delivery'.

[0103] The active ingredient can be incorporated into an erodible matrix formed from a hydrogel blended with a hydrophobic polymer as described in US Pat. No. 7,189,414.

[0104] Suitable oral dosage levels for the adult human being to be treated may range from 0.05 mg to 5 mg, preferably about 0.1 mg to 2.5 mg.

[0105] The frequency of patient dosing is from 1 to 4 times per week, preferably 1 to 2 times per week, and most preferably once per week. Treatment will desirably be maintained for at least 6 weeks, preferably at least 6 months, preferably for an extended period of at least 1 year, and optionally lifelong.

[0106] Combination treatment for related conditions can be performed using separate administration of a composition according to the present disclosure and one or more other therapeutic agents. Alternatively, a composition according to the present disclosure may incorporate one or more other therapeutic agents for co-administration.

[0107] Combination therapies according to the present disclosure include combinations of active compounds as described with insulin, GLP-2, GLP-1, GIP, or amylin, or generally with other antidiabetic agents. Thus, combination therapies, including combination drugs, include biguanides such as metformin, buformin and phenformin, TZDs (PPAR) such as balaglitazone, pioglitazone, rivoglitazone, rosiglitazone and troglitazone, insulin sensitizers including dual PPAR agonists such as aleglitazar, muraglitazar and tesaglitazar; or sulfonylureas such as carbutamide, chlorpropamide, gliclazide, tolbutamide, tolazamide, glipizide, glibenclamide, glyburide, gliquidone, glyclopyramide and glimepiride, meglitinides / glinides (K+) such as nateglinide, repaglinide and mitiglinide, exenatide, lixisenatide, liraglutide, semaglutide, dulaglutide and albiglutide. This may be done with a variety of medications including GLP-1 analogues, secretagogues including DPP-4 inhibitors such as alogliptin, linagliptin, saxagliptin, sitagliptin and vildagliptin; insulin analogues or specialised formulations such as (rapid acting) insulin lispro, insulin aspat, insulin glulisine, (long acting) insulin glargine, insulin detemir, inhaled insulin Exubera and NPH insulin; as well as other medications including α-glucosidase inhibitors such as acarbose, miglitol and voglibose, amylin analogues such as pramlintide, SGLT2 inhibitors such as dapagliflozin, empagliflozin, remogliflozin and sergliflozin, but also benfluorex and tolrestat.

[0108] Further combinations include co-administration or combination with leptin. Leptin resistance is a well-established component of type 2 diabetes. However, injection of leptin has not been able to improve this condition so far. In contrast, there is evidence supporting that amylin, and thus molecules with amylin-like capabilities, as salmon calcitonin mimetics, can improve leptin sensitivity. Amylin / leptin combinations have also shown synergistic effects on body weight and food intake, and insulin resistance [Kusakabe T et al.].

[0109] Further preferred combination therapies include the co-formulation or co-administration of the peptides of the invention with one or more weight loss drugs, including, but not limited to, lipase inhibitors (e.g., pancreatic lipase inhibitors such as orlistat), appetite suppressant amphetamine derivatives (e.g., phentermine), topiramate, Qysmia® (a combination of phentermine / topiramate), 5-HT 2C Receptor agonists (e.g., lorcaserin), Contrave® (naltrexone / bupropion combination), glucagon-like peptide-1 [GLP-1] analogues and derivatives (e.g., liraglutide, semaglutide), sarcoplasmic / endoplasmic reticulum (SR) Ca 2+ These combinations include ATPase (SERCA) inhibitors (e.g., sarcolipin), fibroblast growth factor 21 [FGF-21] receptor agonists (e.g., analogs of FGF-21), and β3 adrenergic receptor agonists (e.g., mirabegron). Such combinations may be used to treat overweight conditions, such as obesity. [Brief description of the drawings]

[0110] Text description of the illustration image024.gif. [Figure 1-1] FIG. 1: Comparison of KBP346, KBP347, KBP349, KBP351, KBP352, KBP353 and KBP089 with respect to food intake and body weight. A) Food intake, 0-4 hours; B) Body weight change, 4 hours; C) Food intake, 4-24 hours; D) Body weight change, 24 hours; E) Food intake 24-49 hours; F) Body weight change, 48 hours. [Figure 1-2] FIG. 1: Comparison of KBP346, KBP347, KBP349, KBP351, KBP352, KBP353 and KBP089 with respect to food intake and body weight. A) Food intake, 0-4 hours; B) Body weight change, 4 hours; C) Food intake, 4-24 hours; D) Body weight change, 24 hours; E) Food intake 24-49 hours; F) Body weight change, 48 hours. [Diagram 2]Figure 2: Single dose studies of KBP375, KBP376 and KBP377. Single doses were administered at t=0. The effect of a single 36 nmol / kg dose of each molecule on food intake and body weight was observed for 168 hours and directly compared to the non-acylated benchmark. A) Food intake; B) Body weight change [Figure 3-1] Figure 3: Dose-response studies of KBP356, KBP358, KBP362, KBP364, KBP368 and KBP370. Single doses were administered at t=0. The effect of a single 36 nmol / kg dose of each molecule on food intake and body weight was observed for 168 hours. AB) Food intake and body weight for acylated KBP-066 mutants; CD) Food intake and body weight for acylated KBP-062 mutants; EF) Acylated KBP-110 mutants [Figure 3-2] Figure 3: Dose-response studies of KBP356, KBP358, KBP362, KBP364, KBP368 and KBP370. Single doses were administered at t=0. The effect of a single 36 nmol / kg dose of each molecule on food intake and body weight was observed for 168 hours. AB) Food intake and body weight for acylated KBP-066 mutants; CD) Food intake and body weight for acylated KBP-062 mutants; EF) Acylated KBP-110 mutants [Figure 3-3] Figure 3: Dose-response studies of KBP356, KBP358, KBP362, KBP364, KBP368 and KBP370. Single doses were administered at t=0. The effect of a single 36 nmol / kg dose of each molecule on food intake and body weight was observed for 168 hours. AB) Food intake and body weight for acylated KBP-066 mutants; CD) Food intake and body weight for acylated KBP-062 mutants; EF) Acylated KBP-110 mutants [Figure 4-1] 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. [Figure 4-2]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. [Diagram 5] Figure 5: 4-hour food intake study of KBP-350 [Figure 6] Figure 6: Cumulative food intake. A) Cumulative food intake over time. Food intake was monitored once daily from the start of the study until day 21. n=3-4 cages. + / - SEM.; B) Total area under the curve for the data shown in Figure 9A. n=9-10. + / - SEM. [Figure 7] Figure 7: ZDF body weight during the study. A) Individual rat body weight in grams; B) Body weight normalized to vehicle weight and expressed as a percentage. Body weight was recorded daily through the first 21 days and 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. [Figure 8] Figure 8: ZDF Fasting Blood Glucose Levels Fasting blood glucose levels after 6 h fasting were measured on days 0, 14, 28, 42 and 62 after the start of the study. n=9-10. + / - SEM. [Figure 9] Figure 9: ZDF HbA1c values ​​A) HbA1c at baseline; B) HbA1c at end of study; HbA1c was measured 3 days prior to study initiation. HbA1c was measured at end of study, day 62. n=9-10. + / - SEM. [Figure 10]FIG. 10: Oral glucose tolerance test (OGTT). A) OGTT over 180 min 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 previously fasted for 11 h at minus 30 min (-30). Blood glucose levels were measured at -30, 0, 15, 30, 60, 120 and 180 min. Glucose was administered orally at 0 min. An upper limit of detection of 33.3 mmol*L-1 was assigned for blood glucose values ​​above 33.3 mmol*L-1. Rats were not previously administered saline or KBP-066 or KBP-066A on the same day. n=9-10. [Figure 11] Figure 11: Single dose studies of KBP-305, KBP-306, KBP-307, KBP-356, 381, KBP-382 and KBP-383. Single doses were administered at t=0 and the effect of a single 3 nmol / kg dose of each molecule on food intake and body weight was observed for 96 hours and directly compared to other molecules and vehicle to determine optimal acylation length. A) Acute food intake in grams (g); B) Body weight change in grams (g); n=4 rats per group. Data as + / - SEM. [Figure 12] Figure 12: 6 week weight loss study in HFD SD rats with KBP-066A11 compounds with different acylation lengths (.03, .04 and .05 acylation). Rats were treated with KBP-066A11.03, KBP-066A11.04, KBP-066A11.05 or vehicle and administered a single subcutaneous injection of 4 nmol compound / kg every third day. Body weight was recorded throughout the study. A) Daily food intake in grams (g); B) Individual rat weight loss in grams (g); n=6 rats per group. Data as + / - SEM. [Figure 13]FIG. 13: Additional parameters from a study of weight loss in HFD SD rats with KBP-066A11 compounds of different acylation lengths (.03, .04 or .05 acylation). Rats were treated with KBP-066A11.03, KBP-066A11.04, KBP-066A11.05 or vehicle. Rats were dosed every third day with a single subcutaneous injection of 4 nmol compound / kg. A) Oral glucose tolerance test; B) Increased area under the curve of OGTT; C) Weight in grams (g) of epididymal WAT at the end of the study; D) Weight in grams (g) of inguinal WAT at the end of the study; E) Weight in grams (g) of perirenal WAT at the end of the study; F) Change in body weight from baseline at the end of the study in grams (g); Body weight was recorded daily throughout the study.; n=6 rats per group. Data as + / - SEM. [Figure 14] Figure 14: Competitive ligand binding assays performed in 2% serum albumin from two different species, rat (Rattus norvegicus) and human (Homo sapiens), using radiolabeled salmon calcitonin (125I-sCT) as tracer. 0.25 nm 125I-sCT was used as tracer; A) Competitive binding assay performed in 2% RSA; B) Competitive binding assay performed in 2% HSA; C) Data as + / - SEM. [Figure 15] Figure 15: Single dose study of KBP-356, KBP-386, KBP-387, KBP-388, KBP-389 and KBP-390 to investigate acylation positions on the KBP-066 backbone. Single doses were administered at t=0 and the effect of a single 3 nmol / kg dose of each molecule on food intake and body weight was observed for 96 hours and directly compared to other molecules and vehicle to determine optimal acylation length. A) Acute food intake in grams (g); B) Body weight change in grams (g); n=4 rats per group. Data as + / - SEM. [Figure 16]Figure 16: Single dose studies of KBP-391, KBP-312, KBP-313, KBP-314, KBP-315, KBP-316, KBP-317 and KBP-318 to investigate the acylation position on the KBP-021 backbone. Single doses were administered at t=0 and the effect of a single 3 nmol / kg dose of each molecule on food intake and body weight was observed for 96 hours and directly compared to other molecules and vehicle to determine the optimal acylation length. A) Acute food intake in grams (g); B) Body weight change in grams (g); n=4 rats per group. Data as + / - SEM. [Figure 17] FIG. 17: 6-week weight loss study in HFD SD rats with KBP-066A11 compound (.03 with the same acylation length, but different acylation positions, A11 and A19). Rats were administered KBP-066A11.03 (KBP-356), KBP-066A19.03 (KBP-389) or vehicle by a single subcutaneous injection of 4 nmol compound / kg every third day. Body weight and food intake were recorded daily throughout the study period. A) Daily food intake in grams (g) over the study period; B) Individual rat weight loss in grams (g); n=6 rats per group. Data as + / - SEM. [Figure 18] Figure 18: Additional parameters from a study of weight loss in HFD SD rats with KBP-066A11 compounds of different acylation lengths (.03, .04 or .05 acylation). Rats were treated with KBP-066A11.03, KBP-066A19.03 or vehicle. Rats were dosed every 3rd day with a single subcutaneous injection of 4 nmol compound / kg. A) Oral glucose tolerance test; B) Increased area under the curve of OGTT; C) Weight in grams (g) of epididymal WAT at the end of the study; D) Weight in grams (g) of inguinal WAT at the end of the study; E) Weight in grams (g) of perirenal WAT at the end of the study; F) Change in body weight from baseline at the end of the study in grams (g); Body weight was recorded daily throughout the study.; n=6 rats per group. Data as + / - SEM. [Figure 19]Figure 19: Investigation of acylation linkages on the KBP-066 backbone with KBP-356, KBP-384 and KBP-385. A single dose was administered at t=0 and the effect of a single 4 nmol / kg dose of each molecule on body weight was observed for 96 hours and directly compared to other molecules to determine the optimal acylation linkage. A) Acute food intake in grams (g); B) Body weight change in grams (g); n=4 rats per group. Data as + / - SEM. EXAMPLES

[0111] Working Example The embodiments of the present disclosure are described in the following examples, which are provided to aid in understanding the present disclosure and should not be considered as limiting the scope of the present disclosure as defined in the claims that follow. The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the disclosure, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0112] Cells and cell lines The following cell lines expressing calcitonin, amylin, and CGRP receptors were purchased and cultured according to the manufacturer's instructions. 1. Calcitonin Receptor (CTR): U2OS-CALCR from DiscoveRx (Cat. No. 93-0566C3) 2. Amylin Receptor (AMY-R): CHO-K1 CALCR+RAMP3 from DiscoveRx (Cat. No. 93-0268C2)

[0113] reagent Thioflavin T (T3516, Sigma). Assay stock ThT was prepared as a 10 mM solution in 5 mM sodium phosphate pH 7. It was aliquoted and stored protected from light at -20°C. Stock ThT was thawed and diluted immediately prior to use. For the calcitonin mimetics under test (hereinafter referred to as "acylated KBPs" or simply "KBPs"), the final buffer conditions were 10 mM Tris-HCL pH 7.5. The final peptide concentration in the well should be 100-200 μM and the final ThT concentration should be 4 μM. ThT is added last (10 μL).

[0114] Animal models In animal model studies, 12-week-old healthy Sprague Dawley (SD) rats were used to evaluate the efficacy of acylated KBPs. In some examples, the rats were fed a normal diet before and during the study, while in other examples, 12-week-old healthy SD rats were fed a high-fat diet (HFD) for 8 weeks before and during the study.

[0115] Acylated calcitonin mimetics Tables 1a and 1b below list the amino acid sequences of the acylated calcitonin mimetics that were tested. As used therein: 1 Acylation is Ac -(glutamic acid linking group)-(C16 fatty acid [palmitate]); 2 Acylation is Ac -(glutamic acid linking group)-(C18 dibasic fatty acid [octadecanedioic acid]); 3 Acylation is K Ac -(2xOEG amino acids linked together with a glutamic acid residue attached to the N-terminus)-(C18 dibasic fatty acid [octadecanedioic acid]); 4 Acylation is K Ac -(2xOEG amino acids linked together with a glutamic acid residue attached to the N-terminus)-(C20 diacid [eicosanedioic acid]); 5 Acylation isAc -(2xOEG amino acids linked together with a glutamic acid residue attached to the N-terminus)-(C22 diacid [docosane diacid]); 6 Acylation is Ac -(2xOEG amino acids linked together with a glutamic acid residue attached to the N-terminus)-(C16 diacid [hexadecanedioic acid]); 7 Acylation is Ac -(3xOEG amino acids linked together with a glutamic acid residue attached to the N-terminus)-(C18 diacid [octadecanedioic acid]); 8 Acylation is Ac -(1xOEG amino acid linked together with a glutamic acid residue attached to the N-terminus)-(C18 diacid [octadecanedioic acid]); 9 Acylation is Ac -(2xOEG amino acids linked together with a glutamic acid residue attached to the N-terminus)-(C24 diacid [tetracosane diacid]); 10 Acylation is Ac -(2xOEG amino acids linked together with a glutamic acid residue attached to the N-terminus)-(C26 diacid [hexacosane diacid]); 11 Acylation is Ac -(2xOEG amino acids linked together with a glutamic acid residue attached to the N-terminus)-(C14 diacid [tetradecanedioic acid])

[0116] The calcitonin mimetics tested are based on the following unmodified core peptide sequence: CSNLSTCMLGRLSQDLHRLQTYPKTDVGANAP (KBP089). CSNLSTC(AiB)LGRLSQDLHRLQTYPKTDVGANAP (KBP066) CGNLSTC(AiB)LGRLTQDLNKFHTFPKTDVGANAP (KBP062) CSNLSTCVLGKLSQELHKLQTYPRTDVGANAP (KBP042) CSNLSTC(AiB)LGRLANFLVHSSNNFGAILPKTDVGANAP (KBP110) CSNLSTCMLGRLSQELHRLQTYPKTDVGANAP (KBP021)

[0117] In Table 1b, the following additional designations are also used: Acylated Amino Acids KBP Revised Name 01 A01 02 A02 03 A03 ... XX AXX ... 31 A31 32 A32

[0118] Acylation type Additional name C16 .01 C18 Diacid .02 C18 Diacid 2*OEG .03 C20 Diacid 2*OEG .04 C22 Diacid 2*OEG .05 C16 Diacid 2*OEG .06 C18 Diacid 3*OEG .07 C18 Diacid 1*OEG .08 C24 Diacid 2*OEG .09 C26 Diacid 2*OEG .10 C14 Diacid 2*OEG .11

[0119] In the examples, the modified name KBP-066A11.03 refers to a peptide modified in that position 11 of the KBP-066 core sequence is substituted with a C18 diacid 2*OEG acylated lysine residue.

[0120] [Table 1]

[0121] [Table 2]

[0122] [Table 3]

[0123] [Table 4]

[0124] [ka]

[0125] [ka]

[0126] [ka]

[0127] [ka]

[0128] Initial Acylation Studies (Examples 1-5) Example 1 (Figures 1 and 5) Comparison of the effects of single-dose administration of monoacylated 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 with the effects of a non-acylated benchmark peptide (KBP-089) in 12-week-old lean SD rats.

[0129] [Table 5]

[0130] Four days before the test, rats were housed individually in cages. They were randomized into six groups according to body weight (vehicle (0.9% NaCl), KBP (dose: 25 nmol / kg (^100 μg / kg)). Rats were fasted overnight and then treated with a single dose of peptide or vehicle via subcutaneous administration in the morning. Food intake was monitored at the following intervals (0-4 h, 4-24 h, 24-48 h). Body weight was measured at baseline and at 24 and 48 h after subcutaneous injection. Acylation of positions “A09”, “A11” and “A32” using monoacylation extended the in vivo response (Figure 1) and therefore merited further testing (see below). Positions 12 (Figure 5), 16 and 18 showed unacceptable results and were not pursued further.

[0131] Example 2: β-arrestin assay PathHunter β-arrestin GPCR assays are whole-cell functional assays that directly measure the ability of ligands to activate GPCRs by detecting the interaction of β-arrestin with activated GPCRs. Because β-arrestin recruitment is independent of G protein signaling, these assays provide a powerful and universal screening and profiling platform that can be used for virtually any Gi-, Gs, or Gq-coupled receptor.

[0132] In this system, the GPCR is inserted in frame. β-arrestin was fused to a small enzyme fragment, ProLink™, and co-expressed in cells stably expressing a fusion protein (termed the enzyme acceptor or EA) of a larger N-terminal deletion mutant of β-gal. Activation of the GPCR stimulates the binding of β-arrestin to the ProLink-tagged GPCR, allowing the two enzyme fragments to complement each other, resulting in the formation of the active β-gal enzyme. This interaction increases enzyme activity, which can be measured using the chemiluminescent PathHunter® Detection Reagent.

[0133] In independent bioassays, CTR and AMY-R cells were treated with increasing doses of KBP (100, 20, 4, 0.8, 0.16, 0.032 nM and vehicle) as specified in Tables 2 and 3 below, for the indicated time points. The assay was 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. To quantify GPCR-mediated β-arrestin recruitment, the assay was performed using the Pathhunter™ Detection Kit (93-0001, DiscoverX) according to the manufacturer's instructions.

[0134] The extended / sustained response was performed using the Calcitonin Receptor (CTR): U2OS-CALCR (Cat# 93-0566C3) cell line from DiscoveRx, and β-arrestin accumulation was assayed and analyzed for 3, 6, 24, 48 or 72 hours, as opposed to the classical 3 hour output. The results of the β-arrestin studies are listed in Table 2 (diacylation) and Table 3 (triacylation).

[0135] [Table 6]

[0136] [Table 7]

[0137] β-arrestin studies have shown that: 1) The efficacy of acylation, in terms of acylation positions in the peptide, is as follows: A11>A32>A09. 2) Di- or tri-acylation at position 11 (A11) is generally the superior acylation / position combination for both peptide cores in terms of activation of the calcitonin receptor (CTR), the amylin receptor (AMY-R), CTR response prolongation, and food intake inhibition. 3) Acylated KBPs with different peptide cores show similar potency and patterns in vitro when modified with the same acylation.

[0138] Example 3 (Figure 2) Comparison of the effects of single doses of the triacylated variants of KBP089, A09 (KBP375), A11 (KBP376), and A32 (KBP377), on food intake and body weight with the nonacylated benchmark peptide (KBP-089) in 12-week-old HFD SD rats.

[0139] [Table 8]

[0140] Four days before the study, rats were housed individually in cages. They were randomized into 11 groups according to body weight (vehicle (0.9% NaCl), KBP (dose: 36 nmol / kg (150-157 μg / kg)). Rats were fasted overnight and then treated with a single dose of peptide or vehicle via subcutaneous administration in the morning. Food intake was monitored at the following intervals (0-4 h, 4-24 h, 24-48 h … 144-168 h). Body weight was measured at baseline and every 24 h after subcutaneous injection. Animal model studies confirmed the results of the β-arrestin studies and demonstrated improved efficacy compared to the unmodified peptide. 1) In terms of the usefulness of the acylation positions using KBP-089 as the core peptide, A11>A32>A09. 2) The diacylation and triacylation are significantly superior to non-acylated KBP-089 at a given dose in terms of in vivo activity and prolonged effect. Animal model studies also showed that acylation at position 9 reduced the potency of the peptide compared to the unmodified peptide, thus eliminating position 9 as a key position for further investigation.

[0141] Example 4 (Figure 3) Comparison of the effects of single doses of triacylated variants A11 and A32 with different peptide cores on food intake and body weight compared with the respective nonacylated benchmark KBPs (KBP-066, KBP-062, and KBP-110) in 20-week-old HFD SD rats.

[0142] [Table 9]

[0143] Four days before the study, rats were housed individually in cages. They were randomized into 11 groups according to body weight: vehicle (0.9% NaCl), KBP (dose: 4 nmol / kg (^17 μg / kg), 12 nmol / kg (^50 μg / kg), or 36 nmol / kg (^150 μg / kg)). Rats were fasted overnight and then treated with a single dose of peptide or vehicle via subcutaneous administration in the morning. Food intake was monitored at the following intervals (0-4 h, 4-24 h, 24-48 h … 144-168 h). Body weight was measured at baseline and every 24 h after subcutaneous injection. The results are as follows: 1) The peptide core did not affect the improvement observed with acylation at positions 11 or 32. 2) A11 was a better acylation site than A32.

[0144] Example 5 (Figure 4) Effects of single high-dose administration of KBP-042 and A11 / 3 acylated mutants of KBP-066 on food intake and body weight in 20-week-old HFD SD rats Four days before the test, rats were individually housed in cages. Rats were randomized into 11 groups according to body weight: vehicle (0.9% NaCl), KBP (dose: 300 nmol / kg (^1000 μg / kg)).

[0145] [Table 10]

[0146] Rats were fasted overnight and then treated with a single dose of peptide or vehicle via subcutaneous administration in the morning. Food intake was monitored at the following intervals (0-4 h, 4-24 h, 24-48 h … 188-312 h). Body weight was measured at baseline and every 24 h after subcutaneous injection. High dose studies with KBP356 and KBP372 demonstrated excellent extension of effects lasting for several days in vivo, therefore making these acylated peptides clear candidates for the development of once-weekly peptide therapeutics.

[0147] Example 6 (Figure 6-10) Compound KBP-356 (KBP-066A11.03) was further studied, which contains an AiB residue at position 8 and a preferred acylation at position 11 of the peptide. Long-term studies were performed in male ZDF rats (obese homozygous recessive (fa / fa) strain: 370) (Charles River, USA). Rats were delivered at 5 weeks of age. Rats were housed 2-3 per cage.

[0148] Long-term treatment of male ZDF rats: Rats were delivered to the Nordic Bioscience animal facility at 5 weeks of age (day -6). Rats were allowed to acclimate for 3 days. HbA1c and BW were recorded (day -3). Rats were randomized on day 4 based on HbA1c (1st place) and BW (2nd place). Study began on day 1.

[0149] Concentration and frequency of administration Animals were administered KBP-066 or saline (vehicle) once daily. KBP-066A11.03 was administered subcutaneously (sc) once every 3 days around noon. Physiological saline: The administration volume was 1 mL / kg. KBP-066: The dose volume was 1 mL / kg, the dose concentration was 5, 50, or 500 μg / kg, and the compound concentration was 5, 50, or 500 mg / L. The dose equivalent in nmol / kg is 1.43, 14.3, or 143 nmol / kg, respectively. KBP-066A11.03: The dose volume was 1 mL / kg, the dose concentration was 25 nmol / kg, and the compound concentration was 25 mmol / L. The dose equivalent in μg / kg is 104 μg / kg.

[0150] [Table 11]

[0151] [Table 12]

[0152] Total weekly dose by treatment group 5 μg / kg KBP-066 is equivalent to 35 μg / kg / week or 10 nmol / kg / week. 50 μg / kg KBP-066 is equivalent to 350 μg / kg / week or 100.4 nmol / kg / week. 500 μg / kg KBP-066 is equivalent to 3500 μg / kg / week or 1004 nmol / kg / week. 25 nmol / kg KBP-066 is equivalent to 243.4 μg / kg / week or 58.3 nmol / kg / week. Compounds were dissolved in saline and stored at −20° C. Aliquots were thawed immediately prior to administration.

[0153] Summary of test results Day 3: HbA1c measurement Day 1: (first day of study), rats were fasted for 6 hours and BG and blood samples were taken before dosing. Day 14: Fasting Blood Glucose (FBG) + Blood Samples (6 hour fast) Day 28: FBG + blood sample (6 hour fast) Day 42: FBG + blood sample (6 hour fast) Day 57: (Groups 1+2) / Day 58 (Groups 3+4) OGTT (11-hour fasting) without prior administration of KBP-066 or KBP-066A11.03. Hb1Ac was measured midway through the OGTT at t=120 or t=180. Day 62: FBG + blood sample (6 hour fast)

[0154] Food intake Food intake was monitored daily. Body weight was monitored daily for the first 3 weeks and then twice weekly from the third week onwards.

[0155] Fasting blood glucose Fasting blood glucose levels were monitored every 2 weeks using the Accu-Check® Avia monitoring system (Roche Rotkreus, Switzerland); measurements were taken from the tail vein.

[0156] HbA1c Rats were non-fasted for the first (randomized) and second (after the second OGTT) HbA1c measurements. A drop of blood was applied to the HbA1c cassette and HbA1c was measured using a DCA Vantage Analyzer. Administration of the compound or saline was continued during the first and second HbA1c measurements.

[0157] Oral glucose tolerance test Oral glucose tolerance tests (OGTTs) were performed after 8 weeks of treatment. Body weight from the previous day was used to calculate glucose doses. Animals were fasted for 11 hours. Heating was performed approximately 45 minutes prior to the -30 minute time point (see diagrams below). Animals were pre-medicated with KBP-066, KBP-066A11.03, or saline during the first OGTT, but not during the second OGTT, hence the (C) in the diagrams below.

[0158] [Table 13]

[0159] result Figure 6A+B, Accumulation of food intake FIG. 6A shows the food intake accumulation over the course of the study. The A11 treatment group consumed less food than the vehicle group. Also, the higher the dose, the greater the decrease in food intake. The acylated KBP-066 A11.03 treatment group had the greatest decrease in food intake compared to all KBP-066 dose groups. At the end of the study, all treatment groups showed a significant decrease in food consumption over the study period compared to the vehicle group, with the acylated KBP-066 A11.03 treatment group showing the greatest decrease in food intake; a decrease of 35% compared to the vehicle group (FIG. 6B).

[0160] Figure 7A+B, Body weight All treatment groups lost weight over the first 3 weeks of the study, and while ZDF vehicle rats became increasingly ill and were unable to maintain their weight / gain rate (FIG. 7A), the treatment groups caught up with the vehicle group in weight. The rate of weight gain compared to the vehicle group was dose-dependent and also dependent on the type of compound used in the treatment group (Figure 7B). The acylated KBP-066A11.03 treatment group had the slowest rate of recovery, followed by the 14.3 nmol / kg and 143 nmol / kg groups, and the fastest weight recovery was observed in the 1.43 nmol / kg group. This indicates that acylated KBP-066A11.03 administered subcutaneously once every three days regimen has an additive pharmacological benefit over non-acylated KBP-066 administered subcutaneously once daily.

[0161] Figure 8. Fasting blood glucose levels Although ZDF vehicle rats progressed to disease progression and were unable to maintain FGB, all treatment groups effectively attenuated FBG over the course of the study compared to the vehicle group. Acylated KBP-066A11.03 treatment was the most effective treatment, and only this treatment was able to increase FBG by only 5 mM during the 62-day study in this highly aggressive animal model of type 2 diabetes. Non-acylated KBP-066 dose-dependently reduced FBG, but was not as potent as the acylated treatment group in attenuating FBG. This also indicates that acylated KBP-066A11.03 has an additional pharmacological benefit over non-acylated KBP-066.

[0162] Figure 9. HbA1c at baseline and end of study As expected, the onset of diabetes in male ZDF rats and HbA1c at baseline before initiating treatment regimens are similar (FIG. 9A). At the end of the study (day 62), all treatment groups had significantly lower HbA1c levels compared to the vehicle group. Interestingly, the acylated KBP-066A11.03 treatment group had the lowest HbA1c values, which were also significantly lower than all non-acylated KBP-066 treatment groups (Figure 9B), further indicating the superiority of acylation over the non-acylated counterpart.

[0163] Figure 10. Oral glucose tolerance test (OGTT) An oral glucose tolerance test (OGTT) was performed after 8 weeks of treatment, and the results are shown in Figure 10A. Due to the treatment-induced FBG differences, the individual OGTT curves are significantly different. This difference is highlighted in the calculated tAUC values ​​(Figure 10B). All treatment groups have significantly lower tAUC than the vehicle group. The acylated KBP-066A11.03 treatment group had the lowest tAUC value, significantly lower than two of the three non-acylated KBP-066 treatment groups, with a p-value of 0.06 when compared to the remaining group, 143 nmol / kg KBP-066 (Figure 10B). In conclusion, the combined data indicate that acylated KBP-066A11.03 administered subcutaneously every 3 days has an additive pharmacological benefit over non-acylated KBP-066 administered subcutaneously once daily in obese diabetic ZDF rats.

[0164] Summary of Results of Examples 1-6 Results of acylation studies by acylation site Position 9 (acylation of the 9th position of a peptide) Acylation of the A09 position using monoacylation resulted in a sustained prolongation of in vivo activity, which merited further testing (Figure 1A-F). Furthermore, acylation of the A09 position using diacylation and triacylation attenuated the EC50 of both the CTR and AMYR receptors and did not result in an extended response of the CTR (Tables 3-4). However, acylation of position A09 using di- and tri-acylations prevented the previously observed extended effect of the core peptide in vivo, rendering the acylated KBPs similar in potency to vehicle. Thus, they were less potent than the nonacylated core peptide (Figure 2A-B) in reducing both food intake and body weight following a single subcutaneous injection of 36 nmol / kg compound. Therefore, position A09 was not considered further.

[0165] A11 position (acylation of the 11th position of the peptide) Acylation of the A11 position using monoacylation resulted in a sustained extension of in vivo activity, which merited further testing (Figure 1A-F). Acylation of the A11 position using diacylation and triacylation yielded the best EC50 value assay results for both the CTR and AMYR receptors and produced the highest extended response values ​​(tAUC) across all core peptides (Tables 3-4). Furthermore, A11 / 3 acylation significantly improved the in vivo activity of the core peptide compared to the nonacylated core peptide in both reducing food intake (Figure 2A) and reducing body weight (Figure 2B) after a single subcutaneous injection of 36 nmol / kg compound, and A11 / 3 acylation was superior in reducing food intake and body weight compared to any of the other acylation positions when using KBP-089 as the core peptide (Figure 2A-B).

[0166] This difference is further understood in the dose-response study, where all three doses (4 nmol / kg, 12 nmol / kg, and 36 nmol / kg) were superior to their corresponding A32 / 3 acylated peptides. In terms of potency, the lowest dose of A11 / 3 at 4 nmol / kg had a similar profile to the A32 / 3 acylated peptide at 36 nmol / kg. This was consistently demonstrated for all core peptides tested (Figure 3A-F). To further explore the potency of A11 position using triacylation, KBP-042 and KBP-066 acylated at position A11 using triacylation were tested at high doses (300 nmol / kg) and compared to the non-acylated counterparts, showing the potential maximum efficacy in terms of prolonged in vivo efficacy combined with prolonged bioavailability (Figure 4A-D). Triacylation at A11 attenuated food intake for over 120 h for both KBP-042 (Figure 4A) and KBP-066 (Figure 4C), with food consumption returning to vehicle levels after ^144 h. Treatment-mediated weight loss peaked after 96 h for both KBP-042 (Figure 4B) and KBP-066 (Figure 4D), with body weight returning to baseline levels after ^240 h. In conclusion, A11 was the best positioned of those tested in terms of preserving ligand potency and maximizing prolonged in vivo effects.

[0167] A12 position (acylation of the 12th position of the peptide) Acylation of the A12 position with monoacylation produced the poorest in vivo results compared to the vehicle group in a 4-h food intake study (Figure 5). Therefore, position A12 was not a good candidate for acylation and was not pursued further.

[0168] A16 position (acylation of the 16th position of the peptide) Acylation of position A16 using monoacylation did not show any extension of in vivo activity (Figure 1A-F). Therefore, position A16 was not a good candidate for monoacylation and was not studied further.

[0169] A18 position (acylation of the 18th position of the peptide) Acylation of the A16 position using monoacylation was effective over the 4-24 hour test period, but the observed effects were not sustained in long-term activity studies (KBP-347, 48 hours, Figure 1F). Therefore, position A18 was not a good candidate for monoacylation and was not studied further.

[0170] A32 position (acylation of peptide position 32) Acylation of the A32 position with monoacylation demonstrated prolonged in vivo efficacy in both food intake and body weight reduction, the best of those tested (Figure 1A-F). Acylation of the A32 position using diacylation and triacylation resulted in inferior EC50 assay values ​​compared to the A11 position for both the CTR and AMYR receptors. Acylation at position A32 slightly attenuated the CTR-mediated prolongation of the response compared with position A11, but still maintained the prolongation of the response (Tables 3-4). Acylation at position A32 improved the in vivo efficacy following a single subcutaneous administration of all core peptides tested compared with the corresponding non-acylated counterparts. However, this position was inferior to A11 in all diacylations and triacylations tested during in vivo studies at equivalent doses (Figures 2A-B, 3A-F). In conclusion, A32 was a neutral position compared to A11 in terms of preserving ligand potency and improving in vivo efficacy using 1, 2, and 3 acylation.

[0171] Further Acylation Studies (Examples 7-12) Example 7: β-arrestin and thioflavin T assays Additional PathHunter β-arrestin GPCR assays were performed using the same procedures as described in connection with Example 2. In independent bioassays, CTR and AMY-R cells were treated with increasing doses of KBP (ranging from 1 μM-0.1 nM, and vehicle) as specified in Tables 4.1-4.4 for the indicated time points.

[0172] A Thioflavin T assay was also performed. Thioflavin T (ThT) is a dye that is widely used to detect amyloid fibrils. In the presence of fibrils, ThT has an excitation maximum at 450 nm and enhanced emission at 480 nm, whereas ThT is essentially non-fluorescent at these wavelengths when not bound to amyloid fibrils. Therefore, ThT in combination with a fluorescent plate reader is an ideal tool for screening large numbers of in vitro samples for the presence of amyloid fibrils. The ThT assay used for KBP is a modification of the procedure described by Nielsen et al. to measure insulin fibril formation (Nielsen L, Khurana R, Coats A, Frokjaer 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).

[0173] The fibril formation screening assay was performed in triplicate with samples in a final volume of 20 μL in 384-well plates (Greiner Bio-One, 784080). The plates were sealed with optical adhesive film to prevent sample evaporation during the course of the assay. The plate is loaded into a fluorescent plate reader such as SpectraMax with SoftMax Pro 7.0.2 software and a template set at 37° C. temperature, 450 nm excitation wavelength, and 480 nm emission wavelength. The plate reader measures every 10 minutes for 24 hours, shaking the plate for 5 seconds before the first reading and 35 seconds before every other reading. Alternatively, plates are read after the following incubation times: 0 hours, 1 hour, 2 hours, 4 hours, and 24 hours.

[0174] Relative fluorescent units (RFU) are plotted as a function of time. Fibrogenesis is determined as an increase in RFU over baseline as described by Nielsen et al. Herein, four fibrillogenesis tiers are defined based on 18h fluorescence signal to obtain a single output reflecting peptide fibrillogenesis capacity: None = <1000RFU, Low = 1000-3000RFU, Medium = 3000-10000, High = >10000 The results of the Thioflavin T assay are also shown in Tables 4.1 through 4.4.

[0175] [Table 14]

[0176] [Table 15]

[0177] [Table 16]

[0178] [Table 17]

[0179] [Table 18]

[0180] Result - Acylation Length In terms of in vitro potency as a function of acylation length, a clear correlation was found between acylation length and in vitro potency: the shortest acylations, 11 (C14 biacid) and 6 (C16 biacid), yielded the lowest EC50 values ​​at both the CTR and AMYR receptors (Table 4.1), whereas the longest acylations, 9 (C24 biacid) and 10 (C26 biacid), recorded some of the highest EC50 values ​​at both receptors. None of the acylated peptides tested in this system using the KBP-066 backbone caused fiber formation problems.

[0181] Results - Acylation sites on the KBP-066 backbone The EC50 values ​​for the CTR and AMYR receptors in this system are listed in Table 4.2. In terms of in vitro potency as a function of acylation position on the KBP-066 backbone, three positions stand out as having calcitonin and amylin receptor dual agonist potential. A11, A19, and A24 all have EC50 values ​​of 5x10 at both receptors. -9 M range and unmatched, whereas the other positions tested did not have sufficient potency in comparison. It is clear that the increased potency at A11, A19 and A24 translates into improved in vivo efficacy for the KBP-066 backbone (see Figures 15, 17 and 18 attached hereto). Interestingly, A09 is the best CTR agonist among them, but has poor AMYR activity, suggesting that acylation at positions close to the N-terminus hinders AMYR activity, forming a biased ligand. Although an unmatched peptide (KBP-066A16.03(387)) gave a "low" score in the ThT assay, fibril formation was not evident for most positions in the KBP-066 backbone.

[0182] Results - Acylation sites on the KBP-021 backbone The EC50 values ​​for the CTR and AMYR receptors in this system are listed in Table 4.3. In terms of in vitro potency as a function of acylation position on the KBP-021 backbone, two positions stand out as having dual agonist capabilities. Both A11 and A19 have EC50 values ​​of 5x10 at both receptors. -9 While it is unique within the M range, the other positions tested did not have sufficient potency in comparison. It is clear that the increased potency at A11 and A19 also translates into improved in vivo efficacy for the KBP-021 backbone (see FIG. 16 attached hereto). Interestingly, fibril formation was evident for the KBP-021 backbone, but A19, unique among the peptides tested, gave good potency both in vitro and in vivo, despite giving a “high” score in the ThT assay, whereas the neighboring position, position 18, also gave a higher “medium” score in the ThT assay, suggesting that the KBP-021 backbone is susceptible to fibril formation when this region of the backbone is acylated. Furthermore, although tetraacylation and pentaacylation on A11 were scored as “low” in the ThT assay, this issue did not affect the favorable A11 acylation, and longer acylations also clearly showed fiber formation for this backbone KBP-021.

[0183] Results - Acylated Linkers The EC50 values ​​for CTR and AMYR in this system are listed in Table 4.4. In terms of in vitro potency as a function of acylation position on the KBP-066 backbone, the OEG-OEG-γGLU linker (356) had a nearly 10-fold higher EC50 in CTR than OEG-OEG-OEG-γGLU (385) and OEG-γGLU (384), however, all linkers had an EC50 of 5 × 10 in AMYR. -9 M range and were very close. In terms of fibril formation, the shortest linker, OEG-γGLU(384), scored “low” in the ThT assay, whereas the other two linkers scored “none.”

[0184] Example 8: (Figure 11) Comparison of the effects of a single dose of several position- and backbone-identical acylated variants (3, 4, 5, 6, 9, 10, 11), i.e., A11 and KBP-066, respectively, on food intake and body weight during acute conditioning in 20-week-old aged SD rats fed a high-fat diet for 8 weeks prior to the experiment.

[0185] [Table 19]

[0186] Rats were housed individually in cages 4 days before the study. They were randomized according to body weight into 8 groups (vehicle (0.9% NaCl), KBP (dose: 3 nmol / kg (^10-11 μg / kg)). They were fasted overnight and then treated with peptide or vehicle in a single dose subcutaneously 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.

[0187] FIG. 11. Results - Food intake and body weight Acylations 6, 10, and 11 were able to attenuate food intake and body weight, with peak suppression at 24 hours and then recovery to vehicle levels. Acylation 9 was able to attenuate food intake and body weight, with peak suppression at 48 hours and then recovery to vehicle levels. Acylation 3 was able to attenuate food intake and body weight, with peak suppression at 72 hours and then recovery to vehicle levels. Acylations 4 and 5 were able to attenuate food intake and body weight, with peak suppression at 96 hours and then recovery. Therefore, acylations 3, 4, and 5 are good candidates for acylation length, since dosing every 3 days in rodents translates to once a week dosing in humans, and the initial goal was to suppress food intake and body weight for at least 72 hours.

[0188] Example 9 (Figures 12, 13 and 14) The best performing variants according to the acute test results, i.e. acylated variants (3, 4, 5), were further investigated in a repeated dosing study to compare the effect of acylation at the same position and backbone, i.e. A11 and KBP-066.Food intake and body weight were investigated by chronic conditioning (5-week study) in 20-week-old SD rats that were fed a high-fat diet for 8 weeks prior to the start of the study.

[0189] [Table 20]

[0190] Rats were housed in pairs and randomized by body weight into treatment groups (vehicle (0.9% NaCl), KBP (dose: 3 nmol / kg (^14 μg / kg)). Food intake and body weight were monitored daily for 35 days. At the end of the study, an OGTT was performed and then the rats were sacrificed for collection and weight measurement of adipose tissue.

[0191] Long-term treatment of male SD rats fed a high-fat diet Rats were delivered to the Nordic Bioscience animal facility at 12 weeks of age and immediately switched to a high fat diet for an additional 8 weeks. Rats were randomized by weight before the start of the study. The study began on day 1.

[0192] Dosage concentration and frequency Animals were administered KBP every third day. Administration was by subcutaneous route (SC) around noon each time. Compounds were dissolved in saline and stored at -20°C. Aliquots were thawed immediately before administration. Physiological saline: The administration volume was 1 mL / kg. KBP: The administration volume was 1 mL / kg, and the administration concentration was 4 nmol / kg. The equivalent dose was ^14μg / kg in μg / kg. Total weekly dose per treatment group: 4nmol / kg KBP was 28nmol / kg / week or ^100μg / kg / week

[0193] Summary of test results Day 1: (Day 1 of Study) Dosing was performed. Days 1-35: Daily measurements of food intake and body weight Day 35: Body weight at end of study Day 35: Oral glucose tolerance test Day 35: Sacrifice + adipose tissue weight measurement

[0194] Oral glucose tolerance test An OGTT was performed after 5 weeks of treatment. Body weight from the previous day was used to calculate glucose dosage. Animals were fasted for 11 hours. Heating was performed approximately 45 minutes prior to the -30 minute time point (see diagram below). Animals were administered KBP or vehicle one day prior to the OGTT.

[0195] [Table 21]

[0196] White adipose tissue (WAT) weight measurement The entire epididymal and perirenal WAT depots were excised and weighed, and for inguinal WAT, a fixed anatomical limited area was excised and weighed.

[0197] FIG. 12. Results of food intake and body weight Figure 12 shows the change in food intake and body weight as a function of treatment over the course of the long-term study. Figure 12A shows the kinetics of food intake for acylations 3, 4 and 5, while Figure 12B shows the weight loss mediated by acylations 3, 4 and 5.

[0198] The results of FIG. 13 show that OGTT and adipose tissue FIG. 13 shows the results obtained from OGTT with the corresponding iAUC(OGTT) (Fig. A+B) and also the weights of three different adipose tissues, epididymal, inguinal and perirenal (Fig. 13C-E) and body weight at the end of the study (Fig. 13F). Treatment with acylation 3 caused a significant decrease in iAUC(OGTT), epididymal WAT size, perirenal WAT size and body weight. Treatment with acylation 4 and acylation 5 caused a significant decrease in iAUC(OGTT), epididymal WAT size and body weight, but not perirenal WAT size. None of the treatments significantly decreased the size of the inguinal WAT. Acylation 3 was slightly better than vehicle when compared to acylation 4 / 5, however there was no significant difference between the treatment groups.

[0199] Results of Figure 14. Competitive I-125 sCT ligand binding To test whether the improved efficacy of acylation 4 and acylation 5 in rapid conditioning could be applied to humans, competitive ligand binding assays were performed to explore the binding of the acylations to rodent and human serum albumin. Figure 14 shows the competitive binding of KBP-066A11.03 and KBP-066A11.05 to radiolabeled I-125 salmon calcitonin (NEX423, Perkin Elmer) in 2% rodent serum albumin (RSA) (Figure 4A) or in 2% human serum albumin (HSA) (Figure 4B). When the assay was performed in 2% HSA, the EC 50 There was no difference between acylation 3 and acylation 5 for IC50. However, when the assay was performed in 2% RSA, the 5-acylation shifted the IC50 further to the right, suggesting a stronger affinity for RSA in the assay. Therefore, it is strongly suspected that the increased efficacy observed in the rapid conditioning is rodent specific and not applicable to humans.

[0200] Example 10 (Figure 15) Comparison of the effects of single administration of three 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 SD rats fed a high-fat diet

[0201] [Table 22]

[0202] Rats were housed singly for 4 days prior to testing. They were randomized by body weight into 8 groups (vehicle (0.9% NaCl), KBP (dose: 4 nmol / kg (^10-11 μg / kg)). They were fasted overnight and then treated with a single dose of peptide or vehicle, administered subcutaneously in the morning. Food intake was monitored at the following intervals (0-4 h, 4-24 h, 24-48 h, 48-72 h, 72-96 h). Body weight was measured at baseline and at 4, 24, 48, 72 and 96 h after subcutaneous injection. Two backbones were tested: KBP-066 and KBP-021.

[0203] FIG. 15 Results - Food intake and body weight With respect to the positions on the backbone, the results for KBP-066 are as follows: At 4 nmol / kg in acute conditioning (FIG. 5), positions A11 and A19 were the two best positions in terms of suppressing both 72-hour food intake and 96-hour body weight. The third best position was A24, followed by A18 and A16. The least potent position for acylation was A12, which appears to be unfavorable for acylation as it slightly interferes with the DACRA-mediated effects on both food intake and body weight. Based on these data, A11 and A19 are preferred positions for acylation of the backbone of KBP-066.

[0204] FIG. 16 Results - Food intake and body weight When a different backbone, KBP-021, was used under the same experimental conditions as those of KBP-066, the positional pattern was slightly different.

[0205] [Table 23]

[0206] At 3 nmol / kg in the rapid conditioning setting (Figure 6), positions A11 and A19 were These were by far the best two positions in suppressing both 72-hour food intake and 96-hour body weight as observed for the KBP-066 backbone. The third best position was A18, but was much worse than A11 and A19. Position 24 was better than vehicle, but not nearly as good as observed for the KBP-066 backbone. Positions A16, A12, and A09 were all indistinguishable from vehicle for both food intake and body weight, impairing peptide activity, suggesting that acylation at these positions is disadvantageous. However, the in vitro characterization in Table 4.3 shows that A19 and A18 have significant problems in terms of fiber formation when combined with KBP-021, making A11 the preferred position for acylation on the KBP-021 backbone.

[0207] Example 11 (FIGS. 17 and 18) The ones showing the best potency based on the rapid assay results, i.e., acylation positions (A11 and A19), were further investigated and a repeat dosing study was performed to compare the effect of acylation with the same acylation and backbone, i.e., 3-acylation and acylation with KBP-066, respectively. Food intake and body weight were investigated by long-term conditioning (5-week study) in aged SD rats aged 20 weeks, which were fed a high-fat diet for 8 weeks prior to the start of the study.

[0208] [Table 24]

[0209] The experimental procedure was followed as described in Example 9. Briefly, rats were housed in pairs and randomized by weight into treatment groups (vehicle (0.9% NaCl), KBP (dose: 4 nmol / kg (^14 μg / kg)). Food intake and body weight were monitored daily for 35 days. At the end of the study, an OGTT was performed followed by sacrifice and adipose tissue harvesting and weighing.

[0210] Results in Figure 17 - Comparison of food intake and body weight for A11 and A19 in chronic conditioning Figure 17 shows the change in food intake (Figure 17A) and body weight (Figure 17B) as a function of treatment over the course of the long-term study. Both A11 and A19 suppressed food intake in a similar manner and significantly reduced body weight after 5 weeks of treatment. However, there was no significant difference between A11 and A19 in terms of their effect on body weight.

[0211] Figure 18 Results – OGTT and adipose tissue FIG. 18 shows the results obtained from OGTT with the corresponding iAUC(OGTT) (Fig. A+B) and also the weights of three different adipose tissues, epididymal, inguinal and perirenal (Fig. 18C-E) and body weight at the end of the study (Fig. 18F). Treatment with A11 in the experiment caused a significant decrease in iAUC(OGTT), epididymal WAT size, perirenal WAT size and body weight. Treatment with A19 in the experiment caused a significant decrease in iAUC(OGTT), epididymal WAT size and body weight, but not perirenal WAT size. None of the treatments significantly decreased the size of the inguinal WAT. A11 was slightly better than the vehicle when compared to A19, however, there was no significant difference between the treatment groups.

[0212] Example 12 (Figure 19) Comparison of the effects of a single dose of three position- and backbone-identical acylated linker variants (3, 7, and 8), i.e., A11 and KBP-066, respectively, on food intake and body weight during acute conditioning in 20-week-old aged SD rats fed a high-fat diet for 8 weeks prior to the experiment.

[0213] [Table 25]

[0214] Rats were housed individually for 4 days prior to testing. They were randomized by body weight into 8 groups (vehicle (0.9% NaCl), KBP (dose: 4 nmol / kg (^13-14 μg / kg)). They were fasted overnight and then treated with peptide or vehicle in a single dose subcutaneously in the morning. Food intake was monitored at the following intervals (0-4 h, 4-24 h, 24-48 h, 48-72 h, 72-96 h). Body weight was measured at baseline and at 4, 24, 48, 72 and 96 h after subcutaneous injection.

[0215] FIG. 19 Results - Food intake and body weight All three linkers tested were good in acute conditioning, with all linkers attenuating food intake up to 72 hours before recovery (FIG. 19A). Acylation 3 was slightly better than acylations 7 and 8 after 96 hours. This was evident from the corresponding weight loss (FIG. 19B), where acylation 3 was differentiated from acylations 7 and 8 early (24 hours) and continued to be differentiated at the 72 and 96 hour time points.

[0216] Summary of Results of Examples 7-12 Data gleaned from Figures 11-14 and Table 4.1 suggest that acylations 3, 4, and 5, ranging from the C18 diacid to the C22 diacid, are alternatives for developing acylated peptides for once-weekly therapy in humans. Therefore, C18, C20 and C22 diacids are preferred acylation lengths for the present invention.

[0217] Acylation position Data collected from Figures 15, 17 and 18 indicate that A19 may have a slight advantage in fast conditioning, whereas A11 has a slight advantage in long-term conditioning using the KBP-066 backbone and 3 acylation. Neither A11 nor A19 caused fiber formation issues when combined with the KBP-066 backbone (Table 4.2). Taken together, these data suggest that acylation at positions A11 and A19, in combination with the KBP-066 backbone, are the two best versatile positions to acylate for the development of a once-weekly therapy in humans. Therefore, A11 and A19 are preferred positions for acylation of KBP-066. Similarly, based on Table 4.3 and FIG. 16, it is apparent that A11 and A19 are the two best acylation positions when combined with KBP-021. However, A19 was highly prone to fiber formation under these conditions, and therefore, based on overall performance, triacylated A11 is the preferred acylation position and length for KBP-021.

[0218] Acylated Linking Group Based on this testing and Table 4.4, the OEG-OEG-γGLU linker is the linker of choice, as shorter linkers can cause fiber formation problems and longer linkers offer no benefit. Additionally, as shown in Figure 19, the OEG-OEG-γGLU linker also exhibits the best in vivo performance in fast-setting conditions, making it the overall preferred acylation linker.

[0219] In this specification, unless expressly indicated otherwise, the word "or" is used to mean an operator that returns a true value if either or both of the stated conditions are met, as opposed to the operator "exclusive-or," which requires that only one of the conditions be met. The word "comprising" is used in the sense of "including" and not "consisting of." All prior teachings acknowledged above are hereby incorporated by reference.

Claims

1. a calcitonin mimetic, wherein the lysine residue at position 11 of the calcitonin mimetic is acylated and / or the lysine residue at position 19 of the calcitonin mimetic is acylated; The side chain ε-amino group of the lysine residue is any one of the following: A fatty acid having a chain length of 16 or more, which may have a linking group, or Divalent fatty acid having a chain length of C16 or more, which may have a linking group A calcitonin mimetic which is acylated with an acyl group selected from:

2. The calcitonin mimetic has formula (I)(a): CX 2 X 3 LSTCX 8 LGK Ac … [During the ceremony, X 2 = A, G or S X 3 = N or S X 8 = M, V or α-aminoisobutyric acid (AiB), K Ac has a side chain ε-amino group which is any one of the following: C which may have a linking group １６ or C which may have a linking group １６ Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from 2. The calcitonin mimetic of claim 1, which is a calcitonin mimetic represented by the formula:

3. The calcitonin mimetic has the formula (I)(b): CX 2 X 3 LSTCX 8 LGX 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 K Ac … [During the ceremony, X 2 = A, G or S X 3 = N or S X 8 = M, V or α-aminoisobutyric 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, K Ac has a side chain ε-amino group which is any one of the following: C which may have a linking group １６ or C which may have a linking group １６ Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from 2. The calcitonin mimetic of claim 1, which is a calcitonin mimetic represented by the formula:

4. The calcitonin mimetic has the formula (II): CX 2 X 3 LSTCX 8 LGX 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 GX 29 X 30 X 31 P [During the ceremony, X 2 = A, G or S X 3 = N or S X 8 = M, V or α-aminoisobutyric 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, X 11 Or X 19 Either or both of Ac and K Ac has a side chain ε-amino group which is any one of the following: C １６ or longer chain length fatty acids C １６ Dibasic fatty acids with a chain length of Linking group -C １６ or Linking group -C １６ Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from 2. The calcitonin mimetic of any preceding claim, which is a calcitonin mimetic represented by the formula:

5. The calcitonin mimetic of formula (II) has the following formula: CX 2 X 3 LSTCX 8 LGX 11 LX 13 X 14 X 15 LX 17 X 18 X 19 X 20 TX 22 PX 24 TDVGANAP [During the ceremony, X 2 = A, G or S X 3 = N or S X 8 = 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, X 11 Or X 19 Either or both of Ac and K Ac has a side chain ε-amino group which is any one of the following: C １６ or longer chain length fatty acids C １６ Dibasic fatty acids with a chain length of Linking group -C １６ or Linking group -C １６ Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from 5. The calcitonin mimetic of claim 4, which is a calcitonin mimetic represented by the formula:

6. X 2 is S and X 3 is N; or X 2 is G and X 3 is N; or X 2 is A and X 3 The calcitonin mimetic of claim 4 or 5, wherein is S.

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

8. X 2 is S and X 3 is N and X 11 K Ac and X 13 is S and X 17 is H and X 18 is K or R, and X 19 is L and X 20 is Q or A, and X 22 is Y; or X 2 is S and X 3 is N and X 11 is R or K, and X 13 is S and X 17 is H and X 18 is K or R, and X 19 K Ac and X 20 is Q or A, and X 22 The calcitonin mimetic of any of claims 4 to 7, wherein is Y.

9. X 2 is A and X 3 is S and X 11 K Ac and X 13 is S and X 17 is H and X 18 is K or R, and X 19 is L and X 20 is Q or A, and X 22 is F; or X 2 is A and X 3 is S and X 11 is R or K, and X 13 is S and X 17 is H and X 18 is K or R, and X 19 K Ac and X 20 is Q or A, and X 22 The calcitonin mimetic of any of claims 4 to 7, wherein is F.

10. X 2 is G and X 3 is N and X 11 K Ac and X 13 is T and X 17 is N and X 18 is K or R, and X 19 is F and X 20 is H or A, and X 22 is F; or X 2 is G and X 3 is N and X 11 is R or K, and X 13 is T and X 17 is N and X 18 is K or R, and X 19 K Ac and X 20 is H or A, and X 22 The calcitonin mimetic of any of claims 4 to 7, wherein is F.

11. The calcitonin mimetic has the formula (III): CSNLSTCX 6 LGX 7 LSQDLHRX 8 QTYPKX 1 TX 5 VGANAP or a 33-mer peptide represented by The calcitonin mimetic has the formula (IV): CSNLSTCX 6 LGX 7 LSQDLHRX 8 QTYPKX 1 X 2 X 3 TX 5 VGANAP or a 35-mer peptide represented by The calcitonin mimetic has the formula (V): CSNLSTCX 6 LGX 7 LSQDLHRX 8 QTYPKX 1 X 2 X 3 X 4 TX 5 VGANAP or a 36-mer peptide represented by The calcitonin mimetic has the formula (VI): CSNLSTCX 6 LG K Ac LZX 1 X 2 X 3 X 4 TX 5 VGANAP A 37-mer peptide represented by [During the ceremony, X 1 ~X 4 Each of X is an amino acid, 1 ~X 4 At least one of X is a basic amino acid residue, and / or 1 ~X 4 At least two of X are independently polar or basic amino acid residues, and / or 1 ~X 4 At least one of X is a Gly residue, provided that 1 ~X 4 are not acidic residues; X 5 is D or N; X 6 is AiB or M; X 7 is K Ac And X 8 is L or X 7 is R or K and X 8 is K Ac either Z is selected from SQDLHRLSNNFGA, SQDLHRLQTYGAI, or ANFLVHSSNNFGA; K Ac has a side chain ε-amino group which is any one of the following: C １６ or longer chain length fatty acids C １６ Dibasic fatty acids with a chain length of Linking group -C １６ or Linking group -C １６ Dibasic fatty acids with a chain length of is a lysine residue acylated with an acyl group selected from 2. The calcitonin mimetic of claim 1, wherein

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

13. X 1 and X 4 At least one of X is a basic amino acid residue. 1 ~X 4 At least two of X are independently polar amino acid residues or basic amino acid residues, and 1 ~X 4 The calcitonin mimetic of claim 11 or 12, wherein none of

14. X 1 ~X 4 At least three of X are independently polar amino acid residues or basic amino acid residues, and 1 ~X 4 14. The calcitonin mimetic of any of claims 11-13, wherein none of the residues are acidic residues.

15. X 1 ~X 4 are independently a polar amino acid residue or a basic amino acid residue, and 1 ~X 4 15. The calcitonin mimetic of any of claims 11-14, wherein none of the residues are acidic.

16. X 1 ~X 4 all of X are independently polar amino acid residues or basic amino acid residues; 1 ~X 4 At least three of X are basic amino acid residues, and 1 ~X 4 16. The calcitonin mimetic of any of claims 11-15, wherein none of the residues are acidic.

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

18. X 1 is selected from Asn, Phe, Val, Gly, Ile, Leu, Lys, His, or Arg; X 2 is selected from Ala, Asn, His, Leu, Ser, Thr, Gly, or Lys; X 3 is selected from Ala, Phe, Ile, Ser, Pro, Thr, Gly or Lys; and / or X 4 is selected from Ile, Leu, Gly, His, Arg, Asn, Ser, Lys, Thr, or Gln; X 1 Or X 4 At least one of X is a basic amino acid residue, and / or 1 ~X 4 at least two of X are independently polar amino acid residues and / or basic amino acid residues, and / or 1 ~X 4 18. The calcitonin mimetic of any one of claims 11 to 17, wherein at least one of said residues is a Gly residue.

19. X 1 is selected from Asn, Gly, Ile, His, or Arg; X 2 is selected from Asn, Leu, Thr, Gly or Lys; X 3 is selected from Phe, Pro, Ile, Ser, Thr, Gly or Lys; and / or X 4 is selected from Gly, His, Asn, Ser, Lys, Thr, or Gln; X 1 Or X 4 At least one of X is a basic amino acid residue, and / or 1 ~X 4 at least two of X are independently polar amino acid residues and / or basic amino acid residues, and / or 1 ~X 4 19. The calcitonin mimetic of any of claims 11 to 18, wherein at least one of the residues is a Gly residue.

20. The calcitonin mimetics of any of the formulae (III) to (V) may contain the following conservative substitutions: The Asp residue at position 15 of the peptide is replaced with Glu; The Arg residue at position 18 of the peptide is substituted with Lys; and / or The Lys residue at position 24 of the peptide is replaced with Arg.

20. The calcitonin mimetic of any one of claims 11 to 19, comprising one or more conservative substitutions selected from:

21. The calcitonin mimetic of formula (VI), wherein the Z moiety of the peptide of formula (VI) is SQDLHRLQTYGAI or SQDLHRLSNNFGA; Conservative substitutions include: The Asp residue at position 15 of the peptide is substituted with Glu; and / or The Arg residue at position 18 of the peptide is replaced with Lys.

20. The calcitonin mimetic of any one of claims 11 to 19, comprising one or more conservative substitutions selected from:

22. the linking group comprises a glutamic acid residue and / or an oligoethylene glycol (OEG) amino acid linking group comprising one OEG amino acid or two or more OEG amino acids linked together; The OEG amino acid has the following formula: 【Chemistry 1】 wherein n is a number from 1 to 10.

22. The calcitonin mimetic of claim 1, wherein

23. 23. The calcitonin mimetic of claim 22, wherein the OEG amino acid linking group comprises a linkage of two to six OEG amino acids together.

24. 24. The calcitonin mimetic of claim 22 or 23, wherein the OEG amino acid linking group further comprises one or more glutamic acid residues at the amino or carboxy terminus of the OEG amino acid linking group.

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

26. The OEG amino acid linking group is any one of the following: 【Chemistry 2】 25. The calcitonin mimetic of any one of claims 22 to 24, selected from:

27. The linking group is as follows: 【Chemistry 3】 27. The calcitonin mimetic of claim 26, wherein

28. The acyl group is C １８ or longer chain length fatty acids C １８ Dibasic fatty acids with a chain length of Linking group -C １８ or Linking group -C １８ Dibasic fatty acids with a chain length of 28. The calcitonin mimetic of any one of claims 1 to 27, selected from:

29. The acyl group may be any one of the following: C １８ ~C ３０ Fatty acids C １８ ~C ３０ Dibasic fatty acids Linking group -C １８ ~C ３０ Fatty acids Linking group -C １８ ~C ３０ Dibasic fatty acids 29. The calcitonin mimetic of any one of claims 1 to 28, selected from:

30. The acyl group may be any one of the following: C １８ ~C ２２ Fatty acids C １８ ~C ２２ Dibasic fatty acids Linking group -C １８ ~C ２２ Fatty acids Linking group -C １８ ~C ２２ Dibasic fatty acids 30. The calcitonin mimetic of any one of claims 1 to 29, selected from:

31. K Ac is acylated with a linking group-divalent fatty acid, The linking group-divalent fatty acid is a divalent fatty acid having a C １８ ~C ２２ and the linking group is as follows: 【Chemistry 4】 31. The calcitonin mimetic of any one of claims 1 to 30, wherein

32. The peptide may be any 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 is selected from The K Ac A calcitonin mimetic according to any one of claims 1 to 31, wherein:

33. The peptide may be any of the following: AcCSNLSTCMLGK Ac LSQDLHRLQTYPKTDVGANAP-NH 2 AcCSNLSTC(AiB)LGK Ac LSQDLHRLQTYPKTDVGANAP-NH 2 AcCGNLSTC(AiB)LGK Ac LTQDLNKFHTFPKTDVGANAP-NH 2 AcCSNLSTCVLGK Ac LSQELHKLQTYPRTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQELHRLQTYPKTDVGANAP-NH 2 AcCASLSTCVLGK Ac LSQDLHKLQTFPKTDVGANAP-NH 2 AcCASLSTCMLGK Ac LSQDLHKLQTFPKTDVGANAP-NH 2 AcCGNLSTCMLGK Ac LSQDLNKFHTFPQTDVGANAP-NH 2 AcCSNLSTC(AiB)LGK Ac LANFLVHSSNNFGAILPKTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHSSTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHSSNTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLSNNFGAILSSTNVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYGAILSPKTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LANFLVHSSNNFGAILPKTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKILSSTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKGLITTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKNNFGTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKRTTQTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHTTNTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHGGQTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHKKNTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQDLHRLQTYPKHKKHTDVGANAP-NH 2 AcCSNLSTC(AiB)LGRLSQDLHRK Ac QTYPKTDVGANAP-NH 2 AcCSNLSTCMLGK Ac LSQELHRLQTYPKTDVGANAP-NH 2 is selected from The K Ac is acylated with a linking group-divalent fatty acid, The linking group-divalent fatty acid is a divalent fatty acid having a C １８ ~C ２２ and the linking group is as follows: 【Chemistry 5】 33. The calcitonin mimetic of any one of claims 1 to 32, wherein

34. 34. A peptide according to any one of claims 1 to 33, formulated for enteral administration.

35. 35. The peptide of claim 34, wherein the peptide is formulated in an oral pharmaceutical composition comprising coated citric acid particles, the coated citric acid particles increasing the oral bioavailability of the peptide.

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

37. 37. The peptide of claim 36, wherein the carrier comprises 5-CNAC, SNAD, or SNAC.

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

39. 39. The peptide of claim 38 formulated for injection.

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

41. 34. A peptide according to any one of claims 1 to 33 for use as a medicament.

42. 34. A peptide according to any one of claims 1 to 33 for use in the treatment of diabetes mellitus (type I and / or type II), overweight, excessive food intake, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, dysregulated blood glucose levels, dysregulated response to a glucose tolerance test or dysregulated food intake.

43. 34. The peptide of any of claims 1 to 33 in combination with metformin or another insulin sensitizer for use in the treatment of diabetes mellitus (type I and / or type II), overweight, excessive food intake, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, dysregulated blood glucose levels, dysregulated response to a glucose tolerance test or dysregulated food intake.

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

45. 45. The peptide of claim 44, wherein the overweight condition is obesity.

46. A combination preparation comprising a peptide according to any one of claims 1 to 33 and an insulin sensitizer.

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

48. 34. A method for the treatment of diabetes mellitus (type I and / or type II), overweight, excessive food intake, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, dysregulated blood glucose levels, dysregulated response to glucose tolerance test or dysregulated 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. 34. A method for the treatment of diabetes mellitus (type I and / or type II), overweight, excessive food intake, metabolic syndrome, rheumatoid arthritis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, alcoholic fatty liver disease, osteoporosis or osteoarthritis, dysregulated blood glucose levels, dysregulated response to glucose tolerance test or dysregulated 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 in combination with metformin or another insulin sensitizer.

50. 34. A method for treating an overweight condition 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 in combination with a weight loss drug.

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