Selective NPY5r agonists with high chemical and metabolic stability

Abstract

The present invention relates to NPY5R agonists with high metabolic and chemical stability as well as their use in the treatment of cachexia or anorexia.

Description

[0001] SELECTIVE NPY5R AGONISTS WITH HIGH CHEMICAL AND METABOLIC STABILITY

[0002] The present invention relates to NPY5R agonists with high metabolic and chemical stability as well as their use in the treatment of cachexia or anorexia.

[0003] BACKGROUND

[0004] Cachexia is a complex syndrome associated with an underlying illness causing ongoing muscle loss that is not entirely reversed with nutritional supplementation. It is estimated that Cachexia affects 6-12 million individuals in Europe, the US, and Japan alone.

[0005] Cachexia is characterized by an unintended weight loss resulting in an overall lowered quality of life and an increased mortality leading to a total of 1.5-2.0 million deaths per year. A range of diseases may cause cachexia, most commonly cancer, congestive heart failure (CHF), chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), and AIDS. Systemic inflammation from these conditions can cause detrimental changes to metabolism and body composition leading to cachexia. It is estimated that cancer cachexia causes 20-30% of deaths of cancer patients globally and that COPD cachexia is associated with approximately a 50% reduction in median survival. Thus, there is a need in the art for effective treatments of cachexia.

[0006] Human neuropeptide Y (hNPY) is a 36 amino acid peptide hormone with the amino acid sequence YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY. hNPY is an abundant neuropeptide of the hypothalamus and one of the most potent orexigenic factors. hNPY acts upon the NPY receptors designated NPY1R (Yl), NPY2R (Y2), NPY4R (Y4), and NPY5R (Y5). This receptor family belongs to a class of G protein- coupled receptors (GPCRs) and is expressed in the CNS, especially in regions of the hypothalamus. The receptors NPY1-NPY5 exhibit both anorectic (NPY2, NPY4) and orexigenic (NPY5, NPY1) effects and are activated by the endogenous peptide hormones hNPY, hPYY, and hPP. In view of the orexigenic effects associated with targeting NPY5R, future treatments for cachexia should focus on the development of NPY5R agonists with high selectivity for NPY5R.

[0007] Albeit selective NPY5R agonists, such as [cPPl-7,NPY19-23,Ala31,Aib32,Gln34]-hPP (i.e.

[0008] (GPSQPTYPGDNATPEQMARYYSALRRYINMA[Aib]RQRY, SEQ ID NO:27), have been described in the prior art (see e.g. Cabrele C., et aL J. BioL Chem, 2000, 275 (46), pp. 36043-36048) and shown to increase body weight in animal models, this polypeptide is not optimal as a drug candidate due to lack of sufficient metabolic stability. Metabolic and chemical stability of polypeptides are of uttermost importance in order to develop successful peptide drugs. High metabolic stability increases the half-life of the drug and allows for less frequent dosing, which is highly important in particular for chronic diseases requiring a lifelong treatment. Chemical stability is equally of high importance as it prolongs the shelf-life of a drug. Lack of sufficient chemical stability may necessitate that the polypeptide is formulated as a lyophilized powder for reconstitution in an aqueous solution prior to administration and / or that the polypeptide must be transported and stored under special storage requirements, such as very low temperature, resulting in increased treatment costs. Thus, the present invention sets forth to provide selective NPY5R agonists with improved chemical and metabolic stability.

[0009] SUMMARY

[0010] In a first aspect, the present invention relates to polypeptides acting as selective NPY5R agonists, said polypeptides comprising an analogue of [cPPl-7,NPY19-23,Ala31,Aib32,Gln34]-hPP. The inventors found that these polypeptides possessed good chemical stability in formulation and increased metabolic stability compared to [cPPl-7,NPY19-23,Ala31,Aib32,Gln34]-hPP.

[0011] In a second aspect, the present invention relates to the medical use of the peptides according to the first and / or second aspect(s) for the treatment of cachexia and / or anorexia, in particular cachexia.

[0012] The invention is set forth in the claims.

[0013] DEFINITIONS AND ABBREVIATIONS

[0014] According to the present invention, unless otherwise stated, the amino acids are all L-amino acids (L- stereoisomer, natural amino acids). In the present context, substitutions in an analogue / derivative may be substitutions to natural amino acids as well as unnatural amino acids, including L- and D-stereoisomers. Preferably, a substitution in a variant is a conservative substitution to a conservative amino acid. The groups of conservative amino acids may be defined as:

[0015] G, A, V, L, I, P (aliphatic or cyclic),

[0016] S, C, T, M (hydroxyl or sulphur containing)

[0017] F, Y, W (aromatic)

[0018] H, K, R (basic)

[0019] D, E, N, Q (acidic or amide)

[0020] According to the present invention, NMeG denotes the amino acid methylglycine (also referred to as N- Methylglycine, NMeGly, or sarcosine). Aib denotes the amino acid 2-amino-2-methylpropanoic acid (also referred to as 2-aminoisobutyric acid). NMeQ denotes the amino acid N-methyl-L-glutamine (also referred to as N-Methylglutamine, or NMeGIn). NMeA denotes the amino acid N-methyl-L-alanine (also referred to as N-Methylalanine, or NMeAla). Tie denotes the amino acid (5)-2-amino-3,3-dimethylbutanoic acid (also referred to as L-2-(tert-butyl)glycine). Phe(4F) denotes the amino acid (S)-2-amino-3-(4- fluorophenyl)propanoic acid (also referred to as 4-fluoro-L-phenylalanine). Phe(4CI) denotes the amino acid (S)-2-amino-3-(4-chlorophenyl)propanoic acid (also referred to as 4-chloro-L-phenylalanine). Phe(4CF3) denotes the amino acid (S)-2-amino-3-(4-(trifluoromethyl)phenyl)propanoic acid (also referred to as 4- (trifluoromethyl)-L-phenylalanine). NMeS denotes the amino acid N-methyl-L-serine (also referred to as (5)-3-hydroxy-2-(methylamino)propionic acid, or NMeSer). bAla denotes the amino acid beta-alanine (also referred to as 3-aminopropanoic acid). GABA denotes the amino acid 4-aminobutanoic acid (also referred to as y-aminobutanoic acid). Nle denotes the amino acid norleucine (also referred to as (2S)-2- aminohexanoic acid). Cha denotes the amino acid L-cyclohexylalanine (also referred to as 3-cyclohexyl-L- alanine or (S)-2-amino-3-cyclohexylpropanoic acid). The amino acid residues are shown below.

[0021] According to the present invention, it should be understood that the polypeptides may have a free amine (-NH2) at the N-terminal, be N-acylated (-NHCOR) with e.g. an acetyl (Ac) group, or be N-methylated (- NHCH3 or -N(CH3)2) at the N-terminal or be deaminated at the N-terminal. Most preferably, the polypeptides have a free amine (-NH2) at the N-terminal. According to the present invention, the polypeptides may have a free carboxylic acid (-COOH) at the C-terminal or be amidated at the C-terminal (-CONH2). Most preferably, the polypeptides are amidated at the C-terminal (-CONH2).

[0022] According to the present invention, ECso values are used as a measure of agonist potency at a given receptor. An ECso value is a measure of the concentration of a compound required to achieve half of that compound's maximal activity in a particular assay. It is well known that ECso values depend on the assay type and assay conditions. Furthermore, variation may also be present within the same assay under apparently identical assay conditions due to variation in e.g. receptor expression in the cells (i.e. receptor density). In order to compare ECso values between different assay runs, hNPY was used as internal standard for measurement of the NPY5R potency. Thus, the NPY5R (Y5) ECso is expressed as a relative ECso compared to hNPY (see e.g. Table 1). The polypeptides according to the present invention have an NPY5R potency relative to hNPY (i.e. ECso-polypeptide / ECso-hNPY) of < 30, more preferably < 20, most preferably < 10.

[0023] Preferably, the polypeptides have a high selectivity for NPY5R over NPY2R. In the present context, the selectivity for NPY5R over NPY2R is given by NPY2R-ECso / NPY5R-ECso.

[0024] According to the present invention, lipidation refers to the covalent attachment of a lipid, optionally through a linker / spacer, to a polypeptide according to the invention. The lipid may be e.g. a C18DA (octadecanedioic acid) or a C20DA (icosanedioic acid). The linker / spacer may consist of one or more covalently connected units commonly used in the art such as but not limited to [yE], [OEG] (also referred to as 2-(2- aminoethoxy)ethoxy]acetic acid or 8Ado), [eLys], [ACHC] (also referred to as trans-4- (Aminomethyl)cyclohexanecarboxylic acid or AMCA), or [Ahx] (also referred to as 6-Aminohexanoic acid or

[0025] EACA) as illustrated below.

[0026] Lipidation is typically performed to improve the pharmacokinetic profile of a polypeptide by e.g. improving metabolic stability, reducing enzymatic degradation, lowering excretion and metabolism, all in all resulting in a prolonged in vivo half-life (ti / 2). The polypeptides according to the present invention may be lipidated or non-lipidated depending on the desired half-life. Most preferably, the polypeptides are lipidated. When lipidated, the lipidation is preferably performed at the lysine residue in position X4, X7, or X10, most preferably in position X4. Preferably, the lipid is selected from the list consisting of C18DA[yE]-, C18DA[yE][OEG][OEG]-, C18DA[yE][eLys]-, C18DA[yE][eLys][eLys]-, C18DA[ACHC][yE][OEG][OEG]-, C18DA[yE][bAla]-, C18DA[yE][yE]-, C18DA[yE][Ahx]-, C18DA[yE][OEG][OEG][OEG][OEG][OEG][OEG]-, C18DA[yE][OEG][OEG][OEG]-, C18DA[yE][OEG][eLys]-, C18DA[yE][OEG][OEG][eLys]-, C20DA[yE]-, C20DA[yE][OEG][OEG]-, C20DA[yE][OEG][OEG][eLys]-, C20DA[yE][OEG][OEG][OEG][OEG]-, and C20DA[yE][yE][OEG][OEG]-. Most preferably, the lipid (and linker) is selected as C18DA[yE][OEG][OEG]- as illustrated herein.

[0027] According to the present invention, a polypeptide or a derivative thereof may be in the form of a pharmaceutically acceptable salt and / or solvate (e.g., a hydrate). Pharmaceutically acceptable salts include both acid addition salts and basic salts, and examples may be found in e.g., Remington's pharmaceutical sciences, 17thedition. DETAILED DESCRIPTION

[0028] In the first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the structure of Formula (I) X1-X2-X3-X4-P-T-X7-P-G-X10-N-A-T-P-E-X16-X17-A-R-Y-Y-S-A-L-R-X26-X27-X28-N-X30-A-Aib-R-X34-R-X36

[0029] (I) wherein,

[0030] X1is selected as G, Aib, NMeG, bAla, GABA, or not present;

[0031] X2is selected as P, G, or not present;

[0032] X3is selected as S, L, NMeS, NMeL, Tie, or P;

[0033] X4is selected as K, Q, or E;

[0034] X7is selected as Y or K;

[0035] X10is selected as D, E, or K;

[0036] X15is selected as E or Q;

[0037] X17is selected as L, M, or Nle;

[0038] X25is selected as R or H;

[0039] X27is selected as Y or Cha;

[0040] X28is selected as I or Y;

[0041] X30is selected as W, M, or Nle;

[0042] X34is selected as NMeQ, NMeA, or Q;

[0043] X35is selected as Phe(4CF3), Phe(4CI), Phe(4F), Y, F, or Cha; with the proviso that X^-X3is not selected as G-P-S or Aib-P-S.

[0044] Chemical and metabolic stability of the N- terminal fragment of the polypeptides

[0045] Poor chemical stability makes peptides suboptimal for long term storage in aqueous formulation and may require low temperature storage to ensure sufficient shelf-life. Although this problem may potentially be overcome by providing the polypeptides as a lyophilized powder for reconstitution in water for injection (WFI) prior to e.g. subcutaneous administration, this is less convenient. The requirement for low temperature storage and / or formulation as a lyophilized powder for reconstitution requires storage and administration by health professionals rather than storage and administration by the patient.

[0046] The selective NPY5R agonist [cPPl-7,NPY19-23,Ala31,Aib32,Gln34]-hPP (i.e.

[0047] (GPSQPTYPGDNATPEQMARYYSALRRYINMA[Aib]RQRY) described in the prior art (see e.g. Cabrele C., et al. J. Biol. Chem, 2000, 275 (46), pp. 36043-36048), possesses good chemical stability at the N-terminal fragment, but was found to be a substrate for DPPIV cleavage resulting in low metabolic stability (see Example 2, Table 2). Poor metabolic stability results in decreased half-life of a peptide requiring more frequent dosing. The present invention sets forth to improve the prior art polypeptides by providing selective NPY5R agonists with improved metabolic stability and high chemical stability. As shown herein, the metabolic stability of the N-terminal of the hNPY analogue could be improved when the amino acid sequence X^-X3was selected as G-P-[Tle], [Aib]-P-[NMeS], [bAla]-P-S, [NMeG]-P-S, G- P-[NMeS], G-P-P, or [GABA]-P-S while simultaneously retaining high chemical stability (see Example 2, Table 2).

[0048] Thus, in a preferred embodiment, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P-[NMeS], [Aib]-P-[NMeS], or [GABA]-P-S. In a more preferred embodiment, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P-[NMeS], or [Aib]-P-[NMeS] . In an even more preferred embodiment, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, or G-P-[NMeS]. In a highly preferred embodiment, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, or [bAla]-P-S. In a more highly preferred embodiment, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, or [NMeG]-P-S. In a most preferred embodiment, the amino acid sequence X^-X3is selected as G-P-[Tle].

[0049] Thus, in a preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the structure of Formula (I) X1-X2-X3-X4-P-T-X7-P-G-X10-N-A-T-P-E-X16-X17-A-R-Y-Y-S-A-L-R-X26-X27-X28-N-X30-A-Aib-R-X34-R-X36

[0050] (I) wherein, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P- [NMeS], [Aib]-P-[NMeS], or [GABA]-P-S, preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P-[NMeS], or [Aib]-P-[NMeS], more preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, or G-P-[NMeS], even more preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, or [bAla]-P-S, yet even more preferably X^-X3is selected as G-P-[Tle], G-P-P, or [NMeG]- P-S, most preferably X^-X3is selected as G-P-[Tle];

[0051] X4is selected as K, Q, or E;

[0052] X7is selected as Y or K;

[0053] X10is selected as D, E, or K;

[0054] X15is selected as E or Q;

[0055] X17is selected as L, M, or Nle;

[0056] X25is selected as R or H;

[0057] X27is selected as Y or Cha;

[0058] X28is selected as I or Y;

[0059] X30is selected as W, M, or Nle;

[0060] X34is selected as NMeQ, NMeA, or Q;

[0061] X35is selected as Phe(4CF3), Phe(4CI), Phe(4F), Y, F, or Cha. Lipidation sites of the polypeptides

[0062] As exemplified herein, position X4, X7, or X10was suitable for introducing a lysine residue (K) as lipidation site in the polypeptides (see Table 1, SEQ ID NO: 87, 88, and 89). In particular, a lipidated lysine residue in position X4was shown to provide the highest selectivity for NPY5R over NPY2R compared to positions X7and X10. Thus, in a most preferred embodiment, X4is selected as lysine (K). As the polypeptides are preferably only mono-lipidated, it follows that when X4is selected as K and used for lipidation, X7is most preferably selected as Y, and X10is most preferably selected as D or E, most preferably D.

[0063] Thus, in a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the structure of Formula (I) X1-X2-X3-K-P-T-Y-P-G-X10-N-A-T-P-E-X16-X17-A-R-Y-Y-S-A-L-R-X26-X27-X28-N-X30-A-Aib-R-X34-R-X36

[0064] (I) wherein, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P- [NMeS], [Aib]-P-[NMeS], or [GABA]-P-S, preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P-[NMeS], or [Aib]-P-[NMeS], more preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, or G-P-[NMeS], even more preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, or [bAla]-P-S, yet even more preferably X^-X3is selected as G-P-[Tle], G-P-P, or [NMeG]- P-S, most preferably X^-X3is selected as G-P-[Tle];

[0065] X10is selected as D or E;

[0066] X15is selected as E or Q;

[0067] X17is selected as L, M, or Nle;

[0068] X25is selected as R or H;

[0069] X27is selected as Y or Cha;

[0070] X28is selected as I or Y;

[0071] X30is selected as W, M, or Nle;

[0072] X34is selected as NMeQ, NMeA, or Q;

[0073] X35is selected as Phe(4CF3), Phe(4CI), Phe(4F), Y, F, or Cha.

[0074] Metabolic stability of the C-terminai fragment of the polypeptides

[0075] The amino acid residues X34 / X35and X35 / X35were identified as metabolic sites in plasma. It was found that substituting the glutamine residue (Q) in X34with N-methyl-alanine (NMeA) or N-methyl-glutamine (NMeQ) prevented cleavage at the identified metabolic sites (see Example 3, Table 3). Thus, in a highly preferred embodiment of the invention, X34is selected as NMeQ or NMeA. NMeQ was found to completely prevent cleavage at the identified metabolic sites. Thus, in the most preferred embodiment of the invention, X34is selected as NMeQ.

[0076] Thus, in an even more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the structure of Formula (I) X1-X2-X3-K-P-T-Y-P-G-X10-N-A-T-P-E-X16-X17-A-R-Y-Y-S-A-L-R-X26-X27-X28-N-X30-A-Aib-R-X34-R-X36

[0077] (I) wherein, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P- [NMeS], [Aib]-P-[NMeS], or [GABA]-P-S, preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P-[NMeS], or [Aib]-P-[NMeS], more preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, or G-P-[NMeS], even more preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, or [bAla]-P-S, yet even more preferably X^-X3is selected as G-P-[Tle], G-P-P, or [NMeG]- P-S, most preferably X^-X3is selected as G-P-[Tle];

[0078] X10is selected as D or E;

[0079] X15is selected as E or Q;

[0080] X17is selected as L, M, or Nle;

[0081] X25is selected as R or H;

[0082] X27is selected as Y or Cha;

[0083] X28is selected as I or Y;

[0084] X30is selected as W, M, or Nle;

[0085] X34is selected as NMeQ or NMeA;

[0086] X35is selected as Phe(4CF3), Phe(4CI), Phe(4F), Y, F, or Cha.

[0087] Improved hNPY5R potency of the polypeptides

[0088] The amino acid position X35was identified as a suitable position to improve the potency of the polypeptides. As shown herein for the matched pairs SEQ ID NO: 28 (reference) and SEQ ID NO: 39-41, the substitution of Y (SEQ ID NO: 28) in position X35with the amino acids F (SEQ ID NO: 41), Phe(4F) (SEQ ID NO: 39) or Phe(4CI) (SEQ ID NO: 40) resulted in improved potency, approximately a factor 4 for F, a factor 10 for Phe(4F) and a factor 14 for Phe(4CI), compared to the amino acid residue Y (see Example 4, Table 4). Hence, small lipophilic substituents on the phenyl ring of the phenylalanine residue improved the potency.

[0089] Thus, in any of the above aspects and embodiments, X35is preferably selected as Y, F, Phe(4F), Phe(4CI), or Phe(4CF3),; more preferably X35is selected as F, Phe(4F), Phe(4CI), or Phe(4CF3); Even more preferably X35is selected as F, Phe(4F), Phe(4CI). Most preferably, X35is selected as Phe(4F) or Phe(4CI). Furthermore, as illustrated herein, the substitution of Y in position X35with F, Phe(4F), or Phe(4CI) resulted in improved selectivity for hNPYR5 over hNPYR2 (see Tabel 1, SEQ ID NO: 39-41 in comparison with SEQ ID NO: 28). Thus, in a most preferred embodiment, X35is selected as Phe(4F) or Phe(4CI) to provide polypeptides with improved hNPYR5 potency and high selectivity.

[0090] Thus, in a highly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the structure of Formula (I) X1-X2-X3-K-P-T-Y-P-G-X10-N-A-T-P-E-X16-X17-A-R-Y-Y-S-A-L-R-X26-X27-X28-N-X30-A-Aib-R-X34-R-X36

[0091] (I) wherein, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P- [NMeS], [Aib]-P-[NMeS], or [GABA]-P-S, preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P-[NMeS], or [Aib]-P-[NMeS], more preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, or G-P-[NMeS], even more preferably X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, or [bAla]-P-S, yet even more preferably X^-X3is selected as G-P-[Tle], G-P-P, or [NMeG]- P-S, most preferably X^-X3is selected as G-P-[Tle];

[0092] X10is selected as D or E;

[0093] X15is selected as E or Q;

[0094] X17is selected as L, M, or Nle;

[0095] X25is selected as R or H;

[0096] X27is selected as Y or Cha;

[0097] X28is selected as I or Y;

[0098] X30is selected as W, M, or Nle;

[0099] X34is selected as NMeQ or NMeA;

[0100] X35is selected as Phe(4CF3), Phe(4CI), Phe(4F), or F, most preferably Phe(4CI) or Phe(4F).

[0101] In any of the above mentioned embodiments, X10is most preferably selected as D; X15is most preferably selected as E; X17is preferably selected as L or Nle, most preferably L; X25is most preferably selected as R; X27is most preferably selected as Y; X28is most preferably selected as I; X30is preferably selected as W or Nle, most preferably W; X34is preferably selected as NMeQ, or NMeA, most preferably NMeQ; X35is most preferably selected as Phe(4F) or Phe(4CI).

[0102] Example 1 - NPY5R agonists

[0103] Example 2 - Chemical stability of N-terminal fragment of NPY5R agonists

[0104] The chemical stability of the different N-terminal fragments was determined at pH 4, 5, 6, and 7 by incubating the peptides in acetate buffer at pH 4 and 5 and in L-His buffer at pH 6 and 7 for 14 days at 40°C. The stability was measured using RP-HPLC as described in the experimental section.

[0105] As can be seen from Table 2, the N-terminal fragment Aib-P-NMeS, NMeG-P-S, bAla-P-S, GABA-P-S, G-P- Tle, G-P-P, and G-P-NMeS provided both good chemical stability compared to the N-terminal fragments Aib-P-S and good metabolic stability compared to the N-terminal fragments G-P-S. Thus, the peptides according to the present invention have a DPPIV stability of at least 100 hours, when measured according to the procedure herein.

[0106] Example 3 - Improved metabolic stability (i.e. position X34) of NPY5R agonists.

[0107] The inventors identified the C-terminal as a metabolic labile spot, resulting in the cleavage of a pentapeptide sequence. In order to identify peptides with improved plasma stability, different amino acids (NMeQ or NMeA) were investigated in position X34. The plasma stability of the different compounds was determined in in vitro mouse plasma by spiking the plasma with the peptide. The degradation of the mother peptide was determined by LC-MS / MS as described in the experimental section (see Table 3).

[0108] As can be seen from the matched pairs SEQ ID NO: 29 (reference), SEQ ID NO: 37 and SEQ ID NO: 38, the substitution of X34= Q (SEQ ID NO: 29) to X34= NMeQ (SEQ ID NO: 37) or NMeA (SEQ ID NO: 38) greatly enhanced the plasma half-life of the peptides.

[0109] Example 4 - Improved efficacy due to the improved metabolic stability (i.e. position X34) of NPY5R agonists.

[0110] The difference in plasma stability of the C-terminal between NMeQ or NMeA at position X34was shown to translate into a clear difference in in vivo efficacy with NMeQ leading to a larger body weight increase compared to NMeA (see Table 4).

[0111] Example 5 - Improved potency of (i.e. position X36) of NPY5R agonists.

[0112] The inventors identified position X35as a position suitable for improving the potency of the peptides by substituting X35= Y with F, Phe(4F) or Phe(4CI) (see Table 5). Based on these findings, other small lipophilic substituents, e.g. trifluoromethyl (-CF3) or bromo (-Br), in the para position of the phenylalanine residue are also expected to increase potency.

[0113] As can be seen from the matched pairs SEQ ID NO: 28 (reference), SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, the substitution of Y (SEQ ID NO: 28 ) with either F (SEQ ID NO: 41), Phe(4F) (SEQ ID NO: 39) or Phe(4CI) (SEQ ID NO: 40) resulted in an approximately factor 4-14 improvement of the potency.

[0114] EXPERIMENTAL SECTION

[0115] Peptide synthesis

[0116] Peptides were synthesized using standard Fmoc (fluorenylmethyloxycarbonyl) chemistry using Rink Amide HMBA Resin (0.30 mmol, 1.00 eq., loading 0.28 mmol / g). Fmoc removal was performed using 20% piperidine in dimethylformamide (DMF) (10 mL) and agitated with nitrogen for 15 min. The resin was washed with dimethylformamide (DMF) five times (10 mL) and filtered. The consecutive amino acid couplings were performed using a solution of 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) (2.85 eq.), N,N-diisopropylethylamine (DIPEA) (6 eq) and Fmoc-protected amino acids (3 eq.) in dimethylformamide (DMF) (5 mL) and agitated with nitrogen bubbling for 30 min at 20°C. The resin was then washed five times with dimethylformamide (DMF) (10 mL). The Fmoc-protected amino acids building blocks used were the standard recommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Dde)-OH, Fmoc- Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, Fmoc-Dpr(octanoyl)-OH, Fmoc-Aib-OH, Fmoc-phPro-OH, Fmoc-Cha-OH, Fmoc-NMeAla-OH, Fmoc-NMeGln(Trt)-OH, Fmoc-NMeLeu-OH, Fmoc-NMeLys(Boc)-OH, Fmoc-NMeVal-OH, Fmoc- NMeSer(tBu)-OH, Fmoc-Phe(4F)-OH, Fmoc-Phe(4CI)-OH, Fmoc-pAla-OH, Boc-pAla-OH, Fmoc-Tle-OH, Fmoc-Nle-OH, Boc-Pro-OH, Boc-Gly-OH, Boc-NMeGly-OH, Boc-Aib-OH, Boc-pAla-OH, Boc-GABA-OH, Fmoc- OEG-OH, Fmoc-Glu-OtBu, Eicosanedioic acid mono-tert-butyl ester, and Octadecanedioic acid mono-tert- butyl ester. If nothing else is specified, the natural L-form of the amino acids was used. Fmoc removal and coupling steps were repeated until the desired peptide sequence was achieved. The resultant linear peptide was used directly in the next step. To remove the Dde protecting group, 3% NzHrEhO in DMF was added and left to react for 30 min - drain and repeat the deprotection. Elongation at the sidechain of the deprotected Lys as described for the standard couplings and Fmoc-deprotection but using 2-(lH-7- Azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronoium hexafluorphosphate (HATU) as coupling reagent instead of HBTU. Peptide cleavage and purification

[0117] The resin was washed with methanol (20 mL x 5) and dried under vacuum to get peptide resin. Subsequently, the peptidyl resin was then treated with the cleavage cocktail (92.5% TFA (trifluoroacetic acid) / 2.5% triisopropyl silane (TIPS) I 2.5% 3-mercaptopropionic acid (Mpr) I 2.5% H2O) (15 mL) for 150 min and the peptide TFA mixture was collected. The peptide was precipitated with cold isopropyl ether and centrifuged (2 min at 3000 rpm). The peptide precipitation was washed with isopropyl ether two more times. The crude peptide was dried over vacuum for 2 hours. The peptide was re-dissolved in an acetonitrile / water mixture and purified by preparative HPLC, and the target peptide fractions were freeze- dried to give a white solid.

[0118] Preparative HPLC purification method:

[0119] System: Gilson GX-281

[0120] Column: Gemini, 5 pm, C18, 110 A followed by a Luna, C18, 10 pm, 100 A.

[0121] Gradient: 21-41% 0-50 min.

[0122] Flow rate: 20 mL / min

[0123] Diode array: 214 / 254 nm

[0124] Column temperature: 30°C

[0125] Solvent A: 0.075% TFA in water

[0126] Solvent B: Acetonitrile

[0127] HPLC QC method:

[0128] System: Agilent Infinity II 1260 HPLC series

[0129] Column: Gemini C18, HOA, 5 pm, 150 x 4.6 mm

[0130] Gradient: Gradient run-time 20 min; 0.00-20.00 min 15-45% B. Column cleaning and equilibration; 20.00-20.10 min 45-95% B, 20.10-23.00 min 95% B, 23.00- 23.10 min 95-15% B, 23.10-28.00 min 15% B

[0131] Flow rate: 1.0 mL / min

[0132] Diode array: 220 / 254 nm

[0133] Column temperature: 30°C

[0134] Solvent A: 0.1% TFA in water

[0135] Solvent B: 0.075% TFA in acetonitrile

[0136] LC-MS method for final products:

[0137] System: Agilent Infinity II 1260 HPLC series

[0138] Column: Xbridge C18, 130A, 3.5 pm, 2.1 x 30 mm

[0139] Detector: Agilent LCMS (G6125C), single quadrupole TIC scan

[0140] Scanning range: m / z min 100, m / z max. 2000, positive mode Gradient: Gradient run-time 1 min; 0.00-1.00 min 10-80% B. Column cleaning and equilibration; 1.00-1.01 min 80-95% B, 1.01-1.60 min 95% B, 1.60-1.61 min 95-10% B, 1.61-2.00 min 10% B

[0141] Flow rate: 1.2 mL / min Diode array: 220 / 254 nm

[0142] Column temperature: Room temperature

[0143] Solvent A: 0.1% TFA in water

[0144] Solvent B: 0.075% TFA in acetonitrile

[0145] In vitro assays

[0146] The in vitro functional measurements were performed by EuroScreen Fast, a CRO unit of Epics Therapeutics S.A., in Gosselies, Belgium or by the University of Leipzig (Professor Annette Beck-Sickinger). The peptides were tested towards hNPY5R using cAMP HTRF assay. The hNPY5R receptor accession numbers (EuroScreen), cellular background, and reference compounds are NPY5R AAC50623.1, CHO-K1, and human NPY, respectively. On each day of experimentation, reference compounds were tested at several concentrations in duplicate (n=2-3) to obtain a dose-response curve and an estimated ECso value. Reference values thus obtained for the test were compared to historical values obtained from the same receptor and used to validate the experimental session. For replicate determinations, the maximum variability tolerated in the test was of + / -20% around the average of the replicates. Dose-response data from test compounds were analysed with XLfit (IDBS) software using nonlinear regression applied to a sigmoidal dose-response model and the following equation:

[0147] XL Fit Model 203: 4 Parameter Logistic Model

[0148] A: Bottom

[0149] B: TOP

[0150] C: LogEC50

[0151] 20 D: Hill fit= (A+((B-A) / (l+(((10-C) / x)-D)))) inv= ((10-C) / ((((B-A) / (y-A))-l)-(l / D))) res = (y-fit) Chemical stability

[0152] The formulation stability (chemical stability) was performed in Pephexia Therapeutics ApS' analytical laboratory, Bioinnovation Institute, Denmark. Each peptide was prepared as 80 nmol aliquots in glass HPLC vials depending on the molecular weight. For each buffer system investigated, 0.5 mL was gently added to an 80 nmol sample. The vail was closed with a lid and turned gently upside down before left to stand for 2 hours to completely dissolve. Subsequently, the sample was transferred to Eppendorf tube and centrifuged at 13,300 rpm for 10 minutes. 50 uL of the sample was transferred to a low volume HPLC vail and analysed by RP-HPLC (time 0). 450 uL was transferred to a HPLC vail and incubated for 2 weeks at 40°C. Subsequently, the sample was centrifuged at 13,300 rpm for 10 minutes before being analysed by RP-HPLC (time 14d). Both target peak AUC and target peak percentage were compared between time 0 and 14d and reported as degradation in percent. The HPLC method was as follows: Flow rate 0.4 mL / min; injection volume 5 pL, detection wavelength 214 nm and 280 nm; gradient 22 min, 14 min 20-60%B (0- 0.2 min 0% B, 0.2-0.5 min 0-20% B, 0.5-14 min 20-90% B, 14-16 min 90-100% B, 18-18.05 min 100-0% B, 18.05-22 0% B). Eluents: A was 89.9% ultrapure water, 10% acetonitrile, 0.1 % TFA and B was 99.9% acetonitrile, 0.1 % TFA. Column: Kinetex 2.6 pm C8 100 A; Size: LC Column 150 x 4.6 mm (Phenomenex). Buffer systems for chemical stability: 1) 20 mM acetate buffer with 5% v / w mannitol, pH 4.0, 2) 20 mM acetate buffer with 5% v / w mannitol, pH 5.0, 3) 20 mM L-His buffer with 5% v / w mannitol, pH 6.0, 4) 20 mM L-His buffer with 5% v / w mannitol, pH 7.0.

[0153] Plasma stability

[0154] Test compounds and control working solution preparation: 1) Test compound working solution was 50 pL of compound stock solution (10 mM in dimethyl sulfoxide (DMSO)) which was diluted with 450 pL of DMSO (working solution concentration: 100 pM, 100% DMSO). 2) Control compound 1 (propantheline bromide) working solution was 5 pL of propantheline bromide stock solution (10 mM in H2O) were diluted with 495 pL of H2O (Working solution concentration: 100 pM, 100% H2O).

[0155] Pooled frozen mouse plasma (C57BI / 6) was thawed in a water bath at 37°C prior to experiment. Afterwards, the plasma was centrifuged at 4000 rpm for 5 min and the clots were removed, if any. Using an Apricot automation workstation, 98 pL / well of blank plasma was added to all 96-well reaction plates. With the Apricot automation workstation, 2 pL / well of working solution (100 pM) was added to all reaction plates except Blank. All reaction plates containing mixtures of compound and plasma were incubated at 37°C in water bath - one plate per time point. At the end of incubation, 800 pL of stop solution (200 ng / mL tolbutamide and 200 ng / mL labetalol in MeOH) was added to each well to precipitate plasma proteins. The plate was sealed and thoroughly mixed by shaking for 20 minutes. After shaking, each plate was centrifuged at 4000 rpm and 4°C for 20 minutes. After centrifugation, an Apricot automation workstation was used to transfer 150 pL of supernatant from each reaction plate to its corresponding bioanalysis plate, and each plate was sealed and shaken for 10 minutes prior to LC-MS / MS analysis. The % remaining of test compound after incubation in plasma was calculated using the following equation: % Remaining = 100 x (PAR at appointed incubation time I PAR at TO time) - where PAR is the peak area ratio of analyte versus internal standard (IS).

[0156] In vivo experiment

[0157] The study was performed by Apigenex s.r.o. (Czech Republic) in vivo pharmacology department. 32 C57BL / 6J mice, males, 10 weeks old at the arrival (Charles River, Germany). The mice were kept under controlled conditions with constant temperature (22 ± 2 °C), relative humidity 45-65 %, fixed dark:light cycle (light off at 7 pm, light on at 7 am) and supplied with standard environmental enrichment (plastic houses, nesting material, aspen blocks). The animals were housed singly and acclimatized for 6 days before the start of the experiment. The bedding (Jeluxyl-Sawi, Germany) was exchanged twice weekly. The mice were supplied with normal chow (Ssniff R / M-H, Germany) ad libitum throughout the study. The animals had free access to UV light-treated drinking water. The water was exchanged twice weekly.

[0158] The compounds were dissolved in 5 mM acetate buffer + 5% w / v mannitol, pH=5. The test items were formulated on day -1 and divided into 7 daily aliquots. The aliquots were stored at -20 °C, thawed at room temperature 60 min prior to morning dosing and kept in refrigerator for the afternoon dosing. The vehicle and the compounds were dosed subcutaneously in volume of 10 ml / kg in the scruff of the neck on days 0- 6 at 7 am and at 6 pm.

[0159] Every day, the clinical observations of all mice were recorded. The body weight was determined on days 0-7 at 6:20 am using calibrated scales Mettler-Toledo PB3002-S. The individual food intake was determined on days 1-7 at 7 am using calibrated scales Mettler-Toledo PB3002-S. The food was equilibrated to animal room humidity for 4 days. At each time point, pre-weighed diet (approximately 25 g per animal) was presented. The food intake was calculated as a difference between the diet amount supplied and the residual amount.

[0160] REFERENCES

[0161] 1. Chiara Cabrele, Michael Langer, Reto Bader, Heike A. Wieland, Henri N. Doods, Oliver Zerbe, and Annette G. Beck-Sickinger. J. Biol. Chem, 2000, 275 (46), pp. 36043-36048.

Claims

CLAIMS1. A polypeptide or a pharmaceutically acceptable salt thereof comprising the structure of Formula (I)X1-X2-X3-X4-P-T-X7-P-G-X10-N-A-T-P-E-X16-X17-A-R-Y-Y-S-A-L-R-X26-X27-X28-N-X30-A-Aib-R-X34-R-X36(I) wherein, the amino acid sequence X^-X3is selected as G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P-[NMeS], [Aib]-P-[NMeS], or [GABA]-P-S;X4is selected as K, Q, or E;X7is selected as Y or K;X10is selected as D, E, or K;X15is selected as E or Q;X17is selected as L, M, or Nle;X25is selected as R or H;X27is selected as Y or Cha;X28is selected as I or Y;X30is selected as W, M, or Nle;X34is selected as NMeQ, NMeA, or Q;X35is selected as Phe(4CF3), Phe(4CI), Phe(4F), F, Y, or Cha.

2. The polypeptide according to claim 1, wherein the amino acid sequence X^-X3is selected from a list consisting of G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, G-P-[NMeS], or [Aib]-P-[NMeS],3. The polypeptide according to any one of the preceding claims, wherein the amino acid sequence X^X2- X3is selected from a list consisting of G-P-[Tle], G-P-P, [NMeG]-P-S, [bAla]-P-S, or G-P-[NMeS].

4. The polypeptide according to any one of the preceding claims, wherein the amino acid sequence X^X2- X3is selected from a list consisting of G-P-[Tle], G-P-P, [NMeG]-P-S, or [bAla]-P-S.

5. The polypeptide according to any one of the preceding claims, wherein the amino acid sequence X^X2- X3is selected from a list consisting of G-P-[Tle], G-P-P, or [NMeG]-P-S.

6. The polypeptide according to any one of the preceding claims, wherein the amino acid sequence X^X2- X3is selected as G-P-[Tle].

7. The polypeptide according to any one of the preceding claims, wherein X34is selected as NMeQ or NMeA, preferably NMeQ.

8. The polypeptide according to any one of the preceding claims, wherein X35is selected as Phe(4CF3), Phe(4CI), Phe(4F), F, or Y, preferably Phe(4CF3), Phe(4CI), Phe(4F), or F.

9. The polypeptide according to any one of the preceding claims, wherein X35is selected as Phe(4F) or Phe(4CI).

10. The polypeptide according to any one of the preceding claims, wherein X4is selected as K; X7is selected as Y; and X10is selected as D or E, preferably D.

11. The polypeptide according to any one of the preceding claims, wherein X15is selected as E; and X17is selected as L or M, preferably L.

12. The polypeptide according to any one of the preceding claims, wherein X25is selected as R; and X27is selected as Y.

13. The polypeptide according to any one of the preceding claims, wherein X28is selected as I; and X30is selected as W or Nle, preferably W.

14. The polypeptide according to any one of the preceding claims, wherein the polypeptide is amidated at the C-terminal (-CONH2).

15. The polypeptide according to any one of the preceding claims, for use as a medicament.

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