IL-4 DERIVED PEPTIDES FOR USE IN THE TREATMENT OF OBESITY - Patent application

Abstract

The present disclosure relates to peptides derived from interleukin-4 (IL-4) for the treatment of obesity.

Description

[Technical field]

[0001] The present disclosure relates to peptides derived from IL-4 for the treatment of obesity. In particular, the present disclosure relates to peptides comprising the amino acid sequence of SEQ ID NO: 1 or a variant thereof for use in the treatment of obesity. [Background technology]

[0002] (background) Overweight and obesity are defined as abnormal or excessive adiposity that presents a risk to health. Body Mass Index (BMI) is calculated based on weight and height. A BMI above 25 is considered overweight and a BMI above 30 is considered obese. Obesity has epidemic magnitude, reaching pandemic levels in the past 50 years. Obesity worldwide has almost tripled since 1975. In 2016, more than 1.9 billion adults aged 18 years and older were overweight. Of these, more than 650 million were obese. More than 340 million children and adolescents aged 5-19 years were overweight or obese in 2016 (Roglic, G. 2016).

[0003] Both overweight and obesity are systemic diseases associated with the leading causes of morbidity and mortality in the United States, including type 2 diabetes, heart disease, stroke, and some types of cancer (Roglic, G. 2016). Obesity is therefore a serious condition, as it is also associated with poor mental health, reduced quality of life, and contributes to reduced life expectancy (Abdelaal 2017).

[0004] Several drugs have been approved for the treatment of obesity. One example is semaglutide, sold under the trade name Wegovy. Semaglutide is the first glucagon-like peptide-1 (GLP-1) agonist developed and approved for the treatment of type 2 diabetes. The most notable effect of GLP-1 agonists is their ability to promote insulin secretion in a glucose-dependent manner by binding to the GLP-1 receptor expressed on pancreatic beta cells. Most importantly, GLP-1 agonists have been shown to inhibit glucagon secretion at blood glucose levels above fasting levels. Importantly, this does not affect the glucagon response to hypoglycemia, making GLP-1 agonists a safe antidiabetic drug with a very low incidence of hypoglycemia compared to insulin.

[0005] In June 2021, semaglutide was approved by the US Food and Drug Administration for chronic weight management in obese or overweight adults with at least one weight-related condition (e.g., hypertension, type 2 diabetes, or high cholesterol). As anti-obesity drugs, GLP-1 agonists act by binding to GLP-1 receptors in the hypothalamus, thereby suppressing appetite. In addition, GLP-1 agonists bind to GLP-1 receptors in the stomach and inhibit gastric emptying, acid secretion, and motility, which collectively promote satiety. As a result, diabetic subjects treated with GLP-1 receptor agonists also often experience beneficial weight loss in addition to controlling their blood glucose levels. However, there remains a need in the art for new and more effective treatments for obesity.

[0006] Human interleukin-4 (IL-4) consists of the following amino acid sequence (signal peptide, i.e., amino acid AA 1-24 It is an anti-inflammatory cytokine that has the following properties: MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS (UniProtKB - P05112; SEQ ID NO: 3)

[0007] Mature IL-4 has 129 amino acid residues and contains three intramolecular disulfide bonds (Cys3-Cys127; Cys46-Cys99; Cys24-Cys65) with the following amino acid sequences: HKCDITLQEI IKTLNSLTEQ KTLCTELTVT DIFAASKNTT EKETFCRAAT VLRQFYSHHE KDTRCLGATA QQFHRHKQLI RFLKRLDRNL WGLAGLNSCP VKEANQSTLE NFLERLKTIM REKYSKCSS (SEQ ID NO:4)

[0008] Like other cytokines, IL-4 exerts its biological activity by binding to receptors on the cell surface. Two receptor types for IL-4 are known, called type I IL-4R and type II IL-4R. Type I IL-4R is composed of the IL-4Rα chain and the IL-2Rγ (γ c) chain, whereas type II IL-4R is composed of IL-4Rα and IL-13α1 chains. IL-4 has many biological roles, including stimulation of activated B and T cell proliferation, and differentiation of B cells into plasma cells. The presence of IL-4 in extravascular tissues promotes alternative activation of macrophages into M2 cells and inhibits classical activation of macrophages into M1 cells, resulting in a reduction of pathological inflammation. IL-4 is mainly secreted by CD4+ T cells (Th2 cells), which play a key role in the pathogenesis of acute and chronic inflammation by controlling the cooperation of specific cytokine inhibitors and soluble cytokine receptors to regulate human immune responses and maintaining an appropriate balance between pro- and anti-inflammatory activities. Therefore, modulation of IL-4 signaling has been greatly studied in autoimmune and inflammatory diseases. Recently, IL-4 has been shown to promote lipolysis via enhancing hormone-sensitive lipase (HSL) activity (see Scientific Reports, 2019 9:11974).

[0009] Small peptide fragments derived from IL-4 that act as IL-4 mimetics have previously been shown to inhibit TNF-α release from macrophages and induce neuritogenic responses from primary neurons (see WO 2010 / 054667). One such example is a peptide with the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1). This peptide can bind to the IL-4 receptor and inhibit macrophage activation, thereby preventing the development of inflammatory responses, and is therefore proposed to be useful in many inflammatory and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, and Huntington's disease. Summary of the Invention [Problem to be solved by the invention]

[0010] (Summary) The present invention relates to the surprising observation that mice administered peptides comprising SEQ ID NO:1 (i.e., treated group) are less susceptible to weight gain compared to the placebo group. Thus, the present invention relates to the recognition that peptides derived from IL-4 and acting as IL-4 mimetics can be used to treat obesity, even though they have previously been associated with anti-inflammatory effects. Thus, peptide fragments derived from IL-4 and acting as IL-4 mimetics may have promising potential as a new generation of anti-obesity drugs with a novel mode of action as the peptide fragment of SEQ ID NO:1 has been shown to be a high affinity partial agonist for type I IL-4R and an antagonist for type II IL-4R (Klementiv 2013). [Means for solving the problem]

[0011] In a first aspect, the present disclosure relates to a peptide, or a pharma- ceutically acceptable salt thereof, for use in the treatment of obesity, comprising 13 to 19 amino acid residues and further comprising a fragment having the amino acid sequence AQAQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having 1, 2 or 3 amino acid substitutions and / or deletions, or a pharma-ceutically acceptable salt thereof.

[0012] In a second aspect the present disclosure relates to a peptide, or a pharma- ceutically acceptable salt thereof, for use in the treatment of obesity, said peptide comprising two or more covalently linked peptide fragments according to the first aspect.

[0013] In a third aspect, the present disclosure relates to a pharmaceutical composition for use in the treatment of obesity, the pharmaceutical composition comprising a peptide according to the first and / or second aspect.

[0014] In a fourth aspect, the present disclosure relates to a method for the treatment of obesity, the method comprising administering to an individual in need thereof an effective amount of a peptide or pharmaceutical composition according to any of the aspects and embodiments disclosed herein.

[0015] (Definitions and Abbreviations) The term "individual" refers to a vertebrate, a particular member of a mammalian species, preferably a primate, including a human. As used herein, "subject" and "individual" may be used interchangeably.

[0016] "Individual in need thereof" refers to an individual who would benefit from the present disclosure. In one embodiment, the individual in need thereof is obese (BMI>30), preferably with at least one weight-related condition (e.g., hypertension, type 2 diabetes, or high cholesterol). In another embodiment, the individual in need thereof is overweight (BMI>25), preferably with at least one weight-related condition (e.g., hypertension, type 2 diabetes, or high cholesterol). Thus, in this context, "obesity" should be understood broadly to include overweight (BMI>25), preferably with at least one weight-related condition (e.g., hypertension, type 2 diabetes, or high cholesterol).

[0017] An "effective amount" of a peptide can be administered in a single dose or via multiple doses totaling an effective amount, for example, within a 24-hour period. It can be determined using standard clinical procedures to determine appropriate amounts and timing of administration. It is understood that an "effective amount" can be the result of empirical and / or individualized (case-by-case) judgment on the part of the treating medical professional and / or individual.

[0018] In the context of the present invention, the present invention provides a method for determining whether a sequence of several consecutive amino acid residues is present in an amino acid region, e.g., the AA 68 -AA 97 When referring to peptide fragments derived from interleukin-4 (SEQ ID NO: 4), it should be understood as small stretches (i.e. contiguous sequences) of amino acids selected from the respective regions of interleukin-4 (SEQ ID NO: 4). Thus, the cytokine IL-4 (SEQ ID NO: 4) does not form part of the present invention. For example, the amino acid region AA 68 -AA 89A peptide fragment containing 16 consecutive amino acid residues derived from can be obtained from the consecutive sequences highlighted in bold below.

[0019] HKCDITLQEI IKTLNSLTEQ KTLCTELTVT DIFAASKNTT EKETFCRAAT VLRQFYSHHE KDTRCLGATA QQFHRHKQLI RFLKRLDRNL WGLAGLNSCP VKEANQSTLE NFLERLKTIM REKYSKCSS (SEQ ID NO:4).

[0020] Thus, the amino acid region AA of interleukin-4 (SEQ ID NO: 4) 68 -AA 89 The 16 consecutive amino acid residues derived from include the fragments / sequences ATAQQFHRHKQLIRFL; TAQQFHRHKQLIRFLK; AQQFHRHKQLIRFLKR, QQFHRHKQLIRFLKRL, QFHRHKQLIRFLKRLD, FHRHKQLIRFLKRLDR, HRHKQLI RFLKRLDRN, and RHKQLI RFLKRLDRNL. The peptide may be a variant of a fragment / sequence derived from interleukin-4 (SEQ ID NO: 4). As a non-limiting example, AQFHRHKQLIRFLKRA (SEQ ID NO: 1) is a variant of the fragment / sequence QQFHRHKQLIRFLKRL, where the terminal amino acids Q and L are replaced with A (i.e., AQFHRHKQLIRFLKRA (SEQ ID NO: 1) has two amino acid substitutions). Thus, in the present context, the present disclosure relates to, for example, the amino acid region AA 68 -AA 97When referring to a peptide comprising a fragment of 13-19 consecutive amino acid residues derived from, it should be understood that the number of consecutive amino acids should not be less than 13 and should not exceed 19 in a single amino acid stretch (i.e., the fragment without considering any linker, if present), but that the peptide may comprise two or more such fragments linked via a linker (spacer). As a non-limiting example, the following peptide of formula (I) comprises two peptide fragments of 16 consecutive amino acids each linked via a linker at their C-termini: [ka]

[0021] The peptide fragments can also be lipidated to alter the pharmacokinetic properties of the peptide.

[0022] As used herein, "sequence identity" refers to the number of exact amino acid residues that match between two different sequences being compared. If a variant is 90% identical to a sequence having 10 amino acid residues, the variant may differ from the sequence by one amino acid substitution.

[0023] As used herein, the terms "treat," "treatment," and "therapy" refer equally to curative, prophylactic, preventative, or ameliorative therapies. The terms include approaches to obtain beneficial or desired physiological results, which may be clinically established, such as, for example, weight loss or fat percentage loss.

[0024] The term "covalently linked" should be understood as at least two peptides / fragments according to the present disclosure covalently linked to each other via a linker or spacer. The covalently linked peptides may have the same sequence or different sequences. Various types of linkers (spacers) can be used to link the peptides together, for example those commonly known in the art for peptide dendrimers, fusion peptides / proteins, or hybrid peptides. For example, the linker (spacer) can be a peptide linker, for example lysine-beta alanine, as exemplified herein, a single amino acid, for example lysine, or a small stretch of amino acids (see, for example, Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369). The linker can also be a non-peptide linker, for example a PEG chain, or a diamine, such as ethylenediamine. Those skilled in the art will understand that the linker (spacer) used can be changed to other linkers (spacers) without departing from the concept of the present invention. Most preferably, the peptides / fragments are covalently linked via their N- or C-terminal units, most preferably via their C-terminal units, as exemplified herein. Most preferably, two peptides / fragments are linked by lysine, as exemplified herein. The beta-alanine of the lysine linker moiety merely acts as a spacer between the link-link linkers used in SPPS to reduce steric hindrance during synthesis. Thus, the beta-alanine moiety may be replaced with other common spacers used in the art of SPPS. Various branched dendrimers based on lysine can be obtained, for example, as described in (Proc. Natl. Acad. Sci. USA, Vol 85, pp. 5409-5413, August 1988). The dimers disclosed herein have been found to perform better than the monomers.

[0025] The term "variant" means that the peptide sequence may be modified, for example, by substitution of one or more amino acid residues. Both L- and D-amino acids may be used, and non-natural amino acids, preferably L-amino acids, are used. In the present context, when referring to a variant having one, two or three amino acid substitutions and / or deletions, it is to be understood that the total of substitutions and / or deletions is up to three. As an example, a variant may have, for example, two substitutions and one deletion (total=3). Similarly, a variant may have three substitutions and zero deletions (total=3), or a variant may have three deletions and zero substitutions (total=3). As an example, if the present disclosure relates to a peptide for use in the treatment of obesity, and said peptide has, for example, 13 amino acid residues, and further said peptide comprises a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1, i.e. 16 amino acids), it would have to be a variant with three deletions (16-3=13 amino acids). Preferably, the substitutions in the variant are conservative substitutions for conservative amino acids. The conservative amino acid groups are as follows: A, G (neutral, weakly hydrophobic), Q, N, S, T (hydrophilic, uncharged) E, D (hydrophilic, acidic) H, K, R (hydrophilic, basic) A, L, P, I, V, M, F, Y, W (hydrophobic, aromatic) C (crosslink formation)

[0026] Conservative substitutions may be introduced at any position in a peptide for use with the present disclosure, however, it may also be desirable to introduce non-conservative substitutions at any position in a peptide for use with the present disclosure.

[0027] The peptide sequences of the present disclosure can be prepared by any conventional synthetic method, such as solid phase peptide synthesis (SPPS).

[0028] The peptide according to the present invention may be in the form of a pharma-ceutically acceptable salt. Thus, the pharma-ceutically acceptable salt is intended to include any salt that is generally used in the formulation of peptides. Such salts include both acid addition salts and basic salts, and can be found, for example, in Remington's Pharmaceutical Sciences, 17th edition. [Brief description of the drawings]

[0029] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] FIG. 1A shows the crystal structure of IL-4 (PDB identifier: 1CYL) with the amino acid residues shown in the region AA71-AA86 which constitute part of α-helix C (AA71-AA94). [Figure 1B] FIG. 1B shows the crystal structure of IL-4 (PDB identifier: 1CYL) with the amino acid residues shown in the region AA71-AA86 which constitute part of α-helix C (AA71-AA94). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] (Detailed Description) The present invention relates to the surprising discovery that small peptide fragments derived from IL-4, and more specifically, the α-helix C of IL-4 (i.e., an IL-4R type I agonist), which have only been associated with anti-inflammatory effects, have been found to significantly reduce the body weight of animals upon administration.

[0031] Thus, the present disclosure relates to peptides that are IL-4 receptor type I agonists, preferably IL-4 receptor type II antagonists, for use in the treatment of obesity.

[0032] Mature IL-4 has 129 amino acid residues and contains three intramolecular disulfide bonds (Cys3-Cys127; Cys46-Cys99; Cys24-Cys65). The peptides used in the experimental section herein have the amino acid sequence of AQFHRHKQLIRFLKRA (SEQ ID NO:1) (i.e., the first fragment) covalently linked via a linker (spacer) (i.e., lysine-beta alanine) to another peptide having the amino acid sequence of AQFHRHKQLIRFLKRA (SEQ ID NO:1) (i.e., the second fragment). The peptide of SEQ ID NO:1 is composed of the amino acid AA 71 -AA 86 (IL-4 without the signal sequence) is a variant of the peptide (highlighted in bold below).

[0033] HKCDITLQEI IKTLNSLTEQ KTLCTELTVT DIFAASKNTT EKETFCRAAT VLRQFYSHHE KDTRCLGATA QQFHRHKQLI RFLKRLDRNL WGLAGLNSCP VKEANQSTLE NFLERLKTIM REKYSKCSS (SEQ ID NO:4)

[0034] Amino acid AA of SEQ ID NO:1 and SEQ ID NO:4 71 -AA 86 As can be seen from the alignment with SEQ ID NO:1, SEQ ID NO:2 is a variant of the peptide fragment derived from this site that has two amino acid substitutions (highlighted in bold).

[0035] QQFHRHKQLIRFLKRL IL-4AA 71 -AA 86 (SEQ ID NO:4) AQFHRHKQLIRFLKRA (SEQ ID NO: 1).

[0036] Region AA of SEQ ID NO:4 71 -AA 86 As shown in the crystal structure of Figure 1A / B, α-helix C (AA 71 -AA 94) (PDB Identifier: 1CYL). This α-helix C interacts with a portion of the ectodomain of IL-4Rα1, and thus α-helix C (AA 71 -AA 94 ) are likely to have similar binding characteristics and pharmacology as SEQ ID NO: 1. 71 -AA 94 , preferably AA of SEQ ID NO:4 71 -AA 86 Peptides derived from or near the region of α-helix C (AA 71 -AA 94 ) are capable of forming an α-helix, at least for a portion of their amino acid sequence. The tendency of short peptides to form α-helices can be predicted using software such as the PROTEUS Structure Prediction Server, or can be determined experimentally, for example using X-ray crystallography. As can be seen, the PROTEUS Structure Prediction Server predicts that the amino acids highlighted in bold AQFHRHKQLIRFLKRA (SEQ ID NO: 1) have a high tendency to form an α-helix (confidence score 9 on a scale ranging from 0 to 9, 9 being the highest confidence). Thus, most preferably, the α-helix C (AA 71 -AA 94 At least a portion of the fragment derived from ) forms an α-helix.

[0037] In particular, the present invention relates to the discovery that a peptide comprising a fragment having the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO:1) significantly reduces weight gain in animals when administered, as shown in the experimental section herein.

[0038] Thus, in a first aspect, the present disclosure relates to a peptide for use in the treatment of obesity, said peptide comprising 13 to 19 amino acid residues, further comprising a fragment having the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having 1, 2 or 3 amino acid substitutions and / or deletions.

[0039] In a preferred embodiment, the present disclosure relates to a peptide for use in the treatment of obesity, comprising 14 to 18 amino acid residues and further comprising a fragment having the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having 1, 2 or 3 amino acid substitutions and / or deletions.

[0040] In a more preferred embodiment, the present disclosure relates to a peptide for use in the treatment of obesity, comprising 15 to 17 amino acid residues and further comprising a fragment having the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having 1, 2 or 3 amino acid substitutions and / or deletions.

[0041] In an even more preferred embodiment, the present disclosure relates to a peptide for use in the treatment of obesity, comprising 16 amino acid residues and further comprising SEQ ID NO:1 (AQFHRHKQLIRFLKRA) or a variant of SEQ ID NO:1 (AQFHRHKQLIRFLKRA) having 1, 2 or 3 amino acid substitutions.

[0042] In another embodiment, the present invention relates to a peptide or a pharma- ceutical acceptable salt thereof for use in the treatment of obesity, wherein the peptide is 68 -AA 97 , preferably AA 71 -AA 94 , more preferably AA 71 -AA 89, most preferably the AA 71 -AA 86 or a variant thereof having 1, 2, 3, or 4 amino acid substitutions. 68 -AA 97 , preferably AA 71 -AA 94 , preferably AA 71 -AA 89 , most preferably AA 71 -AA 86 In a more preferred embodiment, the peptide has 14 to 18 consecutive amino acid residues derived from interleukin-4 (SEQ ID NO: 4) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions. 68 -AA 97 , preferably AA 71 -AA 94 , preferably AA 71 -AA 89 , most preferably interleukin-4 AA 71 -AA 86 In a further preferred embodiment, the peptide has 15 to 17 consecutive amino acid residues derived from (SEQ ID NO: 4) or a variant thereof having 1, 2, 3, or 4 amino acid substitutions. 68 -AA 97 , preferably AA 71 -AA 94 , more preferably AA 71 -AA 89 , most preferably the AA 71 -AA 86 or a variant thereof having 1, 2, 3 or 4 amino acid substitutions. In the above embodiment, it is highly preferred that the peptide comprises the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having 1 or 2 substitutions.

[0043] In any of the above-mentioned embodiments, the variant preferably has two amino acid substitutions and / or deletions, even more preferably one amino acid substitution or deletion.

[0044] Peptides for use according to the invention may be amidated at the C-terminus (-CONH2) or may have a free carboxylic acid (-COOH) or another post-translational modification such as a methyl ester (-COOMe). Peptides according to the invention may be free amine (-NH2), N-acylated (-NHCOR), N-methylated (-NHCH3 or -N(CH3)2), or deaminated at the N-terminus. Peptides may also be lipidated, for example at the N-terminal unit and / or one or more lysine residues, to alter the PK properties of the peptide.

[0045] Thus, in one embodiment of the disclosure, the C-terminal amino acid is present as a free carboxylic acid ("-COOH"). In another embodiment, the C-terminal amino acid is an amidated derivative ("-CONH2"). In another embodiment, the N-terminal amino acid comprises a free amino group ("NH2"). In another embodiment, the N-terminal amino acid is an acetylated derivative (COCH3). Most preferably, it is amidated ("-CONH2") unless the C-terminus is linked to another peptide / fragment via a linker via the C-terminus. Most preferably, the N-terminal unit comprises a free amino group ("NH2").

[0046] In a highly preferred embodiment, the present disclosure relates to a peptide for use in the treatment of obesity, said peptide comprising 16 amino acid residues, and further, said peptide comprises a fragment of SEQ ID NO: 1 (AQFHRHKQLIRFLKRA). Considering the fact that SEQ ID NO: 1 has 16 amino acids, it should be understood from the word "comprising" that the peptide may for example be lipidated and / or may comprise post-translational modifications commonly used in the art.

[0047] In another highly preferred embodiment, the present disclosure relates to a peptide for use in the treatment of obesity, said peptide consisting of SEQ ID NO:1 (AQFHRHKQLIRFLKRA) or a variant of SEQ ID NO:1 (AQFHRHKQLIRFLKRA) having one, two or three amino acid substitutions.

[0048] In a further highly preferred embodiment, the present disclosure relates to a peptide for use in the treatment of obesity, said peptide consisting of Ac-AQFHRHKQLIRFLKRA (SEQ ID NO:2).

[0049] In a most preferred embodiment, the present disclosure relates to a peptide for use in the treatment of obesity, the peptide consisting of AQFHRHKQLIRFLKRA (SEQ ID NO:1).

[0050] The peptides according to the invention are agonists for IL-4R type I and preferably antagonists for IL-4R type II. Agonists can be either superagonists, full agonists or partial agonists.

[0051] The peptides of the present invention may form part of a multimer, such as a dimer, trimer, or tetramer. Multimers incorporating the peptides of the present disclosure, such as peptide dendrimers, may also be used. Most preferably, the peptides form monomers or dimers, most preferably dimers. The peptides according to the present disclosure may be covalently linked to form multimers (e.g., dimers) by covalently linking the peptides via various linkers (spacers), such as those commonly used in the art for fusion peptides or proteins.

[0052] A second aspect of the present disclosure relates to a peptide, or a pharma- ceutically acceptable salt thereof, for use in the treatment of obesity, said peptide comprising two or more covalently linked peptide fragments derived from α-helix C of IL-4.

[0053] Thus, in a second aspect, the present invention relates to a peptide, or a pharma- ceutically acceptable salt thereof, for use in the treatment of obesity, said peptide comprising two or more covalently linked fragments, each fragment comprising an amino acid residue at amino acid position AA 68 -AA 97 , preferably AA 68 -AA 89 , more preferably AA 71 -AA 94 (i.e., α-helix C of IL-4), even more preferably AA 71 -AA 89 , most preferably AA 71 -AA 86 In a preferred embodiment of the second aspect, each fragment comprises 13 to 19 consecutive amino acid residues derived from interleukin-4 (SEQ ID NO: 4) or a variant thereof having 1, 2, 3 or 4 amino acid substitutions. 68 -AA 97 , preferably AA 68 -AA 89 , more preferably AA 71 -AA 94 , and even more preferably AA 71 -AA 89 , most preferably AA 71 -AA 86 In a more preferred embodiment of the second aspect, each fragment consists of 14 to 18 contiguous amino acid residues derived from interleukin-4 (SEQ ID NO: 4), or a variant thereof having 1, 2, 3 or 4 amino acid substitutions. 68 -AA 97 , preferably AA 68 -AA 89 , more preferably AA 71 -AA 94 , and even more preferably AA 71 -AA 89 , most preferably AA 71 -AA 86In an even more preferred embodiment of the second aspect, each fragment consists of 15 to 17 contiguous amino acid residues derived from interleukin-4 (SEQ ID NO: 4), or a variant thereof having 1, 2, 3 or 4 amino acid substitutions. 68 -AA 89 , more preferably AA 71 -AA 94 , and even more preferably AA 71 -AA 89 , most preferably AA 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions. In the above embodiment of the second aspect, it is highly preferred that each fragment comprises the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having one or two substitutions. Most preferably, the covalently linked fragments are identical. Most preferably, the peptide is a dimer (i.e. two covalently linked fragments).

[0054] In a preferred embodiment of the second aspect, the present disclosure relates to a peptide for use in the treatment of obesity, said peptide comprising two or more covalently linked fragments, each fragment consisting of 13 to 19 amino acid residues, and further wherein said peptide comprises the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1) or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having one, two or three amino acid substitutions and / or deletions.

[0055] In a more preferred embodiment of the second aspect, the present disclosure relates to a peptide for use in the treatment of obesity, said peptide comprising two or more covalently linked fragments, each fragment consisting of 14 to 18 amino acid residues, and further wherein each fragment comprises the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1) or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having one, two or three amino acid substitutions and / or deletions.

[0056] In an even more preferred embodiment of the second aspect, the present disclosure relates to a peptide for use in the treatment of obesity, said peptide comprising two or more covalently linked fragments, each fragment consisting of 15 to 17 amino acid residues, and further wherein each fragment comprises the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1) or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having one, two or three amino acid substitutions and / or deletions.

[0057] In an even more preferred embodiment of the second aspect, the present disclosure relates to a peptide for use in the treatment of obesity, said peptide comprising two or more covalently linked fragments, each fragment consisting of 16 amino acid residues, and further, said peptide comprising SEQ ID NO:1 (AQFHRHKQLIRFLKRA) or a variant of SEQ ID NO:1 (AQFHRHKQLIRFLKRA) having 1, 2 or 3 amino acid substitutions.

[0058] In any of the above-mentioned embodiments of the second aspect, the variant preferably has two amino acid substitutions and / or deletions, even more preferably one amino acid substitution or deletion. In any of the above-mentioned embodiments of the second aspect, the peptide most preferably comprises two covalently linked fragments (i.e. a dimer), each fragment consisting of the same sequence.

[0059] In a most preferred embodiment of the second aspect, the present invention relates to a peptide for use in the treatment of obesity, said peptide having formula (I): [ka] or a variant thereof having one or two substitutions in each peptide fragment.

[0060] Each fragment of SEQ ID NO:1 in the dimeric peptide of formula (I) (ie, AQFHRHKQLIRFLKRA) is covalently linked at their C-termini via a linker.

[0061] In another most preferred embodiment of the second aspect, the present invention relates to a peptide for use in the treatment of obesity, said peptide being a dimer of SEQ ID NO:1 linked via a linker (lysine-beta alanine) as shown in the following structure (Compound A): [ka]

[0062] In another embodiment of the second aspect the invention relates to a peptide for use in the treatment of obesity, said peptide forming part of a tetrameric dendrimer comprising four copies of SEQ ID NO: 1 and / or SEQ ID NO: 2, or a variant thereof comprising one, two or three amino acid substitutions. In another preferred embodiment the peptide is a dimeric dendrimer comprising two copies of SEQ ID NO: 1, or a variant thereof comprising one, two or three amino acid substitutions of SEQ ID NO: 2, or two copies of SEQ ID NO: 2, or a variant thereof comprising one, two or three amino acid substitutions. In another embodiment the peptide is a tetrameric dendrimer comprising four copies of SEQ ID NO: 1, or a variant thereof comprising one, two or three amino acid substitutions, or four copies of SEQ ID NO: 2, or a variant thereof comprising one, two or three amino acid substitutions.

[0063] In a third aspect, the present disclosure relates to a pharmaceutical composition for use in the treatment of obesity, said pharmaceutical composition comprising one or more peptides according to the aspects and embodiments disclosed herein. The pharmaceutical composition may comprise an acceptable pharmaceutical carrier and, optionally, one or more excipients. The pharmaceutical composition (i.e., formulation) includes, but is not limited to, a tablet, pill, capsule, emulsion, suspension, sustained release formulation, solution, or lyophilized powder intended to be dissolved prior to administration. In some embodiments, the formulation may be a depot formulation providing sustained release. It is understood that different routes of administration may be used depending on the choice of formulation and chemical and / or metabolic stability of the polypeptide. Such routes of administration may include, but are not limited to, oral administration, parenteral administration (intravenous (IV), subcutaneous (SC), intradermal (ID) and intramuscular (IM)), or inhalation. In a preferred embodiment of the present invention, the route of administration is parenteral administration. In an even more preferred embodiment, the route of administration is subcutaneous.

[0064] In a fourth aspect, the present disclosure relates to a method for the treatment of obesity, comprising administering to an individual in need thereof an effective amount of a peptide according to any of the aspects and embodiments disclosed herein. EXAMPLES

[0065] Working Example Example 1: Effect of IL-4 derived peptides on body weight Materials and Methods (animal) Thirty female mice (BKS.Cg-Dock7m + / + Leptin db / db BLKS, 5 weeks old) were transferred to the Gubra Research Animal Unit in Denmark. Throughout the habituation and study periods, animals were housed in a light-, temperature-, and humidity-controlled room with free access to food and water. All animal experiments were performed in accordance with the Gubra bioethical guidelines, which fully adhere to internationally accepted principles for the care and use of laboratory animals. Animal weights were recorded at the start of the study (baseline), and on days 13, 27, 34, 43, 60, and 77 (study termination).

[0066] (compound) Compound A was prepared using standard solid phase peptide synthesis (SPPS) procedures. Compound A was provided by Phlogo ApS, Copenhagen, Denmark (diluted to a dose formulation of 10 mg / ml in miliQ water). The vehicle was miliQ:PBS 1:5.

[0067] In vivo procedures Mice were randomized into vehicle group (n=15) and 10 mg / kg Compound A (n=15). Compound and vehicle were administered subcutaneously once daily. On day 27, the dose was reduced to 5 mg / kg Compound A (n=15), and on day 43, the dose was reduced to 2.5 mg / kg Compound A (n=15).

[0068] (result) Compared to vehicle, Compound A tested significantly reduced weight in the treatment group receiving Compound A. Weight results (mean values) are shown in Table 1 below.

[0069] [Table 1]

[0070] (Consideration) Interestingly, weight gain is significantly smaller in the treated group compared to the placebo group.The average weight of animals in the placebo group increased by 5.76g (12.8%) during the test period.In contrast, the treated group only increased their weight by 3.51g (7.8%).Therefore, compound A (SEQ ID NO: 1) reduces weight gain.

[0071] (Array Overview) SEQ ID NO:1 AQFHRHKQLIRFLKRA

[0072] SEQ ID NO:2 Ac-AQFHRHKQLIRFLKRA

[0073] SEQ ID NO:3 (signal peptide, IL-4 peptide containing 153 amino acids) MGLTSQLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS

[0074] SEQ ID NO:4 (IL-4 peptide without signal peptide, 129 amino acids) HKCDIT LQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS

[0075] (References) Roglic, G. World Health Organization. (2016). Global report on diabetes. ISBN 9789241565257. Abdelaal M. et al. Morbidity and mortality associated with obesity. Ann Transl Med. 2017 Apr;5(7):161, p.1-12. Klementiev B. et al. Antiinflammatory properties of a peptide derived from interleukin-4. Cytokine, Vol. 64, No. 1, 10.2013, p. 112-21.

[0076] (item) 1. AA 68 -AA 97 , preferably the AA 68 -AA89 or a variant thereof having 1, 2, 3 or 4 amino acid substitutions, for use in the treatment of obesity, or a peptide or a pharma- ceutically acceptable salt thereof.

[0077] 2. The peptide is preferably an AA of interleukin-4. 68 -AA 89 Amino acid region AA of (SEQ ID NO: 4) 68 -AA 97 2. The peptide for use according to any one of the preceding claims, comprising a fragment consisting of 14 to 18 consecutive amino acid residues derived from, or a variant thereof having 1, 2, 3 or 4 amino acid substitutions.

[0078] 3. The peptide is preferably an AA of interleukin-4. 68 -AA 89 Amino acid region AA of (SEQ ID NO: 4) 68 -AA 97 2. The peptide for use according to any of the preceding claims, comprising a fragment consisting of 15 to 17 consecutive amino acid residues derived from, or a variant thereof having 1, 2, 3, or 4 amino acid substitutions.

[0079] 4. The peptide is preferably an AA of interleukin-4. 68 -AA 89 Amino acid region AA of (SEQ ID NO: 4) 68 -AA 97 5. A peptide for use according to any of the preceding claims, comprising a fragment of 16 consecutive amino acid residues derived from, or a variant thereof having 1, 2, 3 or 4 amino acid substitutions.

[0080] 5. The amino acid residue is in the amino acid region AA 71 -AA 94 (i.e., α-helix C of IL-4), preferably AA 71 -AA 89 , most preferably AA 71 -AA 862. A peptide for use according to any of the preceding claims, derived from:

[0081] 6. A peptide for use according to any of the preceding clauses, wherein the peptide comprises a fragment consisting of the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO:1), or a variant thereof having one or two substitutions.

[0082] 7. A peptide for use according to any of the preceding clauses, wherein the peptide comprises two or more identical fragments covalently linked via a linker.

[0083] 8. A peptide or a pharma- ceutically acceptable salt thereof for use in the treatment of obesity, said peptide being a dimer comprising two fragments, each fragment comprising the amino acid region AA of interleukin-4 (SEQ ID NO: 4). 71 -AA 94 or a variant thereof having 1, 2, 3, or 4 amino acid substitutions, wherein each fragment is covalently linked via a linker, or a pharma- ceutically acceptable salt thereof, wherein the peptide or the pharma- ceutically acceptable salt thereof consists of 13 to 19 consecutive amino acid residues derived from (i.e., α-helix C of IL-4), or a variant thereof having 1, 2, 3, or 4 amino acid substitutions, wherein each fragment is covalently linked via a linker, or a pharma- ceutically acceptable salt thereof.

[0084] 9. The amino acid residue is in the amino acid region AA 71 -AA 89 and most preferably AA 71 -AA 86 9. The peptide for use according to item 8, which is

[0085] 10. The peptide for use according to any of items 8 to 9, wherein each fragment consists of 14 to 18 consecutive amino acid residues, preferably 15 to 17 consecutive amino acid residues, most preferably 16 amino acid residues.

[0086] 11. A peptide for use according to any one of items 8 to 10, comprising two identical fragments.

[0087] 12. A peptide for use according to any of the preceding claims, having the structure of formula (I): [ka] or a variant thereof having one or two substitutions in each peptide fragment.

[0088] 13. The peptide according to any one of the preceding paragraphs, wherein the peptide is an agonist for type I IL-4R and an antagonist for type II IL-4R.

Claims

1. 1. A peptide or a pharma- ceutically acceptable salt thereof for use in the treatment of obesity, said peptide being a dimer comprising two fragments, each fragment comprising the amino acid region AA of interleukin-4 (SEQ ID NO: 4). ７１ -AA ９４ or a variant thereof having 1, 2, 3, or 4 amino acid substitutions, each of which is covalently linked via a linker, or a pharma- ceutically acceptable salt thereof.

2. A peptide for use according to claim 1, wherein each fragment consists of 14 to 18 consecutive amino acid residues, preferably 15 to 17 consecutive amino acid residues, most preferably 16 consecutive amino acid residues.

3. The amino acid residues are in the amino acid region AA ７１ -AA ８９ , most preferably AA ７１ -AA ８６ 2. A peptide for use according to any one of the preceding claims, derived from:

4. 2. The peptide according to any of the preceding claims, wherein the peptide is an agonist for type I IL-4R and an antagonist for type II IL-4R.

5. 2. A peptide according to any preceding claim, wherein at least a portion of the peptide forms an α-helix.

6. The peptide is a dimer comprising two fragments, each fragment comprising the amino acid region AA of interleukin-4 (SEQ ID NO: 4). ７１ -AA ９４ 2. The peptide for use according to claim 1, wherein the peptide is a fragment of 13 to 19 consecutive amino acid residues derived from IL-4R (i.e., the α-C helix of IL-4), or a variant thereof having 1, 2, 3 or 4 amino acid substitutions, each fragment being covalently linked via a linker, the peptide being an agonist for type I IL-4R and an antagonist for type II IL-4R, and further wherein at least a portion of the peptide forms an α-helix.

7. The peptide has the formula (I): 【Chemistry 1】 2. A peptide for use according to any of the preceding claims, having the structure:

8. The peptide has the formula (II): 【Chemistry 2】 2. A peptide for use according to any of the preceding claims, having the structure:

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