IL-4 derived peptide fragments for use in the treatment of diabetic nephropathy - Patent Application 20070233334

Abstract

The present disclosure relates to peptide fragments derived from interleukin-4 (IL-4) for the treatment of diabetic nephropathy.

Description

[Technical field]

[0001] The present disclosure relates to peptide fragments derived from IL-4 for use in the treatment of diabetic nephropathy. In particular, the present disclosure relates to peptides comprising one or more fragments of IL-4 having the amino acid sequence of SEQ ID NO: 1 or variants thereof for use in the treatment of diabetic nephropathy.

[0002] (background) Diabetic nephropathy has not historically been considered a low-grade inflammation (i.e., an innate immune inflammatory disease) because metabolic and hemodynamic factors have been thought to be the primary causes of renal damage. However, recent studies support a role for the innate immune system in both the development and progression of diabetic nephropathy. Progressive non-infectious inflammation involves glomerular and tubular cells resulting in progressive loss of renal function. To slow the loss of renal function, blood glucose and blood pressure must be kept as normal as possible. However, specific treatments for innate immune inflammation have not been developed, and the decline in glomerular filtration and tubular function does not stop even with adequate control of blood glucose and blood pressure. In end-stage renal failure, the treatment is chronic dialysis and, in some cases, kidney transplantation. Thus, interventions are needed to prevent or stop the expected inflammation-induced decline in all functions of the nephron.

[0003] Patients are diagnosed with diabetic nephropathy by measuring the biomarker albumin in urine. Urinary albumin is often measured in a spot urine sample and corrected for creatinine (ACR mg / g) in the same urine sample. ACR is divided into three groups: normoalbuminuria (ACR<30mg / g), microalbuminuria (ACR>30mg / g and <300mg / g) and macroalbuminuria (ACR>300mg / g). Clinical evidence indicates that the higher the ACR, the more advanced the kidney damage.

[0004] Urinary albumin and kidney injury molecule-1 (KIM-1) are both powerful biomarkers of renal innate immune inflammation, and human kidney biopsies have confirmed the presence of inflammatory cells in both glomeruli, tubules, and interstitium at all stages of diabetic nephropathy.

[0005] 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)

[0006] 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)

[0007] 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.

[0008] 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]

[0009] (Summary) The present invention relates to the finding that ReninAAV UNx db / db mice (i.e., treated group) administered with a compound comprising a peptide fragment of SEQ ID NO: 1 can significantly reduce urinary albumin corrected for creatinine (ACR) and can reduce kidney damage molecules corrected for creatinine (KIM-1 / CR) (see Figures 2 and 3). Both urinary albumin and KIM-1 are biomarkers of renal innate immune inflammation found in various diseases involving the kidney, including diabetic nephropathy. [Means for solving the problem]

[0010] Therefore, small peptide fragments derived from IL-4 and acting as IL-4 mimetics may have promising potential as a new generation of drugs for the treatment of diabetic nephropathy, since the peptide 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).The present invention is described in the claims.

[0011] (Definitions and Abbreviations) ACR: Albumin corrected for creatinine IL-4: Interleukin-4 KIM-1: Kidney injury molecule-1 KIM-1 / CR: Creatinine-corrected KIM-1 ReninAAV UNx mice: Renin AAV uninephrectomized db / db mice

[0012] "Diabetic nephropathy" is a type of progressive kidney disease that can occur in individuals with diabetes. It affects individuals with type 1 and type 2 diabetes. The risk of diabetic nephropathy increases with the duration of diabetes, as well as other risk factors such as high blood pressure and a family history of kidney disease.

[0013] The term "individual" refers to a vertebrate, a particular member of a mammalian species, preferably a primate, and most preferably a human. As used herein, "subject" and "individual" may be used interchangeably.

[0014] "Individual in need thereof" refers to an individual who would benefit from the present disclosure. In one embodiment, the individual in need thereof is an affected individual, particularly an individual with diabetic nephropathy and / or diabetes mellitus, type 1 or type 2.

[0015] The amino acids disclosed herein are naturally occurring amino acids (L-amino acids) unless otherwise specified.

[0016] 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.

[0017] 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 a peptide fragment derived from interleukin-4 (SEQ ID NO: 4), it should be understood as a small stretch (i.e., a contiguous sequence) of amino acids selected from the respective region of interleukin-4 (SEQ ID NO: 4). For example, the amino acid region AA 68 -AA 89 A peptide fragment containing 16 consecutive amino acid residues derived from can be obtained from the consecutive sequences highlighted in bold below.

[0018] HKCDITLQEI IKTLNSLTEQ KTLCTELTVT DIFAASKNTT EKETFCRAAT VLRQFYSHHE KDTRCLGATA QQFHRHKQLI RFLKRLDRNL WGLAGLNSCP VKEANQSTLE NFLERLKTIM REKYSKCSS (SEQ ID NO:4).

[0019] 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 97 When 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]

[0020] The peptide fragments can also be lipidated to alter the pharmacokinetic properties of the peptide.

[0021] 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.

[0022] As used herein, the terms "treat," "treatment," and "therapy" refer equally to curative, prophylactic, preventative, or ameliorative therapy. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in extent of disease, stabilized (i.e., not worsening) condition, slowing or delaying the progression or worsening of a condition / symptom, improvement or alleviation of a condition or symptom, and remission (whether partial or complete), whether detectable or not (whether partial or complete).

[0023] 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, preferably the same sequence. 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.

[0024] 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 understood that the total of the substitutions and / or deletions is up to three. As an example, the variant may have, for example, two substitutions and one deletion (total=3). Similarly, the variant may have three substitutions and zero deletions (total=3), or the 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 diabetic nephropathy, and the peptide has, for example, 13 amino acid residues, and further the 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)

[0025] 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.

[0026] The peptide sequences of the present disclosure can be prepared by conventional synthetic methods commonly used in the art, such as solid phase peptide synthesis (SPPS) using coupling reagents. [Brief description of the drawings]

[0027] [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). [Diagram 2] FIG. 2 shows the effect of Compound A in reducing ACR (mg(g)) in the treatment relative to the vehicle group. [Diagram 3] FIG. 3 shows the effect of Compound A on KIM-1 / CR (pg / g) in treatment relative to the vehicle group.

[0028] (Detailed Description) The present invention relates to the surprising discovery that small peptide fragments derived from IL-4, more specifically α-helix C of IL-4, are IL-4R type I agonists and can reduce ACR and KIM-I in ReninAAV UNx db / db mice.

[0029] 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., 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., second fragment). The peptide fragment 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).

[0030] HKCDITLQEI IKTLNSLTEQ KTLCTELTVT DIFAASKNTT EKETFCRAAT VLRQFYSHHE KDTRCLGATA QQFHRHKQLI RFLKRLDRNL WGLAGLNSCP VKEANQSTLE NFLERLKTIM REKYSKCSS (SEQ ID NO:4)

[0031] 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).

[0032] QQFHRHKQLIRFLKRL IL-4AA 71 -AA 86 (SEQ ID NO:4) AQFHRHKQLIRFLKRA (SEQ ID NO: 1).

[0033] 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 Peptide fragments derived from or near this region are suitable for use in the treatment of diabetic nephropathy.

[0034] Thus, in a first aspect the present invention relates to a peptide for use in the treatment of diabetic nephropathy, the peptide comprising the amino acid region AA 68 -AA 97, preferably the AA 68 -AA 89 , most preferably the AA of interleukin-4 (SEQ ID NO: 4) 71 -AA 86 or a variant thereof having 1, 2, 3, or 4 amino acid substitutions. 68 -AA 97 (SEQ ID NO: 4), preferably the AA of interleukin-4 68 -AA 89 (SEQ ID NO: 4), most preferably the AA 71 -AA 86 In a more preferred embodiment of the second aspect, the peptide comprises a fragment of 14 to 18 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 (SEQ ID NO: 4), preferably the AA of interleukin-4 68 -AA 89 (SEQ ID NO: 4), most preferably the AA 71 -AA 86 In a further preferred embodiment, the peptide comprises a fragment of 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 (SEQ ID NO: 4), preferably the AA of interleukin-4 68 -AA 89 (SEQ ID NO: 4), most preferably the AA 71 -AA 86(SEQ ID NO: 4), 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 a fragment of the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant of AQFHRHKQLIRFLKRA (SEQ ID NO: 1) having 1 or 2 substitutions. In a preferred embodiment, the present disclosure relates to a peptide for use in the treatment of diabetic nephropathy, comprising the following structure: [ka] or a variant thereof having one or two amino acid substitutions.

[0035] In a most preferred embodiment the disclosure relates to a peptide for use in the treatment of diabetic nephropathy, the peptide consisting of two fragments, each linked via their C-terminus by a linker, having the following structure: [ka] or a variant thereof having one or two amino acid substitutions in each fragment.

[0036] In any of the above embodiments, the variant preferably has two amino acid substitutions and / or deletions, even more preferably one amino acid substitution or deletion.

[0037] 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.

[0038] 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").

[0039] In a highly preferred embodiment, the present disclosure relates to a peptide for use in the treatment of diabetic nephropathy, said peptide comprising 16 amino acid residues, and further, said peptide comprises SEQ ID NO: 1 (AQFHRHKQLIRFLKRA). In view of 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.

[0040] In another highly preferred embodiment, the present disclosure relates to a peptide for use in the treatment of diabetic nephropathy, 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.

[0041] In a further highly preferred embodiment, the present disclosure relates to a peptide for use in the treatment of diabetic nephropathy, said peptide consisting of Ac-AQFHRHKQLIRFLKRA (SEQ ID NO:2).

[0042] In a highly preferred embodiment the present disclosure relates to a peptide for use in the treatment of diabetic nephropathy, said peptide consisting of AQFHRHKQLIRFLKRA (SEQ ID NO:1).

[0043] 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).

[0044] The peptides according to the invention are agonists for type I IL-4R and preferably antagonists for type II IL-4R. The agonists can be either superagonists, full agonists or partial agonists. Most preferably, the peptides are linked via the C-terminal or N-terminal unit, most preferably the C-terminal unit.

[0045] In a second aspect of the present invention, the disclosure relates to a peptide, or a pharma- ceutically acceptable salt thereof, comprising two or more covalently linked peptide fragments derived from α-helix C of IL-4. Most preferably, the peptides are linked via the C-terminus as exemplified herein.

[0046] Thus, in a second aspect, the present invention provides a peptide or pharma- ceutically acceptable salts thereof comprising two or more covalently linked fragments, each of which corresponds to the amino acid region AA of interleukin-4 (SEQ ID NO: 4). 68 -AA 97 , preferably the AA 68 -AA 89 , most preferably the AA of interleukin-4 (SEQ ID NO: 4) 71 -AA 86 or a variant thereof having 1, 2, 3, or 4 amino acid substitutions. 68 -AA 97 (SEQ ID NO: 4), preferably the AA of interleukin-4 68 -AA 89 (SEQ ID NO: 4), most preferably the AA 71 -AA 86In a more preferred embodiment of the second aspect, each fragment comprises 14 to 18 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 (SEQ ID NO: 4), preferably the AA of interleukin-4 68 -AA 89 (SEQ ID NO: 4), most preferably the AA 71 -AA 86 In an even more preferred embodiment of the second aspect, each fragment comprises 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 89 (SEQ ID NO: 4), preferably the AA of interleukin-4 68 -AA 89 (SEQ ID NO: 4), most preferably the AA 71 -AA 86 (SEQ ID NO: 4), or a variant thereof having 1, 2, 3 or 4 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.

[0047] In a preferred embodiment of the second aspect, the present disclosure relates to a peptide, or a pharma- ceutically acceptable salt thereof, for use in the treatment of diabetic nephropathy, wherein said peptide comprises two or more covalently linked fragments, each fragment consisting of 13 to 19 amino acid residues, and further wherein said fragments comprise 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.

[0048] In a more preferred embodiment of the second aspect, the present disclosure relates to a peptide, or a pharma- ceutically acceptable salt thereof, for use in treating diabetic nephropathy, wherein the peptide comprises 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 1, 2, or 3 amino acid substitutions and / or deletions, or a pharma- ceutically acceptable salt thereof.

[0049] In an even more preferred embodiment of the second aspect, the present disclosure relates to a peptide, or a pharma- ceutically acceptable salt thereof, for use in the treatment of diabetic nephropathy, said peptide comprising two or more covalently linked fragments, each fragment consisting of 15-17 amino acid residues, and further each fragment comprising 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.

[0050] In an even more preferred embodiment of the second aspect, the present disclosure relates to a peptide or a pharma- ceutically acceptable salt thereof for use in the treatment of diabetic nephropathy, 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 one, two or three amino acid substitutions.

[0051] In any of the above 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 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.

[0052] In a most preferred embodiment of the second aspect, the present disclosure relates to a peptide, or a pharma- ceutically acceptable salt thereof, for use in the treatment of diabetic nephropathy, said peptide having the formula: [ka] or a variant thereof having one or two amino acid substitutions in each peptide fragment.

[0053] Each fragment of SEQ ID NO:1 in the dimeric peptide of formula (I) (ie, AQFHRHKQLIRFLKRA) is covalently linked at its C-terminus via a linker.

[0054] In another most preferred embodiment of the second aspect, the present invention relates to a peptide or a pharma- ceutically acceptable salt thereof for use in the treatment of diabetic nephropathy, 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]

[0055] In another embodiment of the second aspect, the disclosure relates to a peptide for use in the treatment of diabetic nephropathy, 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.

[0056] In a third aspect, the present disclosure relates to a pharmaceutical composition for use in treating diabetic nephropathy, said pharmaceutical composition comprising one or more peptides or pharma- ceutically acceptable salts thereof according to the aspects and embodiments disclosed herein. The pharmaceutical composition may comprise an acceptable pharmaceutical carrier, and optionally one or more excipients.

[0057] Pharmaceutical compositions (i.e., formulations) include, but are not limited to, tablets, pills, capsules, emulsions, suspensions, sustained release formulations, solutions, or lyophilized powders 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 invention, the route of administration is parenteral administration. In an even more preferred embodiment, the route of administration is subcutaneous.

[0058] In a fourth aspect, the present disclosure relates to a method for treating diabetic nephropathy, comprising administering to an individual in need thereof an effective amount of a peptide or a pharma- ceutically acceptable salt thereof according to any of the aspects and embodiments disclosed herein. EXAMPLES

[0059] (Example: Effect of IL-4-derived peptides on markers of diabetic nephropathy) Materials and Methods (animal) Twenty-eight female mice (BKS.Cg-Dock7m + / + Lepradb / db BLKS, 5 weeks old) were transferred to the Gubra Research animal unit. Throughout the habituation and study period, 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 Gubra bioethical guidelines in full compliance with internationally accepted principles for the care and use of laboratory animals. Animals were group-housed until unineflectomy and then single-housed.

[0060] On day 77, the animals were terminated, blood was collected, body weights were recorded, and spot urine was collected for later analysis.

[0061] (compound) Compound A was provided by Phlogo ApS, Copenhagen, Denmark (10 mg / ml miliQ water diluted to dose formulations). Compound A was provided as a dimeric peptide containing two copies of the sequence AQFHRHKQLIRFLKRA (SEQ ID NO:1). Synthesis of compound A was performed with a Rink linker bound to resin. To reduce steric repulsion between the bulky Rink linker and the dimeric peptide, beta-alanine was selected as the spacer between the Rink linker and lysine on which the dimeric peptide was constructed. The vehicle was miliQ:PBS 1:5.

[0062] In vivo procedures After one week of acclimation, mice were IV-injected with Renin-Adenosine-Associated Virus (AAV). One week later, all mice were uni-neflectomized. Two weeks after Renin-AAV IV injection, mice were randomized according to blood glucose levels into two groups: vehicle group (n=14) and 10 mg / kg Compound A (n=14). Compound and vehicle groups were administered subcutaneously once daily.

[0063] (Biochemical analysis) Urine samples were stored at -80°C until analysis. Urine for albumin and creatinine measurements was centrifuged at 2000g for 2 min prior to analysis. Urinary albumin was measured using a commercial ELISA kit (Bethyl Laboratories, Inc) according to the manufacturer's instructions, and urinary creatinine was measured using a commercial kit (Roche Diagnostics) on a Cobas c 501 automated analyzer according to the manufacturer's instructions. Urinary KIM-1 was measured using a commercial ELISA kit (R&D Systems) according to the manufacturer's instructions.

[0064] (result) Compared to vehicle, the tested compound A significantly reduced both ACR mg / g (Figure 1) and KIM-1 / CR pg / g (Figure 2).

[0065] (Consideration) The ReninAAV UNx db / db mouse model describes the development of diabetic nephropathy and is therefore described as one of the best pharmacological models to elucidate the effect of compounds on disease progression (Sembach 2020). Both urinary albumin and KIM-1 are powerful biomarkers of renal innate immune inflammation.

[0066] The results demonstrated herein show that Compound A mimics IL-4 protein stimulation of IL-4Ra1 and induces anti-inflammatory effects. Compound A significantly reduced both urinary albumin, a biomarker primarily from glomerular endothelial and podocyte injury, and KIM-1, a biomarker from the apical membrane of proximal tubule cell injury. These biomarkers are undetectable in urine from normal kidneys, but are detectable in response to ischemic or toxic injury, including diabetes.

[0067] (Array Overview) SEQ ID NO:1 AQFHRHKQLIRFLKRA

[0068] SEQ ID NO:2 Ac-AQFHRHKQLIRFLKRA

[0069] 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

[0070] 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

[0071] (item) 1. A method for treating diabetic nephropathy comprising administering a therapeutically effective amount of interleukin-4 (SEQ ID NO: 4), preferably AA of interleukin-4 (SEQ ID NO: 4). 68 -AA 89 , most preferably the AA 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions 68 -AA 97 The method comprises administering a pharmaceutical comprising a peptide or a pharma- ceutical agent comprising a fragment consisting of 13 to 19 consecutive amino acid residues derived from the above peptide or a pharma- ceutical agent comprising a pharma-ceutical agent comprising a ... peptide or a pharma-ceutical agent comprising a pharma-ceutical agent comprising a peptide or a pharma-ceutical agent comprising a pharma-ceutical agent comprising a peptide or a pharma-ceutical agent comprising a

[0072] 2. The peptide is selected from the group consisting of interleukin-4 (SEQ ID NO: 4), preferably the AA 68 -AA 89, most preferably the AA 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions 68 -AA 97 A method for producing a fragment of 14 to 18 consecutive amino acid residues derived from the above-mentioned compound, the use of which is described in item 1.

[0073] 3. The peptide is selected from the group consisting of interleukin-4 (SEQ ID NO: 4), preferably the AA 68 -AA 89 , most preferably the AA 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions 68 -AA 97 The use according to any one of the preceding items, comprising a fragment consisting of 15 to 17 consecutive amino acid residues derived from

[0074] 4. The peptide is selected from the group consisting of interleukin-4 (SEQ ID NO: 4), preferably the AA 68 -AA 89 , most preferably the AA 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions 68 -AA 97 The use according to any of the preceding claims, comprising a fragment consisting of 16 consecutive amino acid residues derived from

[0075] 5. The peptide comprises the amino acid region AA of interleukin-4 (SEQ ID NO: 4). 71 -AA 86 2. The 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.

[0076] 6. The method of any of the preceding paragraphs, wherein the peptide comprises a fragment of 16 amino acid residues having the structure AQFHRHKQLIRFLKRA (SEQ ID NO:1), or a variant thereof having one or two substitutions.

[0077] 7. The method of any of the preceding claims, wherein the peptide comprises two or more peptide fragments covalently linked via a linker.

[0078] 8. The method of any of the preceding paragraphs, wherein the peptide comprises two or more peptide fragments covalently linked via a linker, each peptide fragment comprising or consisting of the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO:1), or a variant thereof having one or two substitutions.

[0079] 9. The method according to item 7 or 8, wherein the two or more peptide fragments are covalently linked via their C-terminal units or N-terminal units, preferably via their C-terminal units.

[0080] 10. The peptide has the structure of formula (I): [ka] or a variant thereof having one or two substitutions in each peptide fragment.

[0081] 11. The peptide has the structure of formula (II): [ka] or a variant thereof having one or two substitutions in each peptide fragment.

[0082] 12. The method according to any of the preceding claims, wherein the peptide is N-terminally acylated.

[0083] 13. The method according to any of the preceding paragraphs, wherein the peptide is an agonist for type I IL-4R and an antagonist for type II IL-4R.

[0084] 14. A peptide or a pharma- ceutically acceptable salt thereof for use in the treatment of diabetic nephropathy, said peptide comprising interleukin-4 (SEQ ID NO:4). , , preferably the AA 68 -AA 89 , most preferably the AA 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions 68 -AA 97 A peptide or a pharma- ceutically acceptable salt thereof, comprising a fragment consisting of 13 to 19 consecutive amino acid residues derived from the above.

[0085] 15. A peptide for use according to item 14, wherein the peptide is selected from the group consisting of interleukin-4 (SEQ ID NO: 4), preferably the AA 68 -AA 89 , most preferably the AA of interleukin-4 (SEQ ID NO: 4) 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions 68 -AA 97 A peptide comprising a fragment consisting of 14 to 18 consecutive amino acid residues derived from.

[0086] 16. The peptide is selected from the group consisting of interleukin-4 (SEQ ID NO: 4), preferably the AA 68 -AA 89 , most preferably the AA of interleukin-4 (SEQ ID NO: 4) 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions 68 -AA 9716. The peptide for use according to any one of items 14 to 15, comprising a fragment consisting of 15 to 17 consecutive amino acid residues derived from

[0087] 17. The peptide is selected from the group consisting of interleukin-4 (SEQ ID NO: 4), preferably the AA 68 -AA 89 , most preferably the AA of interleukin-4 (SEQ ID NO: 4) 71 -AA 86 or a variant thereof having one, two, three or four amino acid substitutions 68 -AA 97 17. The peptide for use according to any one of items 14 to 16, comprising a fragment consisting of 16 consecutive amino acid residues derived from

[0088] 18. The peptide is selected from the group consisting of the amino acid region AA of interleukin-4. 71 -AA 86 18. The peptide for use according to any one of items 14 to 17, comprising a fragment consisting of 16 consecutive amino acid residues derived from (SEQ ID NO: 4), or a variant thereof having 1, 2, 3, or 4 amino acid substitutions.

[0089] 19. The peptide for use according to any one of items 14 to 18, wherein the peptide comprises a fragment consisting of 16 amino acid residues having the structure AQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant thereof having one or two substitutions.

[0090] 20. A peptide for use according to any one of items 14 to 19, wherein the peptide comprises two or more peptide fragments covalently linked via a linker.

[0091] 21. A peptide for use according to any of items 14 to 20, wherein the peptide comprises two or more peptide fragments covalently linked via a linker, each peptide fragment comprising or consisting of the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1), or a variant thereof having one or two substitutions.

[0092] 22. A peptide for use according to any one of items 20 to 21, wherein the two or more peptide fragments are covalently linked via their C-terminal units or N-terminal units, preferably via their C-terminal units.

[0093] 23. The peptide has the structure of formula (I): [ka] or a variant thereof having one or two substitutions in each peptide fragment.

[0094] 24. The peptide has the structure of formula (II): [ka] 2. A peptide for use according to any of the preceding claims, having the formula:

[0095] 25. A peptide for use according to any of the preceding clauses, wherein the peptide is N-terminally acylated.

[0096] 26. A peptide for use according to any of the preceding items, wherein the peptide is a partial agonist for type I IL-4R and an antagonist for type II IL-4R.

[0097] 27. A pharmaceutical composition for use in the treatment of diabetic nephropathy, said pharmaceutical composition comprising a peptide according to any of the preceding paragraphs.

[0098] (References) Klementiev B. et al.: Antiinflammatory properties of a peptide derived from interleukin-4. Cytokine, Vol. 64, No. 1, 10.2013, p. 112-21. Frederikke E Sembach, Mette V Ostergaard, Niels Vrang, Bo Feldt-Rasmussen, Keld Fosgerau, Jacob Jelsing, Lisbeth N Fink: Rodent models of diabetic kidney disease: human translatability and preclinical validity. Review Drug Discov Today. 2021 Jan;26(1):200-217. James P. Tam: Synthetic peptide vaccine design: Synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. USA, Vol 85, pp. 5409-5413, August 1988.

Claims

1. 1. A peptide, or a pharma- ceutically acceptable salt thereof, for use in the treatment of diabetic nephropathy, wherein the peptide is a dimer comprising two fragments, each fragment consisting of the amino acid sequence AQFHRHKQLIRFLKRA (SEQ ID NO: 1), and each fragment is covalently linked at its C-terminus via a linker, or a pharma-ceutically acceptable salt thereof.

2. The peptide has the formula (II): 【Chemistry 1】 【change】 2. A peptide for use according to claim 1 having the structure:

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

4. 13. A pharmaceutical composition for use in the treatment of diabetic nephropathy, said pharmaceutical composition comprising a peptide according to any of the preceding claims.

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