Orexin receptor agonists

Abstract

The present invention relates to potent hOX1R / hOX2R agonists, as well as their use as a medicament, in particular for the treatment of narcolepsy. In particular, the present invention relates to potent hOX1R / hOX2R agonists, with prolonged half-life and good physiochemical properties.

Description

[0001] OREXIN RECEPTOR AGONISTS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to hOXlR / hOX2R agonists and their use as a medicament for the treatment of narcolepsy.

[0004] BACKGROUND OF THE INVENTION

[0005] Orexin B (hypocretin 2) is a neuropeptide hormone primarily produced in the lateral hypothalamus of the brain with the amino acid sequence RSGPPGLQGRLQRLLQASGNHAAGILTM (SEQ ID NO: 32). It is part of the orexin system, which also includes Orexin A (hypocretin 1). Orexin B binds to and exerts its action through the human Orexin receptor 1 (hOXIR) and human Orexin receptor 2 (hOX2R).

[0006] One of the key functions of Orexin B is its involvement in the regulation of wakefulness. It interacts with orexin receptors in the brain to promote wakefulness and maintain a state of alertness. Dysfunction of the orexin system, including deficiencies in Orexin B, has been implicated in various sleep disorders, such as narcolepsy, where individuals experience excessive daytime sleepiness and disruptions in sleep-wake cycles.

[0007] Narcolepsy is a chronic neurological disorder that involves a decreased ability to regulate sleep-wake cycles and has debilitating impact on the quality of life. Narcolepsy afflicts 1 in 2000 individuals worldwide and onset may occur during adolescence for a lifelong duration. There are two major types of narcolepsy: type 1 and type 2, differentiated by whether a person experiences cataplexy. The most common form of narcolepsy is type 1 in which the individual experiences brief loss of muscle tone ("drop attacks" or cataplexy) and is caused by a lack of orexin in the brain due to the loss of neurons that produce orexin. Other symptoms of narcolepsy include periods of excessive daytime sleepiness, disturbed nighttime sleep, hypnagogic and hypnopompic hallucinations, inappropriately timed rapid-eye-movement (REM) sleep, and brief involuntary sleep episodes that make persons with narcolepsy fall asleep without warning. Current approved drugs for treatment of the symptoms include e.g. armodafinil (Nuvigil), modafinil (Provigil) and methylphenidate (Ritalin) to improve wakefulness and serotonin-norepinephrine reuptake inhibitors (SNRIs), such as venlafaxine (Effexor), to treat the cataplexy.

[0008] However, patients may benefit from treatments that aim at restoring the orexin neurotransmission. Thus, the development of therapeutics to restore lost orexin-signalling is critically important for the treatment of the root cause of narcolepsy. Thus, it is an object of the present invention to provide hOXlR / hOX2R agonists with high potency, prolonged half-life and good physiochemical properties.

[0009] SUMMARY OF THE INVENTION

[0010] The present disclosure relates to hOXlR / hOX2R agonists as well as their use as a medicament, in particular for the treatment of narcolepsy. In particular, the present invention relates to potent hOXlR / hOX2R agonists, with prolonged half-life and good physiochemical properties, such as an isoelectric point (pl) that allows for formulating the peptides near physiological pH. The invention is set forth in the claims.

[0011] BRIEF DESCRIPTION OF THE FIGURES

[0012] Fig. 1 shows the results of the KEFTA scan by introducing a K, E, F, T, or A amino acid residue in one of the positions P1-28. A positive mean SHAP value indicates an improved potency on hOxlR (grey columns) and / or hOx2R (black columns), and a negative SHAP value indicates a decreased potency on hOxlR (grey columns) and / or hOx2R (black columns).

[0013] Fig. 2 shows the results from the subset of data from the KEFTA scan obtained by mutating glutamate (E) into one or more of positions P1-28(i.e. a glutamate scan). A positive A mean SHAP value indicates an improved potency on hOxlR (grey columns) and / or hOx2R (black columns), and a negative A mean SHAP value indicates a decreased potency on hOxlR (grey columns) and / or hOx2R (black columns).

[0014] Fig. 3 shows the results from the lipidation scan, wherein each of the positions P1-28were mutated into a lysine residue covalently connected to an HLE group selected from C18DA-yGlu-, C18DA-yGlu- Ahx-, C18DA-yGlu-OEG-OEG-, from C20DA-yGlu-, C20DA-yGlu-Ahx-, C20DA-yGlu-OEG-OEG-, and Ac- (referred to as "none" in Fig. 3). A positive A mean SHAP value indicates an improved potency on hOxlR and / or hOx2R, and a negative SHAP value indicates a decreased potency on hOxlR and / or hOx2R.

[0015] Fig. 4 shows the results from the library aimed at identifying chemically labile spots and preventing deamidation of the glutamine (Q) residues in position P8and P16and the asparagine (N) residue in position P20. In Fig. 4, a positive A mean SHAP value indicates an increased deamidation, whereas a negative SHAP value indicates a decreased deamidation. Fig. 5 shows the results from the Aib scan, wherein selected positions P1 11and P13-28were mutated into a Aib residue. In Fig. 5, a positive A mean SHAP value indicates an increased fibrillation, whereas a negative SHAP value indicates a decreased fibrillation.

[0016] Fig. 6 shows the number of cataplectic attacks in Ataxin-3 transgenic mice during the first 10 hours of active dark phase upon treatment with SEQ ID NO: 77, 124, or 129.

[0017] DEFINITIONS

[0018] In the present context, mutations or substitutions are used interchangeably.

[0019] The polypeptides according to the present invention are preferably amidated at the C-terminal (- CONH2), like the native peptide Orexin B. However, the polypeptides according to the present invention may also have either a free carboxylic acid (-COOH) or another post-translational modification. It is most preferred that polypeptides are amidated at the C-terminal as the polypeptides exemplified herein. The polypeptides according to the present invention may have a free amine (-NH2), be N-acylated (-NHCOR, preferably acetylated -NHAc), N-methylated (-NHCH3 or -N(CH3)2) or deaminated at the N-terminal. It is most preferred that the polypeptides are acetylated (-NHAc) at the N-terminal as the polypeptides exemplified herein.

[0020] In the present context, lipidation refers to the covalent attachment of a lipid, optionally through a linker / spacer to a polypeptide according to the invention. The lipid (and spacer / linker) may also be referred to as a half-life extending (HLE) group. Any lipid commonly used in the art may be used, e.g. a C18DA (octadecanedioic acid), C20DA (icosanedioic acid), C18 (stearic acid), or C16 (palmitic acid) optionally connected through a linker / spacer consisting of one or more covalently connected units commonly used. Linker / spacer units commonly used in the art may be but is not limited to e.g. [YGIU], [OEG] and / or [AHX] as illustrated below.

[0021] Lipidation is typically performed to improve the pharmacokinetic profile of a polypeptide by e.g. improving metabolic stability, reducing enzymatic degradation and lowering excretion and metabolism, all in all resulting in a prolonged in vivo half-life (ti / 2). The polypeptides herein are lipidated at the epsilon (E) amino group of a lysine (K) residue in one of the positions P1 19or P23. Preferably, the lipidation is performed at a lysine residue (K) in a position selected from P12, P14or P16. Preferably, the lipid (and linker / spacer) is selected from the list consisting of C18DA-yGlu-, C18DA-yGlu-yGlu-, C18DA-yGlu-yGlu-yGlu-, C18DA-yGlu-OEG-OEG-, C18DA-yGlu-yGlu-OEG-OEG-, C18DA-yGlu-yGlu-yGlu-OEG-OEG-, ClS-yGlu-yGlu-yGlu-yGlu-, Cie-yGlu-yGlu-yGlu-yGlu-, C18-yGlu- yGlu-yGlu-yGlu-OEG-OEG-, ClS-YGIu-YGIu-yGlu-yGlu-yGlu-, C18-YGIU-YGIU-YGIU-YGIU-YGIU-OEG- OEG-, C16-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, (C18-YGIU- YGIU-OEG-OEG-)2K-OEG-OEG-, (C16-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, (C18-YGIU-YGIU-OEG- OEG-)2DAP-OEG-OEG-, (C16-YGIU-YGIU-OEG-OEG-)2DAP-OEG-OEG-, (C18-YGIU-YGIU-OEG-OEG- )2Orn-OEG-OEG-, (C16-YGIu-YGIu-OEG-OEG-)2Orn-OEG-OEG-, (C18-YGIU-YGIU-)2K-OEG-OEG-, (C16- YGIU-YGIU-)2K-OEG-OEG-, (C18-YGIU-YGIU-OEG-OEG-)2K-, (C16-YGIU-YGIU-OEG-OEG-)2K-. Most preferably, the lipid (and linker / spacer) is selected from C18-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, or (C18-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, (C16-YGIU- YGIu-OEG-OEG-)2K-OEG-OEG-.

[0022] As shown in the list, some of the lipids (and linkers / spacers) are branched by comprising a lysine or related moiety (e.g. Orn, DAP) as part of the linker / spacer, wherein both the alpha (a) amino group and side chain amino group are functionalized as illustrated below for (C18-YGIU-YGIU-OEG-OEG-)2K- OEG-OEG-. The * denotes the point of attachment to the epsilon (E) amino group of a lysine residue in a polypeptide according to the invention.

[0023] The amino acids herein refer to the natural amino acids (i.e. L amino acids) unless otherwise stated. The abbreviation Aib refers to 2-aminoisobutyric acid. The abbreviation Cha refers to L- cyclohexylalanine. The abbreviation Orn refers to L-ornithine; The abbreviation hPhe refers to L- homophenylalanine; The abbreviation DAP refers to L-2,3-Diaminopropionic acid. The abbreviation Nle refers to L-Norleucine.

[0024] ECso values are used as a measure of agonist potency at hOxlR and / or hOx2R. 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. The polypeptides according to the invention are at least as potent as Orexin B on hOxlR (i.e. ratio of (hOxlR ECso-polypeptide) / (hOxlR ECso-Orexin B) is < 1) as shown herein. Likewise, the polypeptides according to the invention do not lose more than a factor 4 on hOx2R potency compared to Orexin B (i.e. ratio of (hOx2R ECso-polypeptide) / (4 x hOxlR ECso-Orexin B) is < 1) as shown herein. The skilled person will appreciate that Orexin B has been used as internal standard for comparison between different assays runs or different assays. It should be understood, that the potency assay described herein is made without the presence of high abundance proteins like albumin, to which lipidated molecules bind, in order to compare with Orexin B, which is non- lipidated. Thus, when stated herein that a polypeptide according to the invention has an hOxlR ECso ratio (hOxlR ECso-polypeptide) / (hOxlR ECso-Orexin B) of < 1, and / or an hOx2R ECso ratio (hOx2R ECso-polypeptide) / (4 x hOx2R ECso-Orexin B) is < 1), it is to be understood that this ratio has been determined using the assay herein or a similar assay without the presence of high abundance proteins like albumin.

[0025] The polypeptides according to the invention may be in the form of a pharmaceutically acceptable salt and / or solvate. Pharmaceutically acceptable salts are intended to include any salts that are suitable for formulation of the peptides. Such salts include both acid addition salts and basic salts, and examples may be found e.g. in Remington's Pharmaceutical Sciences, 17th edition.

[0026] In the present context, a substitution in a derivative may be a substitution to natural amino acid residue or an unnatural amino acid residue, including L-and D-stereoisomers. Preferably, a substitution in a derivative is a conservative substitution (i.e. a substitution to a conservative amino acid residue within the same group) to a natural amino acid. The groups of conservative amino acids may be defined as e.g.: A, V, L, I, P, M (hydrophobic side chain); S, T, N, Q (polar uncharged side chain); F, Y, W (aromatic side chain); H, K, R (positively charged side chain / basic); D, E (negatively charged side chain / acidic). Thus, it will be evident to the skilled person that a substitution is preferably a substitution to an amino acid belonging to one of the groups of amino acids that was identified as being tolerated or beneficial in the KEFTA scan in order to maintain the highest hOxlR and hOx2R potency.

[0027] In the present context, the numbers 1-28 in Formula (I) below refer to the amino acid position:Rl.S2.G3.p4.p5.G6.L7.Q8.G9.R10.Lll.Q12.R13.L14.L15.Q16.A17.s18.G19.N20.H21.A22.A23.G24.I25.L26.T27.N|e28

[0028] (I)

[0029] Hence, R1refers to an arginine residue in the first position (P1); S2to refers to a serine residue in second position (P2); G3refers to a glycine residue in the third position (P3); P4refers to a proline residue in the fourth position (P4) and so forth. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention relates to polypeptides based on a novel Orexin B scaffold having a potency enhancing hydrophobic residue in the C-terminal. In particular, the present invention relates to identification of optimal amino acid positions to extend the half-life of such a scaffold and to optimal amino acid positions to lower the pl, to provide soluble peptides around physiological pH, of such a scaffold, without major adverse effect on OxlR and Ox2R potency. Furthermore, the present invention relates to the identification of amino acid positions to increase the physical and chemical stability of such a scaffold.

[0031] Orexin B analogues with improved potency

[0032] As shown in Table 2 herein, the potency of Orexin B scaffold can be improved by substituting the methionine (M) in position P28in Orexin B (SEQ ID NO: 32) with a norleucine (Nle) (see comparison between SEQ ID NO: 32 and 33, in Table 2). In an attempt to identify further positions and amino acids to improve the potency, a KEFTA scan was performed based on the Orexin B scaffold, having a Nle substitution in position P28(see Example 1). For position P28, the KEFTA scan showed that hydrophobic amino acids, such as phenylalanine (F), provided a beneficial effect in terms of OxlR and Ox2R potency (i.e. that highly hydrophobic residues may improve the potency). Thus, the polypeptides according to the invention possess a hydrophobic amino acid residue in the C-terminal (i.e. position P28) selected from Nle, Cha, or F. Most preferably, the hydrophobic amino acid in P28is selected as Nle.

[0033] Orexin B analogues with improved half-life

[0034] As shown in Example 2 and Fig. 3, the positions P1 19and P23were identified to be suitable for introducing a lipidated lysine residue to extend the half-life of the polypeptides without major adverse effect on hOXIR and hOX2R potency. As can be seen from Fig. 3, the hOXIR pECso is in the range of 8-9 for the majority of HLE groups in the positions P1 19and P23, whereas the hOXIR pECso is in the range of 6.5-7.5 for the majority of HLE groups in the remaining positions. Likewise, the hOX2R pECso is in the range from 8-9 for the majority of HLE groups in the positions P1 19and P23, whereas the hOX2R pECso is in the range of 6.75-7.75 for the majority of HLE groups in the remaining positions. Thus, the random forest model shows that HLE groups should be introduced in one of the positions P1 19or P23to alter the PK properties of the polypeptides while maintaining the highest possible potency at hOXIR and hOX2R.

[0035] The exemplified polypeptides in Table 2 confirm the findings from the random forest model, wherein a lipidated lysine in one of the positions selected from P20-22P24-27and P28were associated with a pronounced decrease in hOXIR and hOX2R potency (see SEQ ID NO: 53-55 and 57-61) compared to a position selected from P1 19and P23(see SEQ ID NO: 34-52 and 56). Thus, one technical effect of the invention resides in the identification of optimal positions for introducing a lipidated lysine residue to extend the half-life of the polypeptides with a minimum sacrifice in hOXIR. and / or hOX2R potency. Preferably, the lipidated lysine residue is present in one of the positions P9, P12, P14, P16or P23. Most preferably, the lipidated lysine residue is present in one of the positions P12, P14or P16.

[0036] Orexin B analogues with decreased pl

[0037] As shown in the random forest model in Example 1 and Fig. 2, the subset of data for the glutamate (E) mutations in the KEFTA scan (i.e. glutamate scan) shows that the positions P1-6, P8-9, P12 13, P16-19and P23are suitable for introducing glutamate residues to lower the isoelectric point (pl) of the peptides to provide soluble peptides around physiological pH, as glutamate mutations in these positions had minor effect on hOXIR and hOx2R potency.

[0038] The exemplified peptides in Table 1 confirm these findings. As can be seen, the polypeptides having a glutamate (E) residue in one of the positions selected from P7, P10 -11, P14 15, P20-22, and P24-28had a pronounced decrease in hOXIR and hOX2R potency (see SEQ ID NO: 7, 10-11, 14-15, 20-22, 24-28 in Table 1) compared to one of the positions selected from P1-6, P8-9, P12 13, P16 19and P23(see SEQ ID NO: 1-6, 8-9, 12-13, 16-19, and 23 in Table 1). Thus, another technical effect of the invention resides in the identification of optimal positions for introducing glutamate residues in the polypeptides to lower the pl to provide soluble peptides around physiological pH, with a minimum sacrifice in hOXIR and hOX2R potency.

[0039] The number of positions in Formula (I) substituted with glutamate (E) residues are 2 or 3; most preferably 3. It is further preferred that it is the positions P4, P9, and / or P23of Formula (I) that are substituted with the glutamate residues. Thus, in a preferred embodiment, the amino acid residue in two of the positions of Formula (I) selected from P4, P9, and P23are substituted with glutamate residues (i.e. P4and P9= E; P4and P23= E; or P9and P23= E). In a most preferred embodiment, the amino acid residue in the three positions P4, P9, and P23in Formula (I) are substituted with glutamate residues.

[0040] It will be apparent to the skilled person that any combination of the above findings is contemplated.

[0041] Thus, in a first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),Rl.S2.G3.p4.p5.G6.L7.Q8.G9.R10.Lll.Q12.R13.L14.L15.Q16.A17.s18.G19.N20.H21.A22.A23.G24.I25.L26.T27.N|e28

[0042] (I) wherein the amino acid residue in two or three of the positions selected from P1-6, P8-9, P12 13, P16 19and P23is substituted with a glutamate (E) residue; the amino acid residue in one of the positions selected from P1 19and P23is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid residue in one of the positions selected from P17-23is substituted with an Aib residue; optionally the amino acid residue in position P28(i.e. Nle) is substituted with a Cha, F, or hPhe residue; and optionally the amino acid residue in position P20(i.e. N) is substituted with a Q or A residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution (s) in the derivative is / are not present in position P20or in any position substituted with an E, K, or Aib residue.

[0043] Most preferably, the amino acid residue in position P28is selected as Nle.

[0044] Thus, in a preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0045] Rl.S2.G3.p4.p5.G6.L7.Q8.G9.R10.Lll.Q12.R13.L14.L15.Q16.A17.s18.G19.N20.H21.A22.A23.G24.I25.L26.T27.N|e28

[0046] (I)

[0047] , wherein the amino acid residue in two or three of the positions selected from P1-6, P8-9, P12 13, P16 19and P23is substituted with a glutamate (E) residue; the amino acid residue in one of the positions selected from P1 19and P23is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid residue in one of the positions selected from P17-23is substituted with an Aib residue; and optionally the amino acid residue in position P20(i.e. N) is substituted with a Q or A residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution (s) in the derivative is / are not present in position P20or in any position substituted with an E, K, or Aib residue.

[0048] Most preferably, the amino acid residue in three of the positions, in Formula (I), selected from P1-6, P8-9, p12 13, P16 19and P23is substituted with a glutamate (E) residue. Thus, in a preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0049] Rl.S2.G3.p4.p5.G6.L7.Q8.G9.R10.Lll.Q12.R13.L14.L15.Q16.A17.s18.G19.N20.H21.A22.A23.G24.I25.L26.T27.N|e28

[0050] (I)

[0051] , wherein the amino acid residue in three of the positions selected from P1-6, P8-9, P12-13, P16 19and P23is substituted with a glutamate (E) residue; the amino acid residue in one of the positions selected from P1 19and P23is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid residue in one of the positions selected from P17-23is substituted with an Aib residue; and optionally the amino acid residue in position P20(i.e. N) is substituted with a Q or A residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution (s) in the derivative is / are not present in position P20or in any position substituted with an E, K, or Aib residue.

[0052] Most preferably, the amino acid residue in the three positions P4, P9, and p23in Formula (I) is substituted with a glutamate (E) residue.

[0053] Thus, in a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0054] R1.S2.G3.E4-p5-G6.L7.Q8.E9.R10.L11.Q12.R13-L14-L15-Q16-A17-S18-G19-N20-H21-A22-E23-G24-I25-L26-T27-Nle28

[0055] (I)

[0056] , wherein the amino acid residue in one of the positions selected from P1 19and P23is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid residue in one of the positions selected from P17-23is substituted with an Aib residue; and optionally the amino acid residue in position P20(i.e. N) is substituted with a Q or A residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution(s) in the derivative is / are not present in position P4, P9, P20, P23or in any position substituted with a K, or Aib residue. Preferably, the amino acid residue in one of the positions selected from P9, P12, P14, P16and P23is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker.

[0057] Thus, in a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0058] R1.S2.G3.E4-p5-G6.L7.Q8.E9.R10.L11.Q12.R13-L14-L15-Q16-A17-S18-G19-N20-H21-A22-E23-G24-I25-L26-T27-Nle28

[0059] (I)

[0060] , wherein the amino acid residue in one of the positions selected from P9, P12, P14, P16and P23is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid residue in one of the positions selected from P17-23is substituted with an Aib residue; and optionally the amino acid residue in position P20(i.e. N) is substituted with a Q or A residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution(s) in the derivative is / are not present in position P4, P9, P20, P23or in any position substituted with a K, or Aib residue.

[0061] Most preferably, the amino acid residue in one of the positions selected from P12, P14and P16is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker.

[0062] Thus, in an even more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0063] R1.S2.G3.E4-p5-G6.L7.Q8.E9.R10.L11-Q12-R13-L14-L15-Q16-A17-S18-G19-N20-H21-A22-E23-G24-I25-L26-T27-Nle28

[0064] (I)

[0065] , wherein the amino acid residue in one of the positions selected from P12, P14and P16is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid residue in one of the positions selected from P17-23is substituted with an Aib residue; and optionally the amino acid residue in position P20(i.e. N) is substituted with a Q or A residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution(s) in the derivative is / are not present in position P4, P9, P20, P23or in any position substituted with a K, or Aib residue. Orexin B analogues with improved chemical stability

[0066] As shown in Example 3, Table 3, and Fig. 4, position P20was found to be a chemical labile residue prone to deamidation. The inventors found that deamidation in position P20could be minimized by introducing an alanine (A) or a glutamine (Q) residue (see Table 3) with maintained hOxlR and hOx2R potency. Thus, it is highly preferred that position P20in Formula (I) is substituted with a Q or A residue. It is most preferred that position P20in Formula (I) is substituted with an A residue. Thus, in some embodiments, yet another technical effect of the invention resides in polypeptides with increased chemical stability.

[0067] Orexin B analogues with high physical stability

[0068] As shown in Example 4 and Fig. 5, an Aib residue in one of the positions selected from p17-p23were found to decrease fibrillation. Table 4, SEQ ID NO: 66, and 67, in comparison with SEQ ID NO: 68- 98, supports the results from the random forest model summarized in Fig. 5, wherein the physical stability of the peptides was improved by introduction of a Aib residue in one of positions selected from X17-X22, such that no fibrillation was observed. Thus, in some embodiments yet another technical effect of the invention resides in polypeptides with increased physical stability. The Aib residue (if present) is preferably present in position P19.

[0069] It is highly preferred that the polypeptides according to the present invention, in addition to the findings relating to extended half-life (i.e. the optimal positions to incorporate a lysine residue with a HLE group), and the findings relating to lowering of the isoelectric point (pl) of the peptides, to provide soluble peptides around physiological pH (i.e. the optimal positions to incorporate glutamate residues), incorporate the findings relating to chemical stability (i.e. the amino acids and position to prevent deamidation), and / or the findings relating to physical stability (i.e. amino acids and positions to minimize or prevent fibrillation).

[0070] Thus, it is highly preferred that the amino acid residue in position P20is substituted with a Q or A residue.

[0071] Thus, in an even more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0072] R1-S2-G3-E4-P5-G6-L7-Q8-E9-R10-L11-Q12-R13-L14-L15-Q16-A17-S18-G19-N20-H21-A22-E23-G24-I25-L26-T27-Nle28

[0073] (I)

[0074] , wherein the amino acid residue in one of the positions selected from P12, P14and P16is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid residue in position P19is substituted with an Aib residue; and the amino acid residue in position P20(i.e. N) is substituted with a Q or A residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution(s) in the derivative is / are not present in position P4, P9, P20, P23or in any position substituted with a K, or Aib residue.

[0075] It is most preferred that the amino acid residue in position P20is substituted with an A residue.

[0076] Thus, in a more highly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0077] R1.S2.G3.E4-p5-G6.L7.Q8.E9.R10.L11.Q12.R13-L14-L15-Q16-A17-S18-G19-A20-H21-A22-E23-G24-I25-L26-T27-Nle28

[0078] (I)

[0079] , wherein the amino acid residue in one of the positions selected from P12, P14and P16is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; and optionally the amino acid residue in position P19is substituted with an Aib residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution(s) in the derivative is / are not present in position P4, P9, P20, P23or in any position substituted with a K, or Aib residue.

[0080] In yet a highly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0081] R1-S2-G3-E4-P5-G6-L7-Q8-E9-R10-L11-K12-R13-L14-L15-Q16-A17-S18-Aib19-A20-H21-A22-E23-G24-I25-L26-T27-Nle28

[0082] (I)

[0083] , wherein the lysine (K) residue in position P12is lipidated, optionally through a linker; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution(s) in the derivative is / are not present in position P4, P9, P12, P19, P20, P23.

[0084] Most preferably, the amino acid residue in one of the positions selected from P14and P16is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker.

[0085] Thus, in yet an even more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0086] R1.S2.G3.E4-p5-G6.L7.Q8.E9.R10-L11-Q12-R13-L14-L15-Q16-A17-S18-G19-A20-H21-A22-E23-G24-I25-L26-T27-Nle28

[0087] (I) , wherein the amino acid residue in one of the positions selected from P14and P16is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; and optionally the amino acid residue in position P19is substituted with an Aib residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution(s) in the derivative is / are not present in position P4, P9, P19, P20, P23or in the position substituted with the lysine (K) residue.

[0088] In any of the above aspects and embodiments, it is most preferred that the polypeptide is amidated (-CONH2) in the C-terminal and / or acetylated (AcNH-) in the N-terminal. In any of the above aspects and embodiments, it is most preferred that substitution(s) in the derivative is a substitution to a conservative amino acid. In any of the above aspects and embodiments, it is highly preferred that the lysine (K) residue is lipidated with a lipid (and linker), selected from the list consisting of C18DA- yGlu-, C18DA-yGlu-yGlu-, C18DA-yGlu-yGlu-yGlu-, C18DA-yGlu-OEG-OEG-, C18DA-yGlu-yGlu-OEG- OEG-, C18DA-yGlu-yGlu-yGlu-OEG-OEG-, ClS-yGlu-yGlu-yGlu-yGlu-, Cie-yGlu-yGlu-yGlu-yGlu-, ClS-yGlu-yGlu-yGlu-yGlu-OEG-OEG-, ClS-YGIu-YGIu-yGlu-yGlu-yGlu-, C18-YGIU-YGIU-YGIU-YGIU- YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, (C18-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, (C16-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, (C18-YGIU- YGIU-OEG-OEG-)2DAP-OEG-OEG-, (C16-YGIU-YGIU-OEG-OEG-)2DAP-OEG-OEG-, (C18-YGIU-YGIU- OEG-OEG-)2Orn-OEG-OEG-, (C16-YGIu-YGIu-OEG-OEG-)2Orn-OEG-OEG-, (C18-YGIU-YGIU-)2K-OEG- OEG-, (C16-YGIU-YGIU-)2K-OEG-OEG-, (C18-YGIU-YGIU-OEG-OEG-)2K-, and (C16-YGIU-YGIU-OEG- OEG-)2K-; most preferably the lysine (K) residue is lipidated with a lipid (and linker), selected from the list consisting of C18-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-YGIU-OEG- OEG-, (C18-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, and (C16-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-.

[0089] Thus, in a most preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising or consisting of the structure of SEQ ID NO: 75, 76, 77, 78, 79, 80, 123, 124, 128 or 129, more preferably having the structure of SEQ ID NO: 77, 78, 124, or 129.

[0090] EXPERIMENTAL SECTION

[0091] General procedure for peptide synthesis

[0092] The peptides were synthesized using a SyroII fully automated parallel peptide synthesizer (MultiSynTech GmbH, Germany), equipped with heating block, on Tentagel S RAM with a loading of 0.23-0.25 mmol / g (Rapp polymer GmbH, Germany). No-Fmoc deprotection was performed in two stages by treating the resin with 40 % piperidine / DMF (0.2 M HOBt (1-hydroxybenzotriazole)) for 3 min at 45°C followed by 20 % piperidine / DMF (0.1 M HOBt) for 7 min at 75°C. Except Asp, Cys and His residues which were No-Fmoc deprotections at room temperature; i.e. 40 % piperidine / DMF (0.2 M HOBt) for 3 min followed by 20 % piperidine / DMF (0.1 M HOBt) for 15 min. The coupling chemistry was DIC (N,N'-diisopropyl-carbodiimide) / Oxyma (ethyl cyano(hydroxyimino)acetate) in DMF using amino acid solutions of 0.5 M in DMF and a molar excess of 5-fold. Standard Fmoc protected amino acids were used. Coupling conditions were single or double couplings for 15 min at 75°C, except for His and Cys residues, which were double coupled for 15 min at 50°C. Also, amino acids coupled after Aib were double coupled. The Fmoc-amino acids were dissolved at 0.5 M in DMF containing 0.5 M Oxyma, except His which was dissolved in NMP. The resin was washed 5x with DMF after No-Fmoc deprotection and 3x after couplings.

[0093] For N-terminal lipidated examples, the lipidation was conducted on-resin as the last step in the peptide synthesis. The N-terminal lipidated peptides optionally contained linker residue(s), such as [YE], [OEG], [OEG]-[OEG] etc. The linker residues were introduced by coupling Fmoc-OEG-OH, Fmoc-OEG-OEG and / or Fmoc-Glu-OtBu to the N-terminal amino acid elongating the peptide prior to coupling the fatty diacid, such as tert-butyl protected fatty diacids, such as tBu-C18-diacid, tBu-C20- diacid etc. The linker residues were double- or triple coupled using standard conditions. The fatty acid was double coupled using 2 eq. building block.

[0094] For other lipidated examples, Boc-protected amino acid was incorporated as the N-terminal residue, and the lipidation position was incorporated as orthogonal protected Lysine, here Lys(Mtt). The Mtt group (4-methyl-trityl) was removed by treating the resin with 75% HFIP (1,1, 1,3,3, 3-hexafluoro- propan-2-ol) in DCM (dichloromethane) plus 5% TIPS for 10 minutes. The procedure was repeated 3 times. The resin was washed with 10% DIPEA in DCM, followed by 3x DMF wash.

[0095] The lipidated peptides optionally contained linker residue(s). The linker residue(s) were coupled to the epsilon-amino group of the deprotected lysine prior to coupling the fatty acid, such as tert-butyl protected fatty diacids, such as tBu-C18-diacid, tBu-C20-diacid etc. The linker residues were double- or triple coupled using standard conditions. The fatty acid was double coupled using 2 eq. building block.

[0096] After synthesis, the resin was washed with DCM and dried, and the polypeptide was cleaved from the resin by a 45 min treatment with TFA (trifluoroacetic acid) / TES (triethylsilane) / DODT / water (93 / 2.5 / 2.5 / 2.0) at 40°C, followed by precipitation with 3 volumes of cold diethyl ether, further washing with diethyl ether and left to dry. The peptides were characterized by LC-MS (Waters, Denmark) and quantified by LC-CAD (ThermoFisher scientific, Denmark). Finally, the peptides were freeze-dried to give a white powder using a Telstar benchtop freeze drier.

[0097] Genera! purification of peptides

[0098] Crude peptide was dissolved in acetonitrile / water and purified by reverse phase HPLC using a Waters preparative HPLC with C18 column (Reprosil Gold 200 A, 5pm, 30 mm x 250 mm), preparative pumps (waters 2545), UV / VIS detector (Waters 2489) and a Waters fraction collector III. The mobile phase was run with a gradient of buffer A (0.1% TFA in H2O) and buffer B (0.1% TFA in acetonitrile at a flow rate of 20 mL / min at room temperature. Relevant fractions were analyzed, pooled, and lyophilized. Finally, the peptide was freeze-dried using a Telstar benchtop freeze drier. Peptide purity and mass were determined by analytical RP-HPLC-MS on a ACQUTTY UPLC Peptide CSH C18 column (Waters, ACQUITY UPLC Peptide CSH, C18, 130 A, 1.7 pm, 2.1 mm x 100 mm) using a Waters Acquity HPLC System equipped with 3100 Mass Detector. Analysis was performed by gradient elution with buffer A (0.3% TFA in H2O) and buffer B (0.3% TFA in acetonitrile) at a temperature of 40 °C (gradient used 40-60%B over 14 min).

[0099] Genera! procedure for determination ofhOxlR and hOx2R potency

[0100] HTRF (CisBio) IP1 assay 1 (Potency Assay 1)

[0101] The Homogenous Time Resolved Fluorescence (HTRF) technology optimized for Gq coupled receptors has thoroughly been described in the CisBio IP1 kit manual (#62IPAPEC). In the absence of cellular IP1, the anti-cryptate conjugate may get into proximity to the d2 conjugate and energy (FRET) can be transferred from cryptate to d2.

[0102] CHO-K1 cells stably expressing the human Orexin 1 receptor (hOXIR) or Orexin 2 receptor (hOX2R) (Charles River Laboratory (Cat A674 RW-10-19-17 and A675 RW 10-19-17, respectively) were used with cells brought to life from frozen stocks immediately before assay performance and applied as cells in suspension. 384-Well (Corning, #4513) assay formats with a total assay volume of 20 pl were applied. 15.000 cells / well were incubated with purified peptides for 30 min at 37 °C using IP1 kit stimulation buffer supplemented with 0.05% casein (Sigma, # C4765-10ml). After the addition of HTRF® detection reagents and incubation with shaking (2400 rpm) for one hour at room temperature, signals at 620 and 665nm (raw counts: ratio of 665 / 620) were detected at a ClarioStar (BMG) plate reader. Concentration-response evaluation of purified peptides was performed with 11 concentrations of peptides (covering 3 decades), n=2 times, and with tip change for all serial dilutions. Compound starting concentration (top) was adjusted throughout the study. If the top concentration was adjusted or a 5-fold difference in calculated EC50 values was observed, n was increased by 1. EC50 values were calculated by nonlinear regression using sigmoid concentrationresponse with variable slope.

[0103] HTRF (CisBio) IP1 assay 2 (Potency Assay 2)

[0104] The Homogenous Time Resolved Fluorescence (HTRF) technology optimized for Gq coupled receptors has been thoroughly described in the CisBio IP1 kit manual (#62IPAPEC). In the absence of cellular IP1, the anti-cryptate conjugate may get into proximity to the d2 conjugate and energy (FRET) can be transferred from cryptate to d2.

[0105] CHO-K1 cells stably expressing the human Orexin 1 receptor (hOXIR) or Orexin 2 receptor (hOX2R) (Charles River Laboratory (Cat A674 RW-10-19-17 and A675 RW 10-19-17, respectively) were used with cells brought to life from frozen stocks immediately before assay performance and applied as cells in suspension. 384-Well (Corning, #4513) assay formats with a total assay volume of 20 pl were applied. 15.000 cells / well were incubated with 190 crude library peptides for 30 min at 37 °C using IP1 kit stimulation buffer supplemented with 0.05% casein (Sigma, # C4765-10ml). After the addition of HTRF® detection reagents and incubation with shaking (2400 rpm) for one hour at room temperature, signals at 620 and 665nm (raw counts: ratio of 665 / 620) were detected at a ClarioStar (BMG) plate reader. Crude library peptides were screened in 10-fold serial dilutions of 5 concentrations (n =1) and with the same top concentration for alle peptides. Tip change was done for the first two serial dilutions. EC50 values were calculated by nonlinear regression using sigmoid concentration-response with variable slope.

[0106] Genera! procedure for determination of deamidation

[0107] Chemical stability was tested for a library of 190 peptides at a concentration of 1 mg / ml peptide in 50 mM phosphate buffer pH 7.5. Samples were incubated at 40°C for 14 days. After 14 days, an aliquot of the samples was diluted to a final concentration of lOOpM and analyzed by LC-HR-MSMS. Peptides were identified using Byonics (Protein Metrics) and quantified based on AUC integration of extracted ion chromatograms (EICs) using Alchemist.

[0108] Genera! procedure for determination of solubility

[0109] Solubility around pH 7.5 was tested in 100 mM phosphate buffer. The buffer was pH 8.1 to compensate for a drop in pH induced by TFA counterions upon dissolution. For the desired maximal concentration of 2500 pM (approximately 10 mg / ml), 1000 nmol peptide was dissolved in 400 pL buffer in a Whatman Mini-Uniprep filter tube and incubated for at least 2 h at room temperature on a rocking table. The filter was pushed down and the concentration of peptide in the filtrate was determined using CAD (Charged Aerosol Detection). An aliquot (e.g. 100 pl) was transferred to an UPLC vial and this was subjected to the CAD instrument. Injection volume was selected so that a response was obtained within the instrument's reference standard curve. For a target of 2500 pM, an injection volume of 1.0 pl or 0.5 pl was used, with double determination done for each sample. The solubility was recorded as the concentration of soluble peptide in mg / mL. It is well-known that solubility may change depending on the assay employed, even at same pH value, due to e.g. a different buffer system, buffer capacity and / or ionic strength. Therefore, peptide (SEQ ID NO: 33) is used as internal reference herein, to allow determination of solubility at pH 7.5, without the need for identical assay conditions.

[0110] Genera! procedure for determination of chemical stability

[0111] Soluble samples were split into three vials. One vial was analyzed with reversed phase chromatography, and the main peak purity was recorded; this is the purity at TO (time zero). The second and third vial were incubated at 40°C for 14 days, and the samples were then visually inspected (observations noted) and analyzed with reversed phase chromatography; this is the purity at T14. The main peak purity was determined from the integrated chromatogram as the area under the main peak relative to the total peak area. The purity loss was calculated as the difference between the T14 and TO time points:

[0112] Purity loss (%) = Main peak purity (T14) (%) - Main peak purity (TO) (%)

[0113] Genera! procedure for determination of fibril formation

[0114] Peptides were dissolved in buffers (50 mM sodium acetate at pH 4.0 or 50 mM sodium phosphate at pH 7.0) to 267pM and incubated for 1-2 hours at room temperature. The samples were then divided into two replicates of 80 pl in a black 384 well plate (p-clear, Greiner Bio-One) and mixed with Thioflavin T (ThT) to a final concentration of 4 pM. The plate was centrifuged for 2 min at 2000 rpm to remove air bubbles, sealed, and placed in a plate reader (CLARIOstar, BMG). The plate reader temperature was set to 40 °C, and the fluorescence was measured every 10 min for 72 hours by exciting the ThT at 450 nm and measuring the emission at 480 nm. Samples were stressed by shaking the plate at 700 rpm (linear) for five minutes before every measurement, and fibril formation was determined as the average emission for each sample.

[0115] General procedure for calculating pl

[0116] The pl values herein were calculated using the Marvin 20.19 software. The pl may also be experimentally determined using isoelectric focusing. In the present context, wherein a peptide is stated to have a pl value of less than or equal to (<) 5, it should be determined using the same Marvin 20.19 software as herein. In the alternative, it may be experimentally determined. In Vivo Ataxin Mice PD Study Methods

[0117] Thirty-two male Ataxin-3 transgene mice (Charles River), fourteen-week-old, were randomized at study start based on body weight into four treatment groups (n=8 per group), with free access of water and food. The animals were subcutaneously dosed before lights off with Vehicle (5 ml / kg), SEQ ID NO: 124, SEQ ID NO: 129 or SEQID NO: 77 (1000 nmol / kg, 5 ml / kg). After dosing, all animals were video-tracked for 24 hours and recordings were analyzed for the number of cataplectic attacks using Gubra's Al based technological platform.

[0118] Results

[0119] Dosing with 1000 nmol / kg of SEQ ID NO 124, SEQ ID NO 129 and SEQ ID NO 77 significantly decreased the number of cataplectic attacks in Ataxin-3 transgenic mice in the first 10 hours during the active dark phase, while no differences were found between the vehicle group and the compound-treated groups during the light phase, indicating the efficacy of the treatment in the narcolepsy mouse model. The results can be seen in FIG 6.

[0120] EXAMPLE 1 - KEFTA SCAN

[0121] A KEFTA scan was performed on the Orexin B scaffold, having a Nle substitution in position P28, as reference, to examine the influence of the amino acids lysine (K), glutamate (E), phenylalanine (F), Threonine (T), or alanine (A) on hOxlR and hOx2R potency. A library containing 758 peptides where designed and synthesized, wherein each of the positions P1-?27were mutated into the amino acids K, E, F, T or A / G. For positions, wherein an alanine (A) was already present in the reference, a glycine (G) was introduced instead.

[0122] The KEFTA scan provides a representation of the different groups of natural amino acids and their different physio-chemical properties. Hence, lysine (K) represents the group of amino acids having a positive charged side chain like arginine (R) or histidine (H) (i.e. group 1 amino acids). Glutamate (E) represents the group of amino acids having a negative charged side chain like aspartic acid (D) (i.e. group 2 amino acids). Phenylalanine (F) represents the group of amino acids possessing a (aromatic) hydrophobic side chain like tyrosine (Y) or tryptophan (W) (i.e. group 3 amino acids). Threonine (T) represents the amino acids possessing a polar uncharged side chain like serine (S), asparagine (N), or glutamine (Q) (i.e. group 4 amino acids). Alanine (A) represents the amino acids possessing a (aliphatic) hydrophobic side chain like valine (V), isoleucine (I), leucine (L), methionine (M) or proline (P) (i.e. group 5 amino acids). The unnatural amino acid L-homophenylalanine (hPhe) used herein may be considered a group 4 amino acid. The unnatural amino acid norleucine (Nle) and L-cyclohexylalanine (Cha) used herein may be considered a group 5 amino acids. The ECso values on hOxlR and hOx2R were determined for the 758 peptides and SHAP values calculated from a random forest model, where ECso values are fitted to the peptide amino acid sequences. SHAP values were used to determine the level of contribution of each amino acid mutation on the endpoints hOxlR and hOx2R potency (Breiman, L. (2001), Random Forests, Machine Learning 45(1), 5-32.; Lundberg, S. M., & Lee, S. I. (2017). A unified approach to interpreting model predictions. Advances in neural information processing systems, 30.). The contribution of each substitution was determined as the difference in average SHAP value between the substitution and the reference (i.e. Orexin B, having a Nle substitution in position P28). Substitutions with positive SHAP values increased the endpoint (i.e. improved potency), while negative SHAP values decreased the endpoint (i.e. decreased potency).

[0123] A mutation was defined as being tolerated if the delta mean SHAP values for hOxlR and hOx2R were < 0.1 and > -0.1, which suggests that the mutation has little effect on hOxlR and hOx2R potency. A mutation was defined as being beneficial if the delta mean SHAP values for hOxlR and hOx2R were > 0.1, which suggests that the mutation has a beneficial effect on hOxlR and hOx2R potency. A mutation was defined as being less tolerated or undesired if the delta mean SHAP values for hOxlR and hOx2R were < -0.1, which suggests that the mutation has an adverse effect on hOxlR and hOx2R potency. The results are summarized in Fig. 1.

[0124] As shown in Fig. 1, the amino acid mutation K was found to be tolerated in positions P1-?10, p12-p20and P23, which suggests that other amino acids belonging to group 1 is also tolerated in these positions. The amino acid mutation K was found to be less tolerated in positions P11, P21-22and P24- P28, which suggests that other amino acids belonging to group 1 is likely also less tolerated in these positions.

[0125] As shown in Fig. 1, the amino acid mutation E was found to be tolerated in positions P4-P6, P8-P9, P12-P13, p16-p19and P23, which suggests that other amino acids belonging to group 2 is also tolerated in these positions. The amino acid mutation E was found to be less tolerated in positions P7, P10-Pn, P14-P15, P20-P22, and P24-P28, which suggests that other amino acids belonging to group 1 is likely also less tolerated in these positions.

[0126] As shown in Fig. 1, the amino acid mutation F was found to be tolerated in positions P4-P12, P14, P16- P18, p22-p23and P26, which suggests that other amino acids belonging to group 3 are also tolerated in these positions. The amino acid mutation F was found to be less tolerated in positions P13, P19- P21, P24-P25, and P27, which suggests that other amino acids belonging to group 3 are likely also less tolerated in these positions. The amino acid mutation F was found to be beneficial in positions P15and P28, which suggests that other amino acids belonging to group 3 are likely also beneficial in these positions.

[0127] As shown in Fig. 1, the amino acid mutation T was found to be tolerated in positions P4-P14, P16-P22, and P26-P27, which suggests that other amino acids belonging to group 4 are also tolerated in these positions. The amino acid mutation T was found to be less tolerated in positions P15, p23-p25, and P28, which suggests that other amino acids belonging to group 4 are likely also less tolerated in these positions.

[0128] As shown in Fig. 1, the amino acid mutation A / G was found to be tolerated in positions P4-P14, and P16-P22, which suggests that other amino acids belonging to group 5 are also tolerated in these positions. The amino acid mutation A / G was found to be less tolerated in positions P15, and p23-p28. As the positions P15, P23-26, and P28contain a group 5 amino acid in the reference backbone, this suggests that more hydrophobic amino acids of group 5 are more beneficial.

[0129] Conclusion: Albeit the KEFTA scan shows that the majority of the mutations did not provide any benefit in terms of hOxlR and hOx2R potency, the KEFTA scan renders it plausible that amino acid substitutions may be present in the polypeptides, in particular substitutions to conservative amino acids, without adversely affecting hOxlR and hOx2R potency. However, for position P28, the KEFTA scan suggests that the amino acid phenylalanine (F) may provide a beneficial effect in terms of hOxlR and hOx2R potency (i.e. that highly hydrophobic residues may improve the potency). Furthermore, the subset of data for the glutamate (E) mutations in the KEFTA scan (i.e. glutamate scan) show that position(s) P4-P6, P8-P9, P12-P13, p16-p19and / or P23were suitable for introducing glutamate residues to lower the pl to provide soluble peptides around physiological pH, as glutamate mutations in these positions had very little effect hOxlR and hOx2R potency (see results summarized in Fig. 2).

[0130] Table 1, SEQ ID NO: 1-28, supports the findings of the random forest model created for the glutamate scan. As can be seen P4-P6, P8-P9, P12-P13, p16-p19and P23are preferred positions for introducing glutamate residues to lower the isoelectric point of the peptides without (or with minimum) adverse effect on hOxlR and hOx2R potency compared to P7, P11, P14 15, P20-22, and P24-28. This effect on potency is also illustrated in the in the prior art peptide SEQ ID NO: 29 (i.e. Peptide 20 in WO2024 / 040245), wherein the pl is lowered by introducing three glutamate residues in three preferred positions (SEQ ID NO: 30) versus three non-preferred positions (SEQ ID NO: 31). EXAMPLE 2 - LIPIDATION SCAN

[0131] A lipidation scan was conducted to identify optimal positions for introducing a half-life extending group in Orexin B, without adversely affecting hOxlR and hOx2R potency. The Orexin B scaffold, having a Nle substitution in position P28, was used as reference. A library containing 190 peptides were designed and synthesized, wherein each of the positions Px-P28were mutated into a lysine covalently connected to a HLE group selected from C18DA-yGlu-, C18DA-yGlu-Ahx-, C18DA-yGlu- OEG-OEG-, from C20DA-yGlu-, C20DA-yGlu-Ahx-, C20DA-yGlu-OEG-OEG-, and Ac- (referred to as "none" in Fig. 3).

[0132] The ECso values on hOxlR and hOx2R were determined for the 190 peptides. The results are summarized in Fig. 3.

[0133] Conclusion: As can be seen, the selected positions P1-?19and P23were found to be most suitable for introducing an HLE group. Compared to position p20-p22and P24-P28, these selected positions resulted in up to approximately a 10-fold higher hOxlR and hOx2R potency. Table 2: SEQ ID NO: 33-61 supports the results summarized in Fig. 3. As can be seen, the lipidation in any of the positions P1’19and P23could be performed with only minor effect on hOxlR and hOx2R potency.

[0134] EXAMPLE 3 - CHEMICAL STABILITY

[0135] A library of 190 peptides containing one or more mutations in positions P8, P9, P16, P17, P20and / or P21were designed and synthesized to examine the extent of deamidation of the glutamines (Q) in positions P8and P16and the asparagine (N) in position P20in an attempt to improve the chemical stability of the Orexin B scaffold. The library was constructed based on the reference SEQ ID NO: 107 (i.e. lipidated Orexin B having acylated N-terminal, 3xGlu and Nle). The results are summarised in Fig. 4.

[0136] Deamidation was determined for the 190 peptides and SHAP values calculated from a random forest model, where percent deamidation was fitted to the mutations in P8, P9, P16, P17, P20and / or P21. SHAP values were used to determine the level of contribution of the different mutations on the endpoints on hOxlR and hOx2R potency (Breiman, L. (2001), Random Forests, Machine Learning 45(1), 5-32.; Lundberg, S. M., & Lee, S. I. (2017). A unified approach to interpreting model predictions. Advances in neural information processing systems, 30.). Substitutions with positive SHAP values increased the endpoint (i.e. increased deamidation), while negative SHAP values decreased the endpoint (i.e. decreased potency or deamidation). The results are summarized in Fig. 4. As can be seen from Fig. 4, position P20was found to be suitable for decreasing deamidation, by introducing an alanine (A), whereas the other examined positions had little effect on deamidation, with the exception of glycine (G) in position P21which resulted in increased deamidation as expected.

[0137] Table 3 supports the results from the random forest model summarized in Fig. 4. As can be seen, decreased deamidation was observed in the matched molecular pairs by substituting the asparagine (N) in the reference (SEQ ID NO:62) with an alanine (A) (SEQ ID NO:66) or glutamine (Q) (SEQ ID NO:65) in position P20. The mutation only resulted a minor decrease in hOxlR and hOx2R potency. Furthermore, as can be seen, the substitution of glutamine (Q) with an alanine (A) in positions P8(SEQ ID NO:63) or P16(SEQ ID NO:64) did not result in decreased deamidation as shown in the random forest model.

[0138] EXAMPLE 4 - FIBRILLATION

[0139] The inventors hypothesized that the inherent conformation of the Orexin B analogous could be stabilized, to lower the fibrillation tendency, by introducing 2-Aminoisobutyric acid (Aib) mutations. Thus, an Aib scan was performed in an attempt to minimize the fibrillation tendency of the peptides. A library containing 190 peptides were designed and synthesized, wherein each of the positions X1- X23was substituted with one or more Aib residues. The library was constructed based on the references SEQ ID NO: 66 and 67 (i.e. Orexin B having acylated N-terminal, 3xGlu and Nle). The ECso values on hOxlR and hOx2R were determined, as well as fibril formation, and SHAP values calculated from a random forest model, where ECso values were fitted to the peptide amino acid sequences. SHAP values were used to determine the level of contribution of each Aib substitution on the endpoints on hOxlR, hOx2R and fibril formation (Breiman, L. (2001), Random Forests, Machine Learning 45(1), 5-32.; Lundberg, S. M., & Lee, S. I. (2017). A unified approach to interpreting model predictions. Advances in neural information processing systems, 30.). The contribution of each Aib substitution was determined as the difference in average SHAP value between the Aib substitution and the amino acid in the reference peptides SEQ ID NO: 66 and 67. Substitutions with positive SHAP values increased the endpoint (fibrillation), while negative SHAP values decreased the endpoint (fibrillation). The results are summarized in Fig. 5. As can be seen from Fig. 5, an Aib residue in any of the positions P17-P23were found to decrease fibrillation.

[0140] Table 4, SEQ ID NO: 66, 67, and 68-98, supports the results from the random forest model summarized in Fig. 5, wherein the physical stability of the peptides was improved by introduction of an Aib residue in any of the positions P17-P22. As can be seen from the matched molecular pairs, fibrillation was prevented by introduction of an Aib in position P19in SEQ ID NO:75 compared to SEQ ID NO:66 . Likewise, fibrillation was prevented in SEQ ID NO:76 compared to SEQ ID NO:67.

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] ITEMS

[0150] 1. A polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I),

[0151] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-G-I-L-T-X28

[0152] (I)

[0153] , wherein

[0154] X1is selected as R, E or K; X2is selected as S, E or K; X3is selected as G, E or K; X4is selected as P, E or K; X5is selected as P, E or K; X6is selected as G, E or K; X7is selected as L or K; X8is selected as Q, E or K; X9is selected as G, E or K; X10is selected as R or K; X11is selected as L or K; X12is selected as Q, E or K; X13is selected as R, E or K; X14is selected as L or K; X15is selected as L or K; X16is selected as Q, E, K or Aib; X17is selected as A, E, K or Aib; X18is selected as S, E, K or Aib; X19is selected as G, E, K or Aib; X20is selected as N, Q, A or Aib; X21is selected as H or Aib; X22is selected as A or Aib; X23is selected as A, E, K or Aib; X28is Nle, Cha or F; or a derivative thereof having one or two amino acid substitutions; wherein, in the polypeptide or the derivative thereof, the total number of E residues in Formula (I) is selected as 0, 1, 2, or 3; the total number of Aib residues in Formula (I) is selected as 0 or 1; and wherein only one of the positions Xx-X19or X23is selected as K, and wherein the K is lipidated, optionally through a linker.

[0155] 2. The polypeptide or a derivative thereof according to item 1, wherein the total number of E residues in Formula (I) is selected as 2 or 3; most preferably 3.

[0156] 3. The polypeptide or a derivative thereof according to any one of the preceding items, wherein X4, X9, and X23are selected as E.

[0157] 4. The polypeptide or a derivative thereof according to any one of the preceding items, wherein the Aib residue (if present) is present in position X19.

[0158] 5. The polypeptide or a derivative thereof according to any one of the preceding items, wherein the lysine (K) residue(s) is present in one of the positions X12, X14or X16.

[0159] 6. The polypeptide or a pharmaceutically acceptable salt thereof according to item 1 comprising the amino acid sequence of Formula (I), R-S-G-E-P-G-L-Q-E-R-L-X12-R-X14-L-X16-A-S-X19-X20-H-A-E-G-I-L-T-Nle

[0160] (I) wherein

[0161] X12is selected as Q, or K; X14is selected as L or K; X16is selected as Q, or K; X19is selected as G or Aib; X20is selected as N, Q or A; and wherein only one of the positions X12, X14, or X16is selected as K, and wherein the K is lipidated, optionally through a linker; or a derivative thereof having one or two amino acid substitutions, with the proviso that the substitutions are not present in positions X4, X9, X12, X14, X16, X19, X20, or X23.

[0162] 7. The polypeptide according to any one of the preceding items, wherein X20is selected as A or Q, preferably A.

[0163] 8. The polypeptide according to any one of the preceding items, wherein X12is selected as Q.

[0164] 9. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 comprising the amino acid sequence of Formula (I), R-S-G-E-P-G-L-Q-E-R-L-Q-R-X14-L-X16-A-S-X19-A-H-A-E-G-I-L-T-Nle

[0165] (I)

[0166] , wherein

[0167] X14is selected as L or K; X16is selected as Q, or K; X19is selected as G or Aib; and wherein only one of the positions X14or X16is selected as K, and wherein the K is lipidated, optionally through a linker; or a derivative thereof having one or two amino acid substitutions, with the proviso that the substitutions are not present in positions X4, X9, X12, X14, X16, X19, X20, or X23.

[0168] 10. The polypeptide according to any one of the preceding items, wherein the polypeptide is amidated (-CONH2) in the C-terminal.

[0169] 11. The polypeptide according to any one of the preceding items, wherein the polypeptide is acetylated (AcNH-) in the N-terminal.

[0170] 12. The polypeptide according to any one of the preceding items, wherein the substitution in the derivative is a substitution for a conservative amino acid. The polypeptide according to any one of the preceding items, wherein the K is lipidated with a lipid (and linker), selected from the list consisting of C18DA-yGlu-, C18DA-yGlu-yGlu-, C18DA- yGlu-yGlu-yGlu-, C18DA-yGlu-OEG-OEG-, C18DA-yGlu-yGlu-OEG-OEG-, C18DA-yGlu-yGlu- yGlu-OEG-OEG-, ClS-yGlu-yGlu-yGlu-yGlu-, Cie-yGlu-yGlu-yGlu-yGlu-, C18-yGlu-yGlu-yGlu- yGlu-OEG-OEG-, ClS-YGIu-YGIu-yGlu-yGlu-yGlu-, C18-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, (C18-YGIU- YGIU-OEG-OEG-)2K-OEG-OEG-, (C16-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, (C18-YGIU-YGIU- OEG-OEG-)2DAP-OEG-OEG-, (C16-YGIU-YGIU-OEG-OEG-)2DAP-OEG-OEG-, (C18-YGIU-YGIU- OEG-OEG-)2Orn-OEG-OEG-, (C16-YGIu-YGIu-OEG-OEG-)2Orn-OEG-OEG-, (C18-YGIU-YGIU-)2K- OEG-OEG-, (C16-YGIU-YGIU-)2K-OEG-OEG-, (C18-YGIU-YGIU-OEG-OEG-)2K-, (C16-YGIU-YGIU- OEG-OEG-)2K-. The polypeptide according to any one of the preceding items, wherein the K is lipidated with a lipid (and linker), selected from the list consisting of C18-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, or (C18-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, (C16- YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-.

Claims

CLAIMS1. A polypeptide or a pharmaceutically acceptable salt thereof comprising the amino acid sequence of Formula (I), Rl.S2.G3.p4.p5.G6.L7.Q8.G9.R10.Lll.Q12.R13.L14.L15.Q16.A17.s18.G19.N20.H21.A22.A23.G24.I25.L26.T27.N|e28(I) wherein the amino acid residue in two or three of the positions selected from P1-6, P8-9, P12-13, P16 19and P23is substituted with a glutamate (E) residue; the amino acid residue in one of the positions selected from P1 19and P23is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid residue in one of the positions selected from P17 23is substituted with an Aib residue; and optionally the amino acid residue in position P20(i.e. N) is substituted with a Q or A residue; or a derivative thereof having one or two amino acid substitutions, with the proviso that the one or two substitution (s) in the derivative is / are not present in position P20or in any position substituted with an E, K, or Aib residue.

2. The polypeptide according to claim 1, wherein the amino acid residue in position P20 (i.e. N) is substituted with a Q or an A residue.

3. The polypeptide according to any one of the preceding claims, wherein the amino acid residue in two or three of the positions selected from P4, P9, and P23is substituted with an E residue.

4. The polypeptide according to any one of the preceding claims, wherein the amino acid residue in one of the positions selected from P12, P14and P16is substituted with a lysine (K) residue.

5. The polypeptide according to any one of the preceding claims, wherein optionally the amino acid residue in position P19(i.e. G) is substituted with an Aib residue.

6. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1 comprising the amino acid sequence of Formula (I), R1.s2.G3.E4-p5.G6-L7-Q8-E9-R10-L11-Q12-R13-L14-L15-Q16-A17-S18-G19-A20-H21-A22-E23-G24-I25-L26-T27-Nle28(I), wherein the amino acid residue in one of the positions selected from P12, P14and P16is substituted with a lysine (K) residue, wherein the lysine residue is lipidated, optionally through a linker; optionally the amino acid in position P19(i.e. G) is substituted with an Aib residue; or a derivative thereof having one amino acid substitution, with the proviso that the substitution in the derivative is not present in position P4, P9, P20, P23or in a position substituted with a K, or Aib residue.

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

8. The polypeptide according to any one of the preceding claims, wherein the polypeptide is acetylated (AcNH-) in the N-terminal.

9. The polypeptide according to any one of the preceding claims, wherein the substitution(s) in the derivative is a substitution to a conservative amino acid.

10. The polypeptide according to any one of the preceding claims, wherein the polypeptide has a higher solubility than SEQ ID NO: 33 at pH 7.5.

11. The polypeptide according to any one of the preceding claims, wherein the polypeptide has an isoelectric point (pl) < 5.

12. The polypeptide according to any one of the preceding claims, wherein the hOxlR ECso ratio (hOxlR ECso-polypeptide) / (hOxlR ECso-Orexin B) is < 1, and / or the hOx2R EC50 ratio (hOx2R ECso-polypeptide) / (4 x hOx2R ECso-Orexin B) is < 1.

13. The polypeptide according to any one of the preceding claims, wherein the K is lipidated with a lipid (and linker) selected from the list consisting of C18DA-yGlu-, C18DA-yGlu-yGlu-, C18DA- yGlu-yGlu-yGlu-, C18DA-yGlu-OEG-OEG-, C18DA-yGlu-yGlu-OEG-OEG-, C18DA-yGlu-yGlu- yGlu-OEG-OEG-, ClS-yGlu-yGlu-yGlu-yGlu-, Cie-yGlu-yGlu-yGlu-yGlu-, C18-yGlu-yGlu-yGlu- yGlu-OEG-OEG-, ClS-YGIu-YGIu-yGlu-yGlu-yGlu-, C18-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, C16-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, (C18-YGIU- YGIU-OEG-OEG-)2K-OEG-OEG-, (C16-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, (C18-YGIU-YGIU- OEG-OEG-)2DAP-OEG-OEG-, (C16-YGIU-YGIU-OEG-OEG-)2DAP-OEG-OEG-, (C18-YGIU-YGIU- OEG-OEG-)2Orn-OEG-OEG-, (C16-YGIu-YGIu-OEG-OEG-)2Orn-OEG-OEG-, (C18-YGIU-YGIU-)2K-OEG-OEG-, (C16-YGIU-YGIU-)2K-OEG-OEG-, (C18-YGIU-YGIU-OEG-OEG-)2K-, (C16-YGIU-YGIU- OEG-OEG-)2K-.

14. The polypeptide according to any one of the preceding claims, wherein the K is lipidated with a lipid (and linker), selected from the list consisting of C18-YGIU-YGIU-YGIU-YGIU-YGIU-OEG- OEG-, C16-YGIU-YGIU-YGIU-YGIU-YGIU-OEG-OEG-, or (C18-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG- , (C16-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-, most preferably of C18-YGIU-YGIU-YGIU-YGIU-YGIU- OEG-OEG- or (C18-YGIU-YGIU-OEG-OEG-)2K-OEG-OEG-.

15. The polypeptide according to any one of the preceding claims, having the structure of SEQ ID NO: 75, 76, 77, 78, 79, 80, 123, 124, 128 or 129.

16. The polypeptide according to any one of the preceding claims, having the structure of SEQ ID NO: 77, 78, 124, or 129.

17. The polypeptide according to any one of the preceding claims for use as a medicament for the treatment of narcolepsy.

18. A pharmaceutical composition comprising a polypeptide according to any one of claims 1-16.

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