hcrhr2 selective polypeptides

Abstract

The present invention relates to UCN2 analogs with high solubility, improved potency, high chemical stability, and / or improved selectivity for CRHR2 over CRHR1.

Description

Technical Field

[0001] This invention relates to UCN2 analogs. Specifically, this invention relates to UCN2 analogs having improved potency, improved chemical stability, and / or high selectivity. Background Technology

[0002] Urotropic adrenocorticotropic hormone (UCN) is an endogenous peptide belonging to the urotropic adrenocorticotropic hormone (CRF) family. This family also includes corticotropin-releasing hormone (CRH), which is also known as corticotropin-releasing factor (CRF), urotropic adrenocorticotropic hormone, and sauvagine. The CRF peptide family acts through CRH receptors, namely corticotropin-releasing hormone receptor 1 (hCRHR1) and corticotropin-releasing hormone receptor 2 (hCRHR2), encoded by the hCRHR1 and hCRHR2 genes, respectively. hCRHR1 is expressed in the central nervous system (CNS), while hCRHR2 is also expressed in the CNS and several peripheral tissues. The highest concentration of CRH neurons is found in the paraventricular nucleus (PVN) of the hypothalamus.

[0003] There are three known endogenous urocortins: urocortin I (UCN1), urocortin II (UCN2), and urocortin III (UCN3). Despite their high sequence homology, these peptides bind differently to hCRHR1 and hCRHR2. UCN1 (and CRH) non-selectively activate both hCRHR1 and hCRHR2, while UCN2 and UCN3 are more selective for hCRHR2. UCN2 is a 38-amino acid neuropeptide with the amino acid sequence IVLSLDVPIGLLQILLEQARARAAREQATTNARILARV (SEQ ID NO: 1).

[0004] CRH peptides regulate neuroendocrine stress responses via the hypothalamic-pituitary-adrenal axis (HPA). However, these effects are believed to be mediated solely by activation of hCRHR1. This differs from CRH, where administration of peripheral UCN2 or UCN3 does not increase corticosterone secretion. Furthermore, UCN2 activation of hCRHR2 reduces food intake without inducing behavioral arousal or anxiety-producing effects, as observed in appetite suppression induced by activation of the brain's hCRHR1 signaling pathway. This underscores the potential to separate stress responses and beneficial cardiometabolic effects mediated by the CRH peptide family through the use of selective hCRHR2 agonists.

[0005] Urotropic gonadotropins acting on hCRHR2 have demonstrated significant and beneficial hemodynamic, endocrine, and renal effects in experimental models of heart failure, such as reduced cardiac preload and afterload, increased cardiac output and contractility, decreased blood pressure, increased vasodilation, and improved renal function (i.e., increased creatinine clearance). Clinical studies have shown that UCN2 and UCN3 have direct vasodilatory effects in both healthy volunteers and patients with heart failure. These findings support the potential use of urotropic gonadotropins in the treatment of cardiovascular or renal diseases.

[0006] Furthermore, urinary gonadotropins have recently attracted significant interest in the field of metabolic disorders. Obesity and its associated metabolic disorders, such as insulin resistance and type 2 diabetes (T2D), are major sources of morbidity and mortality and are reaching epidemic proportions. Diet and exercise have proven effective for these diseases; however, adherence to these interventions is often low, and additional treatments are clearly needed to alleviate metabolic dysfunction.

[0007] Intraventricular injection of UCN2 and UCN3 has been shown to suppress food intake. Furthermore, chronic subcutaneous delivery of PEGylated UCN2 has been demonstrated to reduce body weight, suppress food intake, improve glucose tolerance, and increase glucose uptake in skeletal muscle, while simultaneously improving body composition towards a healthier phenotype, namely reducing fat mass and increasing / maintaining lean mass. In addition to its beneficial effects on the regulation of food intake and body weight, UCN2 has also been reported to improve systemic glucose tolerance by improving skeletal muscle insulin sensitivity and increasing insulin-stimulated glucose uptake into skeletal muscle.

[0008] These findings suggest that urinary corticotropin can be used to treat cardiovascular diseases and metabolic disorders such as diabetes and obesity. In these cases, it has the potential to promote healthy and sustained weight loss by reducing adipose tissue mass while selectively targeting hCRHR2 to maintain / increase lean body mass, thereby avoiding potential side effects induced by hCRHR1-mediated HPA axis activation.

[0009] New evidence of the potential benefits of targeting hCRHR2 has led to the development of numerous analogues of UCN2. One such example is WO 2022 / 038179, which discloses analogues of UCN2 and their effects on systolic blood pressure, body weight, and body fat percentage. Other examples of UCN2 analogues can be found, for example, in WO 2023 / 285334, WO2018 / 013803, and WO2023 / 285347.

[0010] Despite the emergence of novel UCN2 analogs, there is still a need in the field for improved UCN2 analogs, particularly those that exhibit high selectivity, high potency, high solubility, and high physical and chemical stability at physiological pH compared to hCRHR1.

[0011] The present invention aims to provide peptides that function as selective hCRHR2 agonists, exhibiting high selectivity for hCRHR2, high solubility, increased chemical stability, and / or improved hCRHR2 potency compared to hCRHR1. Summary of the Invention

[0012] This invention relates to UCN2 analogs with high solubility, improved potency, high chemical stability, and high selectivity for hCRHR2 compared to hCRHR1. The invention is set forth in the claims. Attached Figure Description

[0013] Figure 1 It shows how to do it at position X respectively 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 33 X 35 X 36 X 37 X 39 X 40 or X 41 The effect of introducing a glutamate (E) residue on the potency of hCRHR2 and hCRHR1. A positive SHAP value indicates pEC on hCRHR2 or hCRHR1. 50 Increase. A negative SHAP value indicates pEC on hCRHR2 or hCRHR1. 50 reduce.

[0014] Figure 2A This demonstrates how introducing lipidated lysine (K) residues into a compound with X... 36 =E and X 40 =E in the UCN2 skeleton, position X 4 -X 41 The average hCRHR1-pEC obtained in each of (SEQ ID NO: 228) 50 and hCRHR2-pEC 50 .

[0015] Figure 2B This demonstrates how introducing lipid-modified lysine (K) residues with X... 36=E and X 40 =E's UCN2 skeleton at each position X 4 -X 41 The selectivity ratio (hCRHR1-EC) obtained by (SEQ ID NO: 228) 50 / hCRHR2-EC 50 ).

[0016] Figure 3 The average SHAP values ​​of the substitutions of different amino acid residues in the lipidated UCN2 analog SEQ ID NO: 3 are shown (Ref. 1).

[0017] Figure 4 The X in reference 1 (SEQ ID NO: 205) or reference 2 (SEQ ID NO: 124) is shown to be substituted with different amino acid residues. 20 Improved selectivity of bit acquisition.

[0018] Figure 5 The selected peptides (SEQ ID NO: 16 and 215) reduced the body weight of DIO mice (n = 9, mean ± SEM). Relative body weight relative to day 1 of administration (Study Day 1). Compared with the vector, p < 0.05; Compared with the vector, p < 0.001.

[0019] Figure 6 The mean arterial blood pressure in awake, freely moving healthy mice, measured by wireless telemetry, is shown. The mice were treated with (SEQ ID NO: 3) (n = 4-6 / group, mean ± SEM).

[0020] Figure 7 The heart rate of a conscious, freely moving healthy mouse, measured by radio telemetry, is shown. The mouse was treated with (SEQ ID NO: 3) (n = 4-6 / group, mean ± SEM).

[0021] Figure 8 The hemodynamic effects of treatment with (SEQ ID NO: 3) on awake, freely active, spontaneously hypertensive rats and mice are shown. A) Mean arterial blood pressure; B) Heart rate (n = 8 / group, mean ± SEM)

[0022] Figure 9A-F shows the hemodynamic and safety characteristics of SEQ ID NO: 3 after a single subcutaneous administration in conscious, freely moving healthy pigs using telemetry, following escalating dose regimens (0.03, 0.1, 0.3, and 1.0 mg / kg). A) Heart rate, B) Mean arterial blood pressure, C) Systolic, D) Diastolic, E) PR interval, ECG, F) QT interval; data are averages for each group of n = 4–8 animals.

[0023] Figure 10A -F shows the hemodynamic and safety characteristics of SEQ ID NO: 3 after weekly subcutaneous administration (0.3 mg / kg) in awake, freely moving, healthy pigs, measured using radio telemetry. A) Heart rate; B) Mean arterial blood pressure; C) Systolic; D) Diastolic; E) PR interval, ECG; F) QT interval; Data are mean ± SEM of n = 6–8 animals per group.

[0024] Figure 11A -D shows the hemodynamic effects of SEQ ID NO: 215. A) Contraction (n = 4 to 12, mean ± SEM). B) Coronary blood flow (n = 1 to 6, mean ± SEM). C) Velocity-pressure product (n = 1 to 10, mean ± SEM). D) Velocity-pressure product with contraction.

[0025] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0026] In this context, lipolysis refers to the covalent binding of lipids by the UCN2 agonist according to the invention via a linker / spacer. The lipids may be C18DA (octadecanoic acid) or C20DA (eicosanoic acid), optionally linked via a linker / spacer consisting of one or more covalently linked units commonly used in the art. Commonly used linkers / spacers may be, but are not limited to, those shown below. γ [E], [OEG], [eLys], [ACHC], or [AHX].

[0027]

[0028] Lipidification is often used to improve the pharmacokinetic characteristics of peptides by, for example, improving metabolic stability, reducing enzymatic degradation, and decreasing excretion and metabolism, all of which contribute to the in vivo half-life (t). 1 / 2 The UCN2 agonist according to the invention can be lipidated using various lipids (and linkers / spacers) depending on the desired half-life. Peptides can be lipidated, for example, at the lysine (K) residue illustrated herein. Preferably, at position X... 20 X 25X 28 X 29 X 32 X 33 or X 40 (Optimal Optimization X) 32 or X 33 Lipidification is performed at the lysine residue (K) of the compound. Preferably, the lipid (and linker / spacer) is selected from the list of the following compositions: C16DA (hexadecanoic acid), C18DA, C20DA, C18DA[ γ E]-、C18DA[ γ E][ γ E]-、C18DA[ γ E][OEG]-、C18DA[ γ E][OEG][OEG]-、C18DA[ γ E][ γ E][OEG][OEG]-、C18DA[ γ E][AHX]-、C18DA[ γ E][ γ E][AHX]-、C20DA[ γ E]-、C20DA[ γ E][ γ E]-、C20DA[ γ E][OEG]-、C20DA[ γ E][OEG][OEG]-、C20DA[ γ E][ γ E][OEG][OEG]-、C20DA[ γ E][AHX]-or C20DA[ γ E][ γ E][AHX]-. More preferably, lipids (and linkers) are selected from the list of the following compositions: C18DA[ γ E]-、C18DA[ γ E][ γ E]-、C18DA[ γ E][OEG]-、C18DA[ γ E][OEG][OEG]-、C18DA[ γ E][ γ E][OEG][OEG]-、C20DA[ γ E]-、C20DA[ γ E][ γ E]-、C20DA[ γ E][OEG]-、C20DA[ γ E][OEG][OEG]-or C20DA[ γE][ γ E][OEG][OEG]-. Most preferably, it is lipotropically converted to C20DA[ γ E][ γ E][OEG][OEG]-.

[0029] In this document, amino acids refer to natural amino acids (i.e., L-amino acids) unless otherwise stated. The abbreviation Aib refers to 2-aminoisobutyric acid. The abbreviation Nle refers to L-oroleucine ((2S)-2-aminohexanoic acid). MeI refers to N-methyl-L-isoleucine. Hyp refers to L-hydroxyproline ((2S,4R)-4-hydroxyproline). Cle refers to cycloleucine (1-aminocyclopentane-1-carboxylic acid). c4NHPro refers to cis-4-amino-L-proline ((2S,4S)-L-Pro(4-NH2)-OH). c4FPro refers to cis-4-fluoro-L-proline ((2S,4S)-L-Pro(4-NH2)-OH). In this context, substitution of derivatives is preferably a conservative substitution of the conserved amino acid. Conserved amino acid groups can be defined as: G, A, V, L, I, P (aliphatic or cyclic); S, C, T, M (containing hydroxyl or sulfur); F, Y, W (aromatic); H, K, R (basic); D, E, N, Q (acidic or amide).

[0030] In this document, it should be understood that all peptides according to the invention have a C-terminal carboxamide (-CONH2). In this context, it should be understood that the polypeptide may have a free amine (-NH2) at the N-terminus, may be N-acylated (-NHCOR) at the N-terminus by, for example, an acetyl (Ac) group, may be methylated (-NHCH3 or -N(CH3)2) at the N-terminus, or may be deaminated at the N-terminus. In the most preferred embodiment, the polypeptide is mono-N-methylated (-NHCH3) at the N-terminus.

[0031] EC 50 The value is used as a measure of agonist potency at corticosteroid receptor 1 (hCRHR1) and corticosteroid receptor 2 (hCRHR2). 50 The value is a measure of the concentration of a compound required to achieve half of its maximum activity in a specific assay. In this context, a selective hCRHR2 agonist should be understood as having at least 500 hCRHR1-EC. 50 / hCRHR2-EC 50 An agonist for the ratio. Preferably, the selectivity ratio is higher than that of natural UCN2 (selectivity ratio of 917, see Table 2). Therefore, preferably, when measured using the assays and conditions for purifying peptides described herein, hCRHR1-EC 50 / hCRHR2-EC 50The ratio is at least 1000, more preferably, hCRHR1-EC 50 / hCRHR2-EC 50 The ratio is at least 1500, or even more preferably, hCRHR1-EC 50 / hCRHR2-EC 50 The ratio is at least 2000, and more preferably, hCRHR1-EC 50 / hCRHR2-EC 50 The ratio is at least 2500, most preferably hCRHR1-EC 50 / hCRHR2-EC 50 The ratio must be at least 3000. Due to EC 50 The value can vary depending on the type of measurement performed or the specific measurement conditions; therefore, the EC value for UCN2 is given in this paper. 50 As an internal standard, hCRHR1-EC is used to compare differences between different assay runs or even between different assays. This is demonstrated in this paper. 50 / hCRHR2-EC 50 The ratio can be greater than 5000. hCRHR1-EC 50 / hCRHR2-EC 50 The ratio is also interchangeably called selectivity.

[0032] Furthermore, the selective hCRHR2 agonist of the present invention has at least 10 hCRHR1 potency relative to natural UCN2 [(hCRHR1-EC]]. 50 ) Peptide / Purified Peptide (hCRHR1-EC) 50 [UCN2], for example at least 15, for example at least 20, when measuring purified peptides using the assays and conditions described herein.

[0033] The UCN2 agonist of the present invention may be in the form of pharmaceutically acceptable salts and / or solvates. Therefore, pharmaceutically acceptable salts are defined as any salt commonly used in peptide formulations. Such salts include both acidic addition salts and basic salts, and examples can be found, for example, in Remington's Pharmaceutical Sciences, 17th edition. Detailed Implementation

[0034] This invention relates to UCN2 analogs exhibiting high solubility, improved potency, high chemical stability, and high selectivity for hCRHR2 compared to hCRHR1. More specifically, this invention relates to the discovery of UCN2 analogs by introducing at least two glutamate residues (E) into X. 21 X 22 X 24 X 25 X 26 X27 X 28 X 33 X 35 X 36 X 39 or X 40 The invention relates to the discovery that, in the X position, a UCN2 analog with high solubility is obtained without major adverse effects on the potency and selectivity of hCRHR2 (see Example 1). The invention also relates to the discovery that, in X 20 X 25 X 27 X 28 X 29 X 32 X 33 or X 40 Lipidification at position X provides the highest selectivity ratio of hCRHR2 relative to hCRHR1 (see Example 2). The invention also relates to the discovery that by […] at position X 33 or X 35 Certain amino acid residues at the X site can yield UCN2 analogs with improved chemical stability (see Example 3). Furthermore, this invention relates to methods using X... 6 X 10 X 11 X 12 and / or X 38 Introducing certain amino acids, especially X. 38 The presence of an ortholeucine (Nle) residue at position X enables the production of a UCN2 analog with improved hCRHR2 efficacy (see Example 4). Finally, this invention relates to the discovery that: the position X... 20 X 21 X 26 X 30 and / or X 41 Certain amino acids at a certain position can improve the selectivity ratio, especially when X 38 When an Nle residue is present to enhance the efficacy.

[0035] UCN2 analogs with high solubility and high selectivity for hCRHR2

[0036] As shown in Example 1 of this article, amino acid position X 21 X 22 X 24 X 25 X 26 X 27 X 28 X 33 X 35 X 36 X 39 and X 40The position identified as suitable for introducing glutamate residues (E) to improve peptide solubility. Furthermore, as shown in Example 2 of this document, amino acid position X... 20 X 25 X 28 X 29 X 32 X 33 and X 40 It was identified as a suitable location for introducing lipidated lysine (K) residues to alter the PK properties of the peptide while providing the highest selectivity ratio of hCRHR2 relative to hCRHR1.

[0037] Therefore, in a first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0038] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q, E, or L; X 22 Choose A or E; X 24 Choose A or E; X 25 Choose R, E, or K; X 26 Choose A, E, or L; X 27 Choose A, E, or K; X 28 Choose R, E, or K; X 29 Choose E or K; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 36 Choose R or E; X 38 Choose L or Nle; X 39 Choose A or E; X 40 Choose R, E, or K; X 41 Choose V or I; and where X 20 X 25 X 27 X 28 X 29 X32 X 33 or X 40 Only one of them is selected as K, and said K is lipid-modified, optionally lipid-modified via a linker / spacer; and further wherein X 21 X 22 X 24 X 25 X 26 X 27 X 28 X 33 X 35 X 36 X 39 and X 40 At least two of them should be selected as E.

[0039] Highly preferred, X 22 X 33 X 36 and X 40 At least two of them are selected as E to improve the solubility of the peptide.

[0040] Therefore, in a preferred embodiment of the first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0041] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 22 Choose A or E; X 25 Choose R or K; X 26 Choose A or L; X 27 Choose A or K; X 28 Choose R or K; X 29 Choose E or K; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 36 Choose R or E; X 38 Choose L or Nle; X40 Choose R, E, or K; X 41 Choose V or I; and where X 20 X 25 X 27 X 28 X 29 X 32 X 33 or X 40 Only one of them is selected as K, and said K is lipid-modified, optionally lipid-modified via a linker / spacer; and X is selected as X. 22 X 33 X 36 and X 40 At least two of them should be selected as E.

[0042] In a preferred embodiment of the first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0043] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 22 Choose A or E; X 26 Choose A or L; X 29 Choose E or K; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 36 Choose R or E; X 38 Choose L or Nle; X 40 Choose R, E, or K; X 41 Choose V or I; and where X 20 X 29 X 32 X 33 or X 40 Only one of them is selected as K, and said K is optionally lipid-modified by a linker / spacer; and at least two of them are X 22 X33 X 36 and X 40 Choose E.

[0044] In the most preferred embodiment, X 36 and X 40 It was selected as E to increase the solubility of the peptide.

[0045] Therefore, in a more preferred embodiment of the first aspect, the present invention relates to a polypeptide comprising the structure of formula (I) or a pharmaceutically acceptable salt thereof:

[0046] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 29 Choose E or K; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 38 Choose L or Nle; X 41 Choose V or I; and where X 20 X 29 X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer.

[0047] All amino acid positions X 20 X 25 X 28 X 29 X 32 X 33 and X 40 It was identified as a suitable site for introducing lipid-modified lysine residues to alter the PK properties of the peptide while providing high selectivity for hCRHR2. However, X 32 or X 33 It is best used as a lipidation site.

[0048] Therefore, in a more preferred embodiment of the first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0049] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 38 Choose L or Nle; X 41 Choose V or I; and where X 32 or X 33 Only one is selected as K, and wherein said K is optionally lipidated by a linker / spacer.

[0050] In yet another preferred embodiment of the first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0051] Where X 4 Choose I, MeI, or P; X 20 Choose E, A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Choose Q or L; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 38 Choose L or Nle; X 41 It is V or I; and where X is... 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer.

[0052] In a highly preferred embodiment of the first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0053] Where X 4 Choose I, MeI, or P; X 20 Choose E, A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Choose Q or L; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35 Choose A, L, I, or V; X 38 Choose L or Nle; X 41 It is V or I; and X 32 K is selected, and wherein K is optionally lipidized by a linker / spacer.

[0054] UCN2 analogs with improved chemical stability

[0055] Position X 34 These were identified as chemically unstable residues in the UCN2 backbone. As shown in Example 3 of this paper, in X 35 The amino acid alanine (A) at position X is replaced by amino acids Aib, L, I, or V, or at position X... 33 The substitution of the amino acid threonine (T) at a certain position with amino acids E, K (Lip), or Aib significantly improves chemical stability, with no effect on efficacy or only a slight loss of efficacy.

[0056] Therefore, in a second aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0057] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q, E, or L; X 22 Choose A or E; X 24 Choose A or E; X 25 Choose R, E, or K; X 26 Choose A, E, or L; X 27Choose A, E, or K; X 28 Choose R, E, or K; X 29 Choose E or K; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35 Choose A, E, Aib, L, I, or V; X 36 Choose R or E; X 38 Choose L or Nle; X 39 Choose A or E; X 40 Choose R, E, or K; X 41 Choose V or I; and where X 20 X 25 X 27 X 28 X 29 X 32 X 33 or X 40 Only one of them is selected as K, and wherein said K is optionally lipid-modified by a linker / spacer; and further wherein X 21 X 22 X 24 X 25 X 26 X 27 X 28 X 33 X 35 X 36 X 39 and X 40 At least two of them are selected as E; and further, when X 33 When X is T 35 Choose Aib, L, I, or V, or when X 35 When X is A 33 The choice is E, K, or Aib, wherein the K is optionally lipidized via a linker / spacer.

[0058] In a preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0059] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X21 Choose Q or L; X 26 Choose A or L; X 29 Choose E or K; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35 Choose A, Aib, L, I, or V; X 38 Choose L or Nle; X 41 Choose V or I; and where X 20 X 29 X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer; and further wherein when X 33 When X is T, 35 Choose Aib, L, I, or V, or when X 35 When X is A, 33 The choice is E, K, or Aib, wherein the K is optionally lipidized via a linker / spacer.

[0060] In a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0061] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35 Choose A, Aib, L, I, or V; X 38 Choose L or Nle; X 41 Choose V or I; and where X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer; and further wherein when X 33 When X is T, 35 Choose Aib, L, I, or V, or one of them when X 35When X is A, 33 The choice is E, K, or Aib, wherein the K is optionally lipidized via a linker / spacer.

[0062] In a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0063] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 35 Choose Aib, L, I, or V; X 38 Choose L or Nle; X 41 Choose V or I; and where X 32 K is selected, and wherein K is optionally lipidized by a linker / spacer.

[0064] In yet another preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising a structure of formula (I):

[0065] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose E, K, or Aib; X 38 Choose L or Nle; X 41 Choose V or I; and where X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer.

[0066] In another preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0067] Where X 4 Choose I, MeI, or P; X 20 Choose E, A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Choose Q or L; X 35 Choose Aib, L, I, or V; X 38 Choose L or Nle; X 41 Choose V or I; and where X 32 K is selected, and K is optionally lipidized via a linker / spacer.

[0068] In yet another preferred embodiment of the first aspect, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0069] Where X 4 Choose I, MeI, or P; X 20 Choose E, A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Choose Q or L; X 32 Choose T or K; X 33 Choose E, K, or Aib; X 38 Choose L or Nle; X 41 It is V or I; and where X is... 32 or X 33 Only one of them is selected as K, and K is optionally lipidized by a linker / spacer.

[0070] Optimal Optimum X 33 =T and X 35 =Aib, L, I or V, with Aib being the best choice to ensure high chemical stability.

[0071] UCN2 analogues with enhanced potency

[0072] As shown in Example 4 of this article, at position X 6 X 10 X 11 X 12 and / or X 38 Certain amino acid residues at X have a positive effect (i.e., increase) the hCRHR2 potency. Specifically, at X... 38 The presence of ortholeucine (Nle) residues at the site leads to improved hCRHR2 efficacy.

[0073] Therefore, in a third aspect, the present invention relates to a polypeptide having improved hCRHR2 potency or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0074] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q, E, or L; X 22 Choose A or E; X 24 Choose A or E; X 25 Choose R, E, or K; X 26 Choose A, E, or L; X 27 Choose A, E, or K; X 28 Choose R, E, or K; X 29 Choose E or K; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 36 Choose R or E; X 39 Choose A or E; X 40 Choose R, E, or K; X 41 Choose V or I; and where X 20 X 25 X 27 X 28 X 29 X 32 X 33 or X 40 Only one of them is selected as K, and said K is lipid-modified, optionally lipid-modified via a linker / spacer; and further wherein X 21 X 22 X 24 X 25 X 26 X 27 X 28 X 33 X 35 X36 X 39 and X 40 At least two of them should be selected as E.

[0075] In a preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, comprising the structure shown in formula (I):

[0076] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 41 Choose V or I; and where X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer.

[0077] In a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0078] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35Choose A, Aib, L, I, or V; X 41 It is V or I; and where X is... 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer; and further wherein when X 33 When it is T, X 35 Choose Aib, L, I, or V, or one of them when X 35 When it is A, X 33 The choice is E, K, or Aib, wherein the K is optionally lipidized via a linker / spacer.

[0079] In X 38 In some highly preferred implementations of Nle, by selecting X 6 For T; X 10 For Cle; X 11 For Hyp; and / or X 12 Using I or L can further enhance effectiveness.

[0080] In another preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0081] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 41 Choose V or I; and where X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer.

[0082] UCN2 analogs with improved potency and high selectivity

[0083] As shown in this article, at position X38 The addition of the ortholeucine (Nle) residue resulted in improved hCRHR2 efficacy, but simultaneously reduced selectivity for hCRHR2 (see Example 4, Table 3). Figure 3 As shown, at position X 20 X 21 X 26 X 30 and / or X 41 Certain amino acid residues were found to reduce hCRHR1 efficiency while hCRHR2 efficiency remained unchanged. These positions (i.e., X...) 20 X 21 X 26 X 30 and / or X 41 It can be used when introducing potency-improving substitutions, such as Nle at position X. 38 High selectivity is restored at that time. For example... Figure 4 As shown, at position X 20 At this site, amino acid residues A, F, G, H, I, K, L, N, Q, R, S, T, or V were found to reduce hCRHR1 efficacy without impairing hCRHR2 efficacy (see Example 5). Figure 4 ).like Figure 3 As shown, at position X 21 Leucine (L) at position X; 26 Leucine (L) at position X; 30 Arginine (R) at position X, and arginine at position X 41 Isoleucine (I) at the site was also found to reduce hCRHR1 efficacy without impairing hCRHR2 efficacy.

[0084] In a highly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure of formula (I):

[0085] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 41 Choose V or I; and where X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer, and wherein at least one of X 20 X 21 X 26 X 30 or X 41 Choose from the following: X 20 Choose A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Selected as L; X 26 Selected as L; X 30 Choose R; or X 41 Select I.

[0086] In a highly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0087] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35 Choose A, Aib, L, I, or V; X 41 Choose V or I; and where X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer; and further wherein when X 33 When X is T, 35 Choose Aib, L, I, or V, or one of them when X 35 When X is A, 33Selected as E, K, or Aib, wherein K is optionally lipotropic via a linker / spacer; and further wherein at least X 20 X 21 X 26 X 30 or X 41 One of the options is: X 20 Choose A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Selected as L; X 26 Selected as L; X 30 Choose R; or X 41 Select I.

[0088] More preferably, use position X 20 or X 41 To improve selectivity, and X 21 X 26 and X 30 It was selected as a natural amino acid in UCN2.

[0089] Therefore, in a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0090] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 41 It is V or I; and where X is... 32 or X 33 Only one of them is selected as K, and wherein K is optionally lipidized by a linker / spacer, and further wherein X 20 or X 41 One of the options is as follows: X 20 Choose A, F, G, H, I, K, L, N, Q, R, S, T, or V; or X 41 Select I.

[0091] In yet another preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising a structure of formula (I):

[0092] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35 Choose A, Aib, L, I, or V; X 41 It is V or I; and where X is... 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidated via a linker / spacer; and further wherein when X 33 When it is T, X 35 Choose Aib, L, I, or V, or when X 35 When it is A, X 33 Selected as E, K, or Aib, wherein K is optionally lipid-modified via a linker / spacer; and further wherein X 20 or X 41 One of them is as follows: X 20 Choose A, F, G, H, I, K, L, N, Q, R, S, T, or V; or X 41 Select I.

[0093] Most preferably, at position X, where the enhancing effect exists, Nle... 38 Use position X in the case of 20 To restore selectivity, and most preferably, X 41 V is selected.

[0094] Therefore, in another preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0095] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I, X 20Choose A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Choose Q or L; X 26 Choose A or L; X 30 Choose Q or R; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose from A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; and where X 32 or X 33 Only one of them is selected as K, and K is optionally lipidized by a linker / spacer.

[0096] In a more preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0097] Where X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose L or I; X 20 Choose A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35 Choose A, Aib, L, I, or V; and X 32 or X 33 Only one of them is selected as K, and wherein said K is optionally lipidized by a linker / spacer; and further wherein when X 33 When X is T, 35 Choose Aib, L, I, or V, or one of them when X 35 When X is A, 33 The choice is E, K, or Aib, wherein the K is optionally lipidized via a linker / spacer.

[0098] In any of the foregoing aspects and implementation schemes, unless otherwise stated or shown, X 4 Preferably, it is MeI or P, and most preferably X. 4 It is MeI. In any of the above aspects and embodiments, unless otherwise shown, X 6 Preferably, L is selected. In any of the above aspects and embodiments, unless otherwise shown, X 10Preferably, V is selected. In any of the above embodiments, if not shown, then X 11 P is preferably selected. In any of the above embodiments, unless otherwise shown, X is preferably selected. 12 Option I is selected. In any of the above aspects and implementation schemes, unless otherwise shown, it is preferable when X... 38 When X = Nle, 20 Choose N, F, G, K, Q, S, or T; the optimal choice is when X 38 When X = Nle, 20 Choose S. When X 38 When it is L, X is preferred. 20 Choose E, N, F, G, K, Q, S, or T; the most preferred option is X. 20 Choose E or S. In any of the above aspects and implementations, unless otherwise shown, X 21 Q is preferably selected. In any of the above aspects and embodiments, unless otherwise shown, X... 22 Preferably, option A is selected. In any of the above aspects and embodiments, unless otherwise shown, X... 24 Preferably, option A is selected. In any of the foregoing aspects and embodiments, unless otherwise shown, X... 25 Preferably, R is selected. In any of the above aspects and embodiments, unless otherwise shown, X 26 Preferably, option A is selected. In any of the above aspects and embodiments, unless otherwise shown, option X is preferred. 27 Choose A. In any of the above aspects and implementation schemes, if not already shown, then X 28 R is preferably selected. In any of the above aspects and embodiments, unless otherwise shown, X 29 Preferably, E is selected. In any of the above aspects and embodiments, if not shown, X is preferred. 30 Option Q is preferred. In any of the above aspects and embodiments, unless otherwise shown, X is preferred. 38 Select Nle. In any of the above aspects and implementation schemes, if not already shown, then when X... 33 When it is T, X is the preferred choice. 35 As Aib. In any of the above aspects and implementations, unless otherwise shown, when X 35 When it is A, X 33 Preferably, Aib or K is selected, wherein lipolysis can be performed via a linker / spacer. In any of the above aspects and embodiments, unless otherwise shown, X 39 Preferably, option A is selected. In any of the above aspects and embodiments, if not shown, then X... 41Preferably, V is selected. In any of the above aspects and embodiments, unless otherwise shown, X... 36 and X 40 Preferably selected from E.

[0099] Therefore, in a particularly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising the structure shown in formula (I):

[0100] Where X 4 Choose MeI or P; X 20 Choose from N, F, G, K, Q, S, or T, with X being the most preferred. 20 Selected as S; X 32 Choose T or K; X 33 Choose T, E, K, or Aib; X 35 Choose A, Aib, L, I, or V; and X 32 or X 33 One of them is selected as K, and K is optionally lipidated via a linker / spacer; and further wherein when X 33 When X is T, 35 Choose Aib, L, I, or V, or when X 35 When X is A, 33 Choose E, K, or Aib, wherein K is optionally lipid-modified via a linker / spacer.

[0101] In another particularly preferred embodiment, the present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising a structure of formula (I):

[0102] Where X 4 Choose MeI or P; X 20 Choose from N, F, G, K, Q, S, or T, with X being the most preferred. 20 Selected as S; X 33 Choose T or Aib; X 35 Choose A or Aib; and further, when X 33 When X is T, 35 Choose Aib, or when X 35 When X is A, 33 Selected as Aib; This indicates that the lipid is covalently bound to the ε-amino group of the lysine side chain via an optional linker / spacer.

[0103] In a more particular preferred embodiment, the peptide is selected from the following list: [MeI]VLSLDVPIGLLQILLSQARARAAREQA[ ]TN[Aib]EI[Nle]AEV(NH2)(SEQ IDNO: 215), PVLSLDVPIGLLQILLSQARARAAREQA[ ][Aib]NAEI[Nle]AEV(NH2)(SEQ ID NO:238), [MeI]VLSLDVPIGLLQILLEQARARAAREQAT[ ]NAEILAEV(NH2)(SEQ ID NO: 6), or [MeI]VLSLDVPIGLLQILLEQARARAAREQA[ ]TN[Aib]EILAEV(NH2)(SEQ ID NO:16), in This indicates that the lipid is covalently bound to the ε-amino group of the lysine side chain via a linker / spacer.

[0104] The lipid (and linker / spacer) can be selected from any lipid (and linker / spacer) known in the art. Most preferably, the lipid (and linker / spacer) is C20DA[γE][γE][OEG][OEG]-.

[0105] In a most preferred embodiment, the peptide is [MeI]VLSLDVPIGLLQILLSQARARAAREQA[ ]TN[Aib]EI[Nle]AEV(NH2), where This indicates the covalent linkage of lipids to the ε-amino group of the lysine side chain, wherein the lipid (and linker / spacer) is C20DA[γE][γE][OEG][OEG]-.

[0106] It will be apparent to those skilled in the art that the optimal positions described herein for improving solubility without compromising potency or selectivity (i.e., at least two glutamate residues (E) entering position X) are... 21 X 22 X 24 X 25 X 26 X 27 X 28 X 33 X 35 X 36 X 39 or X 40 ); Modifying PK while ensuring the highest possible selectivity of hCRHR2 relative to hCRHR1 (i.e., lipid-mediated lysine at position X) 20 X25 X 27 X 28 X 29 X 32 X 33 or X 40 One of them); the position that improves chemical stability (i.e., position X) 33 or X 35 (specific amino acids in); the position that enhances hCRHR2 efficacy (i.e., position X) 6 X 10 X 11 X 12 and / or X 38 Specific amino acid residues, especially position X 38 (e.g., the ortholeucine (Nle) residue); and the position that improves the selectivity ratio (i.e., position X). 20 X 21 X 26 X 30 and / or X 41 Specific amino acid residues, particularly at position X 38 In the presence of potency-enhancing Nle residues, these are preferably combined to address multiple issues and provide an optimal peptide. Similarly, these findings can be applied to improve existing peptide UCN2 analogs, such as those described in WO 2023 / 285334, WO 2018 / 013803, and WO 2023 / 285347.

[0107] Experimental Section

[0108] solid-phase peptide synthesis General program

[0109] Peptides were synthesized on a Tentagel S RAM at a loading of 0.23–0.25 mmol / g using a SyroII fully automated parallel peptide synthesizer equipped with a heating block (MultiSynTech GmbH, Germany) (Rapp polymer GmbH, Germany). Nα-Fmoc deprotection was performed in two stages: first, the resin was treated with 40% piperidine / DMF (0.2 M HOBt (1-hydroxybenzotriazole)) at 45°C for 3 min, followed by treatment with 20% piperidine / DMF (0.1 M HOBt) at 75°C for 7 min. Except for Asp, Cys, and His residues, Nα-Fmoc deprotection was carried out at room temperature. α-Fmoc deprotection was performed by treatment with 40% piperidine / DMF (0.2M HOBt) for 3 minutes, followed by treatment with 20% piperidine / DMF (0.1M HOBt) for 15 minutes. The coupling chemical was a DMF solution of DIC (N,N'-diisopropylcarbodiimide) / Oxyma (ethyl cyano(hydroxyimino)acetate), using a 0.5M DMF solution of amino acids and a 5-fold molar excess. Standard Fmoc-protected amino acids were used. Coupling conditions were single or double coupling at 75°C for 15 minutes. Double coupling was performed at 50°C for 15 minutes, except for His and Cys residues. Additionally, amino acids coupled after Aib were double-coupled. Fmoc-amino acids, except for His, were dissolved in 0.5M DMF containing 0.5M Oxyma, while His was dissolved in NMP. The resin was in N... α -Fmoc was deprotected and washed 5 times with DMF, and then washed 3 times with DMF after coupling.

[0110] For the N-terminal lipoylation example, lipoylation is performed on a resin in the final step of peptide synthesis. The N-terminal lipoylated peptide optionally contains linker residues such as [γE], [OEG], [OEG]-[OEG], etc. These linker residues are introduced by extending the peptide prior to coupling with fatty diacids such as tert-butyl-protected fatty diacids such as tBu-C18-diacid, tBu-C20-diacid, etc., by linking Fmoc-OEG-OH, Fmoc-OEG-OEG, and / or Fmoc-Glu-OtBu to the N-terminal amino acid. The linker group residues are double- or triple-linked using standard conditions. Fatty acid double bonds are coupled using 2 equivalents of building blocks.

[0111] For other lipoylation examples, the Boc-protected amino acid was introduced as an N-terminal residue, and the lipoylation site was introduced as an orthogonally protected lysine, here Lys(Mtt). The Mtt group (4-methyltriphenylmethyl) was removed by treating the resin with 75% HFIP (1,1,1,3,3,3-hexafluoropropane-2-ol) in DCM (dichloromethane) with 5% TIPS for 10 minutes. This process was repeated 3 times. The resin was washed with DCM containing 10% DIPEA, followed by 3 DMF washes.

[0112] The lipotropic peptide optionally contains a linker residue. The linker residue is coupled to the ε-amino group of an unprotected lysine residue, and then a fatty acid, such as a tert-butyl-protected fatty dicarboxylic acid (e.g., tBu-C18-dicarboxylic acid, tBu-C20-dicarboxylic acid), is coupled. The linker residue is coupled using standard conditions with either doubly or triple coupling. The fatty acid double bonds are coupled using two equivalents of building blocks.

[0113] Used for library peptide synthesis: Following synthesis, the resin was washed with DCM and dried. The peptide was then lysed from the resin using a mixture of TFA (trifluoroacetic acid) / TES (triethylsilane) / DODT (2,2'-(ethylenedioxy)dithiol) / water (93 / 2.5 / 2.5 / 2.0) at 40°C for 45–60 min. Precipitation was then carried out with 3 volumes of cold diethyl ether, followed by further washing with diethyl ether and drying. The peptide was characterized by LC-MS (Waters, Denmark) and quantified by LC-CAD (ThermoFisher Scientific, Denmark). Finally, the peptide was freeze-dried using a Telstar benchtop freeze dryer.

[0114] Peptide to be purified: After synthesis, the resin was washed with DCM and dried. The peptide was then cleaved from the resin using a TFA (trifluoroacetic acid) / TES (triethylsilane) / DODT (2,2'-(ethylenedioxy)dithiol) / water mixture (93 / 2.5 / 2.5 / 2.0) at 40°C for 120 min, followed by precipitation with cold diethyl ether, further washing with diethyl ether, and air drying. The peptide was dissolved in acetonitrile / water and purified by reversed-phase HPLC using a Waters preparative HPLC system (with a C18 column (Reprosil Gold 200A, 5 μm, 40 mm × 250 mm)), a preparative pump (Waters 2545), a UV / VIS detector (Waters 2489), and a Waters Collector III. The mobile phase consisted of a gradient of buffer A (0.1% TFA in H₂O) and buffer B (0.1% TFA in acetonitrile), run at a flow rate of 40 mL / min at room temperature. The relevant fractions were analyzed, collected, and freeze-dried. Finally, the peptides were freeze-dried using a Telstar benchtop freeze dryer.

[0115] Peptide purity and mass were determined by analytical RP-HPLC-MS on a Waters Acquity HPLC system equipped with a 3100 mass spectrometer detector, using an ACQUITY UPLC peptide CSH C18 column (Waters, ACQUITY UPLC peptide CSH, C18, 130 Å, 1.7 μm, 2.1 mm × 100 mm). Elution was performed using a gradient of buffer A (0.3% TFA in H2O) and buffer B (0.3% TFA in acetonitrile) at 40 °C (using a gradient of 40–60% B over 14 min, or 50–70% B over 14 min).

[0116] Determining the potency of hCRHR1 General program

[0117] HTRF (CisBio) cAMP Assay: This assay technique is fully described in the CisBio HTRF cAMP Assay Kit Handbook (#62AM4PEC). In short, time-resolved fluorescence technology is used to measure cAMP. This technique is based on the use of anti-cAMP antibodies labeled with cryptic compounds and d... 2 - Competitive immunoassay for labeled cAMP. In the absence of cellular cAMP, anti-cAMP cavitation compound conjugates can access cAMP-d2 conjugates, and energy (FRET) can be transferred from the cavitation compound to d2.

[0118] Cells stably expressing human corticotropin-releasing hormone receptor 1 (CHO-K1 cells, Eurofins / DiscoverXCat#95-0047C2 hCRHR1 stable monoclonal cell line) were immediately thawed from frozen cell libraries before assays. A 384-well assay format (Corning, #4513) with a total assay volume of 20 μL was used, and cells were incubated with peptide agonists (2000 cells / well) for 30 minutes at room temperature using DPBS (Sigma, #D8537) containing 0.5 mM IBMX (Sigma, #I5879) and 0.05% casein (Sigma, #C4765-10 ml) as stimulation buffer. Add HTRF detection reagent and incubate on a plate shaker for 1 hour (2400 rpm), then detect the signals at 620 and 665 nm on a ClarioStar (BMG Labtech, Ortenberg, Germany) plate reader (raw count: 665 / 620 ratio).

[0119] For peptide libraries: The concentration-response ratio of the compounds was evaluated using five concentrations of agonist peptides, and the EC was calculated using nonlinear regression by employing sigmoid concentration-response curves with variable slopes. 50 value.

[0120] For purified peptides: The concentration-response ratios of agonist peptides at 11 concentrations were evaluated, and the EC was calculated using nonlinear regression with sigmoid concentration-response curves of varying slopes. 50 value.

[0121] Determining the potency of hCRHR2 General program

[0122] HTRF (CisBio) cAMP Assay: This assay technique is fully described in the CisBio HTRF cAMP Assay Kit manual (#62AM4PEC). In short, it uses time-resolved fluorescence technology to measure cAMP. This technique is based on the use of anti-cAMP antibodies labeled with cryptic compounds and d... 2 - Competitive immunoassay for labeled cAMP. In the absence of cellular cAMP, anti-cAMP cavitation compound conjugates can access cAMP-d2 conjugates and energy (FRET) can be transferred from the cavitation compound to d2.

[0123] Cells stably expressing human corticotropin-releasing hormone receptor 2 (CHO-K1 cells, Eurofins / DiscoverX, Cat#95-0048C2 hCRHR2 stable monoclonal cell line) were immediately thawed from frozen cell libraries before assays. A 384-well assay format (Corning, #4513) with a total assay volume of 20 μL was used, and cells were incubated with peptide agonists (2000 cells / well) for 30 minutes at room temperature using DPBS (Sigma, #D8537) containing 0.5 mM IBMX (Sigma, #I5879) and 0.05% casein (Sigma, #C4765-10 ml) as stimulation buffer. Add HTRF detection reagent and incubate on a plate shaker for 1 hour (2400 rpm), then detect the signals at 620 and 665 nm on a ClarioStar (BMG Labtech, Ortenberg, Germany) plate reader (raw count: 665 / 620 ratio).

[0124] For peptide libraries: The concentration-response ratio of the compounds was evaluated using five concentrations of agonist peptides, and the EC was calculated using nonlinear regression of sigmoid concentration-response curves with variable slopes. 50 value.

[0125] For purified peptides: The concentration-response ratios of the compounds were evaluated using 11 concentrations of agonist peptides, and the EC was calculated by nonlinear regression of the concentration-response ratios using sigmoid curves with variable slopes. 50 value.

[0126] Measurement library Turbidity of peptides and formation of fibrils General program

[0127] The peptide was dissolved in buffer (50 mM sodium phosphate, pH 7.5) to a final concentration of 267 μM and incubated at room temperature for 1–2 hours. The sample was then aliquoted into two 80 μl replicates and mixed with thioflavin T (ThT) to a final concentration of 4 μM in a black 384-well plate (µ-clear, Greiner Bio-One). The plate was centrifuged at 2000 rpm for 2 minutes to remove air bubbles, sealed, and placed in a plate reader (CLARIOstar, BMG). First, the turbidity of the sample was measured at 600 nm as absorbance. Second, the plate reader temperature was set to 40 °C, and fluorescence was measured every 10 minutes for 72 hours by exciting ThT at 450 nm and measuring emission at 480 nm. Stress was applied to the sample by shaking the plate at 700 rpm (linearly) for five minutes before each measurement, and filament formation was determined as the average emission for each sample.

[0128] Assay for protofibrillation of purified peptides General program

[0129] Thioflavin T fibril formation assay: The peptide was dissolved in 50 mM phosphate buffer at pH 7.5 and incubated at room temperature for 2 hours on a rocking shaker to obtain a 267 μM solution. The sample was then aliquoted into three 80 μl / well portions and mixed with 2 μl / well of thioflavin T (ThT) into black-bottomed 384-well microplates (Greiner #781096) to a final concentration of 4 μM ThT. The plates were inserted into a CLARIOstar Plus microplate reader (BMG Labtech), and fibril formation was measured as the increase in fluorescence emission over 96 hours at 40°C, measured at 480 nm under 450 nm excitation, with a 5-minute rest and a 5-minute linear oscillation cycle (at 700 rpm).

[0130] Determination of the solubility of purified peptides General program

[0131] Solubility was tested using the following carrier: 100 mM phosphate buffer, which resulted in target pH values ​​of 6.5, 7.0, and 7.5. Samples were dissolved directly in a 0.45 μm unit (Whatman) of the Mini-UniPrep needle-free filter. For the desired concentration, such as 4000 μM (approximately 20 mg / mL), 1600 nmol of peptide was dissolved in 400 μL of carrier. Samples were incubated on a shaking table at room temperature for at least 1 hour. pH was measured and adjusted. Samples were then placed on the shaking table for another hour, followed by a second pH measurement and adjustment.

[0132] A visual inspection was performed and the results recorded before the filter was introduced. The concentration of peptides in the filtrate was determined using CAD (with electrosol detection), with two measurements performed for each sample. The measured concentrations are expressed in μM. Additionally, the pH of the filtrate was measured and reported. Peptides with measured concentrations within ±20% of the target concentration were considered completely soluble.

[0133] Measurement library A general procedure for the chemical stability of peptides

[0134] Chemical stability was tested using 267 μM peptides in the following carriers: 50 mM phosphate buffer at pH 7.5 and 8.0. The dissolved samples were incubated on a swing stage at room temperature for at least 2 hours. The purity of the major peak at time zero (T0) was determined using reversed-phase chromatography coupled with high-resolution mass spectrometry. Samples were incubated at 40 °C for 7 days (T7) and 14 days (T14). Samples were analyzed using reversed-phase chromatography coupled with high-resolution mass spectrometry.

[0135] Reversed-phase chromatography coupled with mass spectrometry was performed on an Acquity BEH column (1.7 μm, C) connected to a Thermo Exploris 120 high-resolution mass spectrometer. 18 Mass spectrometry was performed on a Thermo Vanquish tandem UHPLC system (130 Å, 50 × 2.1 mm, Waters, 186002350). Analysis was performed by gradient elution using buffer A (0.1% formic acid in H₂O solution) and buffer B (0.1% formic acid in acetonitrile solution), with a column temperature of 40 °C and a gradient from 5% to 70% B over 7.5 min at a flow rate of 0.4 mL / min. The mass spectrometer was run in positive mode with data-dependent acquisition (Top 4) at MS1 resolution of 60,000 and MS2 resolution of 30,000. Peptides were fragmented using a normalized collision energy of 25%. Peptides were quantified by extractive ion chromatography (EIC) based on their highest isotopic peaks. Intact peptides were determined by integral chromatograms, which showed the area of ​​the main peak relative to the total peak area. Purity loss was calculated as the difference between time points T0 and T7 and / or T0 and T14.

[0136] Determination of the chemical stability of purified peptides General program

[0137] Chemical stability was tested using a peptide at a concentration of approximately 1.0 mg / mL in the following carrier: 50 mM phosphate buffer, pH 7.5. The dissolved sample was incubated on a rocking stand at room temperature for at least 2 hours. The purity of the major peak at time zero (T0) was determined using reversed-phase chromatography. The sample was incubated at 40 °C for 14 days (T14) and 28 days (T28). The sample was analyzed using reversed-phase chromatography and size exclusion chromatography.

[0138] Reversed-phase chromatography was performed on a Thermo Dionex Ultimate UHPLC system (UV detection wavelength 215 nm) equipped with a Kinetex column (1.7 μm, C8, 100 Å, 150 x 2.1 mm (00F-4499-AN)). Gradient elution was performed using buffer A (0.1% TFA in 95:5 H₂O:acetonitrile) and buffer B (0.1% TFA in 5:95 H₂O:acetonitrile), analyzed at a column temperature of 50 °C (using 25-55% B in a gradient over 40 min). The flow rate was 0.5 mL / min. The purity of the main peak was determined by the integral chromatogram as the percentage of the main peak area relative to the total peak area. Purity loss was calculated as the difference between time points T0 and T14 and / or T0 and T28.

[0139] Size exclusion chromatography (SEC) was used for the analysis of high molecular weight products (HMWP, covalent dimers, trimers, etc.). SEC analysis was performed on a Thermo Dionex Ultimate UHPLC system equipped with a Waters BEH 125A column (1.7 μm, 300 mm × 4.6), with UV detection at 215 nm. Analysis was performed using isocratic elution with buffer A (0.1% TFA in 95:5 H₂O: acetonitrile) at a column temperature of 60 °C for 20 min, followed by analysis with 60% buffer B (0.1% TFA in 5:95 H₂O: acetonitrile). The flow rate was 0.3 mL / min.

[0140] The chromatogram was integrated as HMWP (all peaks eluted before the main peak), the main peak (assuming this is a monomeric peptide), and, if relevant, LMWP (low molecular weight product, all peaks eluted after the main peak). The amount of HMWP was reported as a percentage of the total area.

[0141] Dynamic light scattering (DLS) study on peptide oligomerization / particle formation

[0142] Samples were prepared by dissolving 800 nmol of each peptide in 400 μl of 50 mM phosphate at pH 8.0 to a concentration of 2000 μM (approximately 10 mg / mL). After complete dissolution on a shaker for 2 hours, the samples were adjusted to pH 7.5 and filtered through a 0.2 μm Whatman Anotop filter. The comparative product was the commercially available liraglutide drug product (Victoza). TM The samples were prepared using Novo Nordisk (pH 8.2) and a diluent of the drug product, adjusted to pH 6.7. These samples were filtered through a 0.02 μm Whatman Anotop filter.

[0143] Samples were transferred in three 30 μl aliquots to Aurora 384-well microtiter plates using pipettes, centrifuged at 2200 rpm for 2 minutes, and then sealed with Thermo sealing tape 235307. DLS measurements were performed using a Wyatt DynaPro plate reader III with the following parameters:

[0144] Prior to subsequent DLS measurements, the microtiter plates were incubated in a Grant-bio Thermo PHMP-4 shaker at 40°C and 700 rpm with continuous shaking. Data analysis was performed using Wyatt Dynamics software ver. 7.10.1.21.

[0145] PK in mice

[0146] Lean male NMRI / RjHan mice were obtained from JanVier (JanVier Labs, France) at 6 weeks of age. Animals were housed individually under a 12-hour light / 12-hour dark cycle, with lights turned off at 3 PM. Room temperature was maintained at 22°C ± 1°C, and humidity at 60% ± 20%. Animals had free access to standard rodent food (Altromin 1324, Brogaarden, Denmark) and tap water.

[0147] After a two-week acclimatization period, animals were randomly assigned to treatment groups (n = 9 per group) based on body weight. Animals were administered one peptide IV and another peptide SC (15 or 50 nmol / kg). In a sparse sampling design, plasma samples were collected at 0.17, 1, 3, 6, 24, 30, 48, 72, and 96 hours post-administration for pharmacokinetic (PK) analysis, with 3 mice per group at each time point. Plasma samples were analyzed using LC-MS, and PK parameters were estimated by non-compartmental analysis (NCA).

[0148] PK in rats

[0149] Lean male Sprague-Dawley rats (RJHan: SD) were obtained from JanVier (JanVier Labs, France) at 7 weeks of age. Animals were housed in pairs until randomization, under a 12 / 12-hour light / dark cycle, with lights turned off at 3 PM. Room temperature was maintained at 22°C ± 1°C, and humidity at 60% ± 20%. Animals had free access to standard rodent food (Altromin 1324, Brogaarden, Denmark) and tap water.

[0150] After a two-week acclimatization period, animals were housed individually and randomly assigned to treatment groups (n = 3 per group) based on their body weight on Study Day 3. On Study Day 1, animals were administered a combination of two peptides at 50 nmol / kg IV and SC. Plasma samples were collected at 0.17 h, 1 h, 3 h, 6 h, 24 h, 30 h, 48 h, 72 h, and 96 h post-administration for PK analysis. Plasma samples were analyzed using LC-MSMS, and PK parameters were estimated by non-compartmental analysis (NCA).

[0151] PK among miniature pigs

[0152] Pharmacokinetic studies were conducted on male Göttingen miniature pigs from Ellegaard GöttingenMinipigs A / S. Animals were acclimatized for at least 7 days prior to the study. Prior to the study, central venous catheters for blood collection were surgically implanted in the pigs. Animals were placed under a 12-hour light-12-hour dark cycle with free access to drinking water and fed a commercial standard maintenance diet twice daily.

[0153] The test compound was administered either as a kit or as a single compound. It was administered via a single intravenous injection or short infusion (5–10 minutes) through an implanted catheter, or via subcutaneous injection. Subcutaneous injection was performed using a needle plug in the middle of the neck between the ear and shoulder blade, allowing a needle insertion of 0.5 cm. Plasma concentration-time curves were obtained from each animal using 12–16 sampling points. For example, blood samples were collected at the following times: 0 hours after administration (before administration), 0.17, 0.33, 0.5, 1, 2, 5, 6, 24, 48, 72, 92, 168, 240, and 336 hours.

[0154] Approximately 2 mL of blood was drawn from each animal's central venous catheter at each sampling point. Blood samples were collected using K2 or K3 EDTA tubes. The blood was placed on ice for up to 30 minutes (10 min, 4°C, 2000 x g) prior to centrifugation. Approximately 200 μL of plasma sample was transferred to Micronic tubes and stored at -20°C or lower.

[0155] Pharmacokinetic parameters were calculated using non-compartmental analysis (NCA) of individual plasma concentration-time curves in animals. The log-linear trapezoidal method was used to estimate AUC and AUMC. Terminal T 1 / 2 It was determined to be ln(2) / λ Z , where λ Z It is the first-order rate constant determined by the logarithmic linear portion of the terminal part of the curve.

[0156] Plasma concentrations were measured using electrospray ionization and multiple reaction monitoring (MRM) via LC-MSMS. Calibration standards and quality control (QC) samples were prepared using a sample-matched matrix. Protein precipitation was performed using 60 μL of methanol to extract 15 μL of calibration standards, QC, and study samples, followed by the addition of 45 μL of ultrapure water. Samples were agitated (800 rpm) at room temperature for 5 min before centrifugation (2570 x g, 40 min, 4 °C), and the supernatant was transferred to a LoBind PCR plate. Samples were analyzed on a Thermo Triscent UHPLC system connected to a Sciex API 6500+ mass spectrometer. Samples were cleaned in-line using SPE on an HLB column (1 × 50 mm, Waters) before loading onto an Aeris peptide XB C18 column (3.6 μm, 100 Å, 2 × 50 mm, Phenomenex). The mobile phase consisted of acetonitrile and ultrapure H2O, both containing 0.1% v / v formic acid. The flow rate was 0.60 mL / min, and the column was kept at room temperature.

[0157] Impact on food intake

[0158] Male NMRI mice were obtained from JanVier (JanVier Labs, France) at 5 weeks of age. Animals were housed in groups of 4 mice per cage under a 12-hour dark-12-hour light cycle, with lights turned off at 1 p.m. Room temperature was maintained at 22°C ± 1°C, and humidity at 60% ± 20%. Animals had free access to standard rodent food (Altromin 1324, Brogaarden, Denmark) and tap water.

[0159] Animals were transferred to the HM-2 real-time food intake monitoring system (MBRose, Denmark) 5–7 days prior to the start of the study to acclimatize to experimental conditions. Since animals are uniquely identified by microchips, each individual animal was identified by its microchip when entering and leaving the food passage. Randomization of mice in each study group (n = 7–8) was based on body weight measured the day before the start of the study. Vector treatment groups were included in each experiment. Animals were fasted 6 hours before the start of the dark period. One hour before the dark period, animals received a single subcutaneous administration of the test peptide. Food intake was reported hourly for 72 hours. The percentage reduction in food intake was normalized to the mean food intake from the vector groups. Statistical significance was assessed using one-way ANOVA (Dunnett's multiple comparison test). P < 0.05 was considered statistically significant.

[0160] Effects on body weight and body fat percentage in a diet-induced obesity (DIO) mouse model

[0161] Evaluate the ability of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 238, SEQ ID NO: 16 and SEQ ID NO: 215 to reduce body weight and body fat content in a diet-induced obesity (DIO) mouse model.

[0162] Male C57BL / 6JRj mice were acquired from JanVier (JanVier Labs, France) at 5 weeks of age. Animals were housed in groups during the induced obesity period, then individually for 2 weeks prior to the start of the study. They were placed under a 12-hour dark-12-hour light cycle, with lights turned off at 3 p.m. Room temperature was maintained at 22°C ± 1°C, and humidity at 60% ± 20%. Animals had free access to a high-fat diet (60% kcal fat, D12492, Research Diets) and tap water before the start of treatment and throughout the study period (weeks 22–23).

[0163] Animals were randomized based on lean mass (EchoMRI) and body weight. Animals were administered a single subcutaneous dose over 30–32 days. In Study 1, animals were administered the drug using either the carrier or SEQ ID NO: 3 (10, 30, 60, or 100 nmol / kg). In Study 2, animals were administered the drug using either the carrier or SEQ ID NO: 6 (10 or 30 nmol / kg), SEQ ID NO: 238 (10 or 30 nmol / kg), SEQ ID NO: 16 (10 or 30 nmol / kg), SEQ ID NO: 215 (10 or 30 nmol / kg), or SEQ ID NO: 3 (30 nmol / kg).

[0164] Weight and food intake were recorded daily until day 28, and body composition was assessed on day 26 using EchoMRI. At the end of the study, blood was collected for HbA1c analysis and the gastrocnemius and soleus muscles (M. soleus) were weighed.

[0165] Statistical significance was assessed using one-way ANOVA (Dunnett's multiple comparison test). P < 0.05 was considered statistically significant.

[0166] mouse telemetry

[0167] Blood pressure and heart rate were monitored in freely moving, awake mice using telemetry (PA-C10, Data Sciences). Briefly, analgesia was administered with mebendazole (100 mg / kg sc). For implantation of the telemetry transmitter, isoflurane was used for anesthesia, with an induction dose of 5% and a maintenance dose of 2%. The neck was opened at the sternum from the laryngeal region to the midline. A subcutaneous pouch for the transmitter housing was prepared. The right carotid artery was then bluntly exposed and clamped. The transmitter was pushed caudally into the carotid artery through the incision. The catheter was secured with a monofilament suture, Prolene, which also closed the incision. The container was also ligated to the cranial side, and the clamp was then removed. The transmitter housing was advanced along the skin into the prepared subcutaneous pouch on the opposite side of the catheter. Finally, the skin wound was closed. Postoperatively, antibiotics (Ursocyclin) were administered. TM 10. Serumwerk Bernburg AG, Germany; 400 mg / L drinking water for 10 days) and pain reliever (Rimadyl) TM (4 mg / kg subcutaneous injection, continued for 3 days) was administered postoperatively.

[0168] For arterial blood pressure measurements, animals were kept in a suitable room for at least one day in a 12-hour diurnal cycle prior to the first measurement. Animals were administered subcutaneously once daily for 7 consecutive days the following substances: 1) placebo; 2) SEQ ID NO: 3, 10 nmol / kg; 3) SEQ ID NO: 3, 30 nmol / kg; or 4) SEQ ID NO: 3, 100 nmol / kg. In a crossover design, there were 2 animals per group per week (n = 6–8), with a 1-week washout period between treatments. Telemetry measurements were performed over 24 hours. Mean data (1-minute to 10-minute grid) are presented graphically.

[0169] For statistical analysis, five time points were calculated starting one hour after application and one hour after lights were turned off. All data were evaluated using a two-way ANOVA with the Dunnett test.

[0170] Remote sensing of rats

[0171] Heart rate and blood pressure were monitored in freely moving, awake animals using radiometry (DSI Data Science International, MN, USA). Transmitters were implanted in female adult hypertensive rats (SHR). Following abdominal wall scraping, an abdominal incision was made at the midline, and a fluid-filled sensor catheter was inserted upstream into the exposed descending aorta between the iliac bifurcation and the renal artery. Following DSI guidelines, the tip of the telemetry catheter was positioned directly posterior to the renal artery and secured with tissue adhesive. The transmitter body was fixed to the small intestinal wall prior to abdominal closure. A two-layer closure was used, with separate sutures to the peritoneum and muscle walls, followed by closure of the outer skin. The surgery was performed under sterile conditions. Postoperative protection against infection and pain included administration of antibiotics (Ursocyclin 10% pro inj., Serumwerk, s.c.) and a dose of analgesic (Rimadyl). TM 4 mg / kg s. c. (Pfizer, Germany). All animals were housed individually at an ambient temperature of 22°C–24°C under a 12-hour dark / 12-hour light cycle, on standard laboratory rat feed with free access to water.

[0172] All animals were treated for 7 days with a daily subcutaneous dose (10 or 30 nmol / kg) (1 ml / kg) of either the carrier or SEQ ID NO: 3. Drug administration occurred at 9:00 AM (=0 hours). For analysis, data were grouped to provide averages for half-hourly intervals.

[0173] For statistical analysis, the average value was calculated over 24 hours from the time of application. All data were evaluated using a two-way ANOVA (Dunnett's test).

[0174] pig telemetry

[0175] The PK / PD / safety properties of SEQ ID NO: 3 were evaluated in freely moving, awake, and healthy pigs using radio measurements.

[0176] In Study A, the effects of escalating dosing regimens of SEQ ID NO: 3 (0.03, 0.1, 0.3, and 1.0 mg / kg) were compared with placebo. The primary endpoints were blood pressure, heart rate, and ECG measurements.

[0177] In Study B, the effects of repeated administration of a single dose of SEQ ID NO: 3 (0.3 mg / kg) (once weekly for 4 weeks) were compared with placebo. The primary endpoints were blood pressure, heart rate, and ECG measurements.

[0178] Acute hemodynamic effects of selective peptides on anesthetized pigs

[0179] Göttingen miniature pigs were anesthetized with an anesthetic and fitted with instruments. Hemodynamics were continuously monitored during baseline measurements (0.5 h), carrier treatment (0.5 h), and at SEQ ID NO: 215 doses (10, 30, 100, and 300 μg / kg / min) with 0.5-hour increments each time. The results were compared with a similar setup using dobutamine (1, 3, 10, and 20 μg / kg / min) and a carrier (NaCl).

[0180] Rat myocardial ischemia model

[0181] Six-week-old male Wistar rats were obtained from JanVier (Janvier Labs, France). Animals were housed in pairs. During the study period, the animals had free access to food (RM1, SDS Dietex) and water.

[0182] Myocardial infarction (MI) was induced by chronic left anterior descending coronary artery (LAD) ligation performed on day zero. Sham-operated animals received the same protocol; after a left lateral sternal incision with the heart exposed, the rib cages were closed without passing through the LAD sutures.

[0183] Treatment was initiated one month after MI in order to assess the potential beneficial effects of the candidate compounds in preventing pathological progression.

[0184] Rats underwent transthoracic echocardiography (ECG) to non-invasively assess cardiac morphology and function. Echocardiography was performed using a digital ultrasound system (Vivid S60, GE Medical Systems) equipped with a 12 MHz phased array and 18 MHz linear array transducers. Standard B-mode and M-mode images of the heart were obtained via two-dimensional (2D) parasternal long axis views (PSLA). LV parameters were measured and calculated as averages over three consecutive cardiac cycles by a single, blinded, trained operator.

[0185] Four ECG examinations were performed on all animals in total. The first examination was performed 5 to 7 days post-surgery and served as a surgical control. The second ECG was performed one month after LAD ligation. Based on the second ECG, the included MI rats were randomly assigned to four homogeneous groups according to diastolic left ventricular diameter (LVIDd), terminal diastolic volume (TeleD), ejection fraction (EF), and fractional shortening (FS): Group 1 (sham surgery): n = 10; Group 2 (MI / carrier): n = 19; Group 3 (MI / atenolol-lisinopril-spironolactone (A+L+S) 1-1-10 mg / kg): n = 19; Group 4 (MI / test peptide 30 nmol / kg): n = 19; and Group 5 (MI / test peptide 100 nmol / kg): n = 19. The carrier and test peptide were administered daily via SC, and A+L+S was administered via drinking water.

[0186] The third and fourth ECGs were performed 2 and 3 months post-surgery and were used to assess cardiac remodeling and function following myocardial infarction (MI). The isovolumetric relaxation time (IVRT) and cardiac output (CO) of the fourth ECG were also assessed by Doppler echocardiography.

[0187] Prior to termination, dynamic blood flow measurements were performed under anesthesia using fluid-filled catheters (BLPR and TBM4m, WorldPrecision Instrument). At termination, the left soleus muscle was dissected and weighed.

[0188] Statistical analysis was performed using GraphPad 9 software. Parametric analysis was performed if the values ​​were normally distributed. Nonparametric analysis was performed if samples were taken from a non-normally distributed population. First, a t-test was used to assess the difference between the sham surgery group and the MI / solvent group. Then, a one-way ANOVA followed by an appropriate post-hoc test was used to compare the treated MI group with the MI / solvent group.

[0189] Subchronic IRI

[0190] Male C57BL / 6jRj mice were obtained from Jan Vier (Jan Vier Labs, France) at 10 weeks of age. Animals were housed individually in individually ventilated cages (IVC from Tecniplast, Typ I SL cages) with free access to water, food, and enrichment. Animals were allowed acclimatization for 7 days prior to the start of the study. After acclimatization, animals were randomly assigned to groups based on body weight: 1) sham-operated group; 2) uIRI-solvent; 3) uIRI-SEQ ID NO: 3, 30 nmol / kg; and 4) uIRI-SEQ ID NO: 3, 100 nmol / kg.

[0191] On study day 0, unilateral ischemia-reperfusion injury (uIRI, occlusion for 25 minutes followed by reperfusion) was performed in groups 2–4 (followed by unilateral nephrectomy (UNx) on study day 6). Animals received subcutaneous administration once daily from before the IRI procedure until day 7 of the study, where the animals were terminated. Plasma was collected here, and the remaining kidney was weighed and sampled for histological analysis. The kidneys were stained, analyzed, and quantified using the following markers: collagen I (col1a1), F4 / 80, and KIM-1.

[0192] Example 1: Exploration of glutamate scanning-solubility and hCRHR2 / hCRHR1 potency

[0193] To identify the optimal sites for improving solubility without adversely affecting the potency and selectivity of hCRHR2, a library of 190 peptides was synthesized, in which 1–6 glutamate residues were introduced compared to the lipidated form of native UCN2 (SEQ ID NO: 2).

[0194] Determining EC on hCRHR2 and hCRHR1 50 The value is calculated from the random forest model, where pEC is the SHAP value. 50 Values ​​were fitted to the peptide amino acid sequence. The Δ-mean SHAP value was used to determine the contribution level of each glutamate substitution to the efficacy of hCRHR2 and hCRHR1 relative to native UCN2 residues (Breiman, L. (2001), Random Forests, MachineLearning 45(1), 5-32.; Lundberg, SM, & Lee, SI (2017). A unified approach to interpreting model predictions. Advances in neural information processing systems, 30). Substitutions with positive Δ-mean SHAP values ​​increased the endpoint relative to native UCN2 residues, while negative Δ-mean SHAP values ​​decreased the endpoint relative to native UCN2 residues. Figure 1 The results are summarized. Location X 21 X 22 X 24 X 25 X 26 X 27 X 28 X 33 X 35 X 36 X 39 and X 40The position X was identified as a suitable location for introducing glutamate residues (E) to increase solubility without significantly adversely affecting the potency and selectivity of hCRHR2. Specifically, position X 22 X 33 X 36 X 39 and X 40 The location identified as the site for introducing glutamate residues to increase solubility while maintaining or improving hCRHR2 potency and selectivity. Location X 36 and X 40 It was identified as the optimal location for introducing glutamate residues to improve solubility and, consequently, the efficacy of hCRHR2.

[0195] Example 2: Lipidification Scan - Exploring Lipidification Sites and hCRHR2 / hCRHR1 Efficacy

[0196] To identify the optimal lipidation sites, in the presence of X 36 =E and X 40 Lipidification scanning was performed on the UCN2 backbone (SEQ ID NO: 228) of E, by measuring the lipidation at position X. 4 -X 41 Each lysine (K) residue is introduced with various lipidation methods. The following lipidation strategies are used: C18DA-γGlu, C18DA-γGlu-OEG-OEG, C18DA-γGlu-γGlu-OEG-OEG, C20DA-gGlu, C20DA-γGlu-OEG-OEG, or C20DA-γGlu-γGlu-OEG-OEG.

[0197] Figure 2A The average activity of each lipidation site for hCRHR1 and hCRHR2 under different lipidation strategies is shown. Aggregates in Figure 2A The lipidation site in the upper left corner provides the highest hCRHR2 potency and the lowest hCRHR1 potency. Figure 2B This demonstrates the selectivity ratio of each lipidation site for hCRHR2 relative to hCRHR1 (i.e., hCRHR1 EC50) under different lipidation strategies. 50 / hCRHR2 EC 50 The average value of ). Figure 2B Location X is displayed. 20 X 25 X 27 X 28 X 29 X 32 X 33 and X 40The optimal site for lipolysis was identified, providing the highest selectivity ratio of hCRHR2 relative to hCRHR1. Most preferably, X 32 or X 33 Used as a lipidation site.

[0198] Table 1 shows that in X 32 or X 33 The lipoylation at this location maintains high hCRHR2 potency and provides excellent selectivity for hCRHR2 compared to natural UCN2. Table 1 also shows that at the optimal position X... 36 and X 40 In order to increase the solubility of glutamate (E), it is necessary to maintain high efficacy against hCRHR2 and high selectivity for hCRHR2 compared to hCRHR1. 4 N-terminal monomethylation or X 4 The substitution of =P has a small effect on the effectiveness of hCRHR2, where X 4 N-terminal monomethylation, i.e., X 4 =MeI had the least effect on hCRHR2 potency. Furthermore, the data confirmed that the desired high solubility could be achieved by incorporating at least two glutamic acids into lipid-modified UCN2. No fibroblasting was observed in any of the peptides. Interestingly, peptides SEQ ID NO: 3 and SEQ ID NO: 4 exhibited higher selectivity ratios compared to prior art peptides SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 disclosed in WO 2018 / 013803A1 and WO 2023 / 285334A1. This indicates that, compared to prior art peptides, at position X... 36 and X 40 It has two glutamic acids at position X to improve solubility, and at position X 32 or X 33 The presence of lipidation to alter PK properties provides superior selectivity.

[0199] Table 1 also shows the site X used in WO 2022 / 038179A1. 15 (SEQ ID NO: 261 in Table 1) (This is site X) 12 Lipidification of ) provides poor hCRHR2 efficacy.

[0200]

[0201] Example 3: Chemical Stability - Identification of Sites and Substitutions that Improve Chemical Stability

[0202] Position X 34The residue was identified as chemically unstable in the UCN2 backbone. To improve the chemical stability of the peptide, a compound was synthesized in which the amino acid threonine (T), present in UCN2, is located at position X. 33 The amino acid alanine (A) present in UCN2 at position X 35 The substitutions are performed at the sites (see Table 2). The effects of each substituent relative to reference 1 (i.e., UCN2, X) are as follows. 32 =K(Lip), X 36 and X 40 =E) is shown.

[0203]

[0204] As shown in Table 2, at position X 35 The amino acid alanine (A) is substituted by amino acids L, I, V, or Aib, or at position X. 33 Substitution of the amino acid threonine (T) with E, Aib, or K (Lip) significantly improved chemical stability with no effect on potency or only a minor loss of potency compared to reference 1. Furthermore, all peptides maintained a higher selectivity ratio than native UCN2. Additionally, data indicate that substitution at position X... 33 Or position X 35 At this time, high physical stability (i.e., no fibrosis) and solubility are maintained.

[0205] Therefore, X is highly preferred. 35 L, I, V, or Aib are selected to provide UCN2 analogs with improved chemical stability. Alternatively, X is highly preferred. 33 Choose E, Aib, or K(Lip) to provide a UCN2 analog with improved chemical stability.

[0206] Example 4: Improvement of hCRHR2 efficacy

[0207] To identify sites that enhance hCRHR2 potency, a library of 1140 peptides was designed. This library was based on lipid-modified UCN2 analogs, see 1, SEQ ID NO: 3, and the goal was to improve potency by optimizing the following: (1) the hCRHR2 binding pocket and the site located at X... 6 X 10 X 11 and X 12 (2) Interactions of different amino acid residues at X; 20-27 The curved section in UCN2 at the location; or (3) by introducing in X 25-27 X 29-30 X 38 and X 41The hCRHR2-ECD:UCN2 interface was optimized by introducing different amino acid residues at each position. The tested amino acid residues studied at each position are summarized in... Figure 3 middle.

[0208] EC on hCRHR2 and hCRHR1 was measured for each library. 50 The value is calculated, and the SHAP value is determined based on the random forest model, where pEC 50 Values ​​were fitted to the peptide amino acid sequence. The Δ-mean SHAP value was used to determine the contribution of each amino acid substitution to hCRHR2 and hCRHR1 relative to the corresponding native UCN2 residues (Breiman, L. (2001), RandomForests, Machine Learning 45(1), 5-32.; Lundberg, SM, & Lee, SI (2017). A unified approach to interpreting model predictions. Advances in neural information processing systems, 30). Substitutions with positive Δ-mean SHAP values ​​increased the endpoint relative to native UCN2 residues, while negative Δ-mean SHAP values ​​decreased the endpoint relative to native UCN2 residues. The contributions of each amino acid are summarized in... Figure 3 middle.

[0209] Figure 3 The results showed that most of the tested amino acids resulted in either reduced or unchanged hCRHR2 potency compared to the native UCN2 residues, except for amino acid site X. 6 X 10 X 11 X 12 and X 38 In addition, these sites exhibit a positive effect (i.e., increase) in hCRHR2 pEC. 50 The substitutions are threonine (T), cyclic leucine (Cle), hydroxyproline (Hyp), leucine (L), and ortholeucine (Nle). However, these substitutions also resulted in hCRHR1 pEC. 50 The increase was greater than that of hCRHR2 pEC 50 Larger, thus reducing overall selectivity.

[0210] However, as Figure 3 As shown, in X 20 X 21 X 26 X 30 and X 41Some substitutions at certain locations were found to reduce hCRHR1 pEC. 50 And hCRHR2 pEC 50 No change. For position X 20 Asparagine (N), serine (S), and threonine (T) were all found to reduce hCRHR1 pEC. 50 Without harming hCRHR2 pEC 50 For X 21 Leucine (L) at position X 26 Leucine (L) at position X 30 Arginine (R) at position X, and its effect on X 41 Isoleucine (I) at position 1 was found to lower hCRHR1 pEC. 50 Without harming hCRHR2pEC 50 Therefore, when a substituent that enhances the effectiveness, such as Nle, is introduced at position X... 38 (See Example 5) Position X can be used 20 X 21 X 26 and / or X 41 To restore high selectivity.

[0211] Table 3 shows the X by replacing UCN2 with the amino acid ortholeucine (Nle). 38 The presence of leucine (L) at the site yields matching molecule pairs and improved potency. It can be seen that when X... 20 and X 41 When the position is a naturally occurring amino acid present in UCN2 (i.e., X) 20 =E and X 41 =V), the replacement of L with Nle improves the efficacy of hCRHR2 but reduces selectivity.

[0212]

[0213] Example 5: Improving hCRHR2 efficacy while maintaining high selectivity, chemical stability, and solubility

[0214] To determine the location of hCRHR2 X 20The ability to recover highly selective alternatives at position X was demonstrated in the synthesis of a library of 74 peptides. 32 Lipidification, at position X 36 and X 40 Replace with glutamic acid (E), and in X 33 or X 35 Position X in the chemically stable UCN2 analogue replaced by Aib (i.e., reference 1 or 2 in Table 4). 20 Eighteen amino acid substitutions were introduced.

[0215] EC on hCRHR2 and hCRHR1 was measured for each library. 50 The value is calculated, and the SHAP value is determined based on the random forest model, where pEC 50 Values ​​were fitted to the peptide amino acid sequence. The Δ-mean SHAP value was used to determine the contribution of each amino acid substitution to hCRHR2 and hCRHR1 relative to the corresponding native UCN2 residues (Breiman, L. (2001), RandomForests, Machine Learning 45(1), 5-32.; Lundberg, SM, & Lee, SI (1997). A unified approach to interpreting model predictions. Advances in neural information processing systems, 30). Substitutions with positive Δ-mean SHAP values ​​increased the endpoint relative to native UCN2 residues, while negative Δ-mean SHAP values ​​decreased the endpoint relative to native UCN2 residues. The contributions of each amino acid are summarized in... Figure 4 middle.

[0216] Figure 4 The results proved X 38 =Nle's power enhancement effect and when X 38 =Nle replaces position X 20 The selective enhancement effect. In X 20 The following amino acids have a negative effect on hCRHR1, but do not significantly adversely affect hCRHR2 pEC. 50 : A, F, G, H, I, K, L, N, Q, R, S, T, or V. Therefore, when X 38 When =Nle, the numerator can be replaced by A, F, G, H, I, K, L, N, Q, R, S, T, or V. 20 The glutamate (E) in hCRHR2 is used to increase the selectivity ratio of hCRHR2 to hCRHR1 (i.e., hCRHR1 EC). 50 / hCRHR2 EC 50 In a preferred embodiment, when X 38 =Nle When choosing X to improve effectiveness 20 As A, F, G, H, I, K, L, N, Q, R, S, T, or V to restore the high selectivity for hCRHR2. In a more preferred embodiment, when X 38 =Nle When choosing X to improve effectiveness 20 To restore the high selectivity for hCRHR2, N, F, G, K, Q, S, or T are used. In the most preferred embodiment, when X... 38 =Nle to improve effectiveness, choose X 20 As S.

[0217]

[0218] The results in Table 5 confirm the potency-enhancing effect of X38 = Nle and the selectivity-enhancing effect of substitution position X20 when X38 = Nle. Furthermore, the selective retention effect of substitution position X41 is also shown. The data further demonstrate that high chemical stability (i.e., obtained through X33 or X35), high physical stability (i.e., no fibrosis), and high solubility (obtained through X36 and X40) are preserved when these substituents are introduced.

[0219]

[0220] Example 6 - Extended biophysical characterization of selected compounds

[0221] Solubility of selected peptides at different pH values

[0222] The solubility of selected compounds with improved chemical stability was tested at different pH values ​​with and without preservatives, up to a maximum concentration of 20 mg / mL, with phenol used as the preservative. Compounds were dissolved to a nominal concentration of 4000 μM and analyzed as described above. Samples containing phenol contained 5.0 mg / mL of phenol. The measured concentrations of peptides in the solutions are shown in Table 6.

[0223]

[0224] In summary, all peptides are soluble up to 20 mg / mL in the pH range of 6.5–7.5. At pH 7.5, all peptides are soluble up to at least 20 mg / mL in the presence of 5.0 mg / mL phenol.

[0225] Chemical stability of the selected peptide

[0226] The chemical stability of the selected peptides was evaluated for 28 days at 40°C and different pH values. Samples were prepared and analyzed according to the method described, and the pH-adjusted samples were added to the target pH values ​​shown below. As a comparison, the commercially available semaglutide drug product (Ozempic) was used. TM Novo Nordisk was included in the sample group. The results are shown in Table 7 below.

[0227]

[0228] All peptides showed less than 10% chemical degradation after 28 days at 40°C. No HMWP formation was observed in any peptide.

[0229] Dynamic light scattering (DLS) measurement of particle size

[0230] Samples were prepared and measured according to the procedure described. The hydrodynamic radius (Rh) and associated polydispersity (PD) obtained by cumulative analysis (assuming a single substance) are shown in Table 8, representing the average of three replicate experiments. T0 represents the measurement at zero time after sample preparation; T5 represents the measurement results after five days of incubation as described above.

[0231]

[0232] In summary, during a five-day incubation period with continuous shaking at 40°C, SEQ ID NO: 6 and liraglutide formed larger particles and several species (multimodal distribution) at pH 6.7, while all other compounds showed no or only a slight increase in hydrodynamic radius, indicating high physical / colloidal stability during incubation.

[0233] Assessment of amyloid fibrillation using ThT assay

[0234] Dissolve the compound in 50 mM phosphate buffer to a concentration of 200 µM (approximately 1.0 mg / mL), following the above instructions. General procedure for determination of fibrilformation of peptides. The method described above was used for preparation and ThT detection. Additionally, the sample was adjusted to the pH value shown below. The compound was dissolved in a commercially available semaglutide drug product (Ozempic). TM Additional co-formulations of the compound with semaglutide were prepared at concentrations up to 200 μM; these co-formulations were adjusted to pH 7.4. Samples showing increased ThT fluorescence and thus no amyloid fibrillation were indicated as "yes," while samples showing no increase in ThT fluorescence and therefore no amyloid fibrillation were indicated as "no." The results are summarized in Table 9.

[0235]

[0236] In summary, none of the tested compounds formed amyloid fibrils in 50 mM phosphate at pH 7.0–8.0. All compounds could be co-formulated with semaglutide without immediate precipitation or the observation of amyloid fibril formation.

[0237] Example 7 - Potential Characterization of Selected Compounds

[0238] Different species

[0239] Using a universal procedure, the potency of selected peptides against different species (mice, rats, and pigs) was tested in transiently transfected CHO-K1 cells.

[0240] Table 10 shows the EC50 of selected peptides tested on CRH1 and 2 receptors in humans, mice, rats, and pigs using the transient transfection CHO-K1 cell system. 50 Values ​​(mean, n=3) (pcDNA3.1(+)-N or C-DYK, provided by Genscript; rCRHR2 NM_022714.1 (rat), rCRHR1 NM_030999.4 (rat), mCRHR2 NM_001288618.1 (mouse), mCRHR1 NM_007762.5 (mouse), pCRHR1 (pig), pCRHR2 (pig), hCRHR1 NM_004382.5 (human), hCRHR2a NM_001883.5 (human), hCRHR2b NM_001202475.1 (human)).

[0241]

[0242] The overall ranking of peptides from these four species was the same. EC50 between humans and the test species in transient transfection cell systems. 50 The difference in values ​​was less than 10-fold. The two isoforms of the human CRHR2 receptor (hCRHR2a and hCRHR2b) showed a high level of correlation (R0). 2 =0.81).

[0243] Example 8 - Pharmacokinetic Characterization of Selected Peptides

[0244] The pharmacokinetic properties of the selected peptides were evaluated in mice, rats, and pigs according to the general procedures described above. The results of these studies are shown in Tables 11-14 below.

[0245]

[0246] In summary, all evaluated peptides showed favorable pharmacokinetic profiles compatible with weekly administration in humans.

[0247] Example 9 - Effects of the selected peptide on acute food intake in mice

[0248] The selected compounds were tested according to a standard procedure for acute food intake in mice to determine the effect of a single subcutaneous dose on food intake in mice over 3 days. The results are shown in Table 15 below.

[0249]

[0250] Dunnett's test for one-way linearity compared to the vector group: p<0.05 p<0.01, p<0.001

[0251] In summary, all selected peptides showed a dose-dependent reduction in food intake compared to vector-treated animals.

[0252] Example 10: Assessment of body weight and body composition in DIO mice

[0253] The effects of the selected peptides on the body composition of DIO mice were assessed. Results from the two DIO mouse model studies are shown in Tables 16 and 17. Figure 5 middle.

[0254] Compared with animals treated with the vector, treatment with SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 238, SEQ ID NO: 16, and SEQ ID NO: 215 for 28 days resulted in a sustained dose-dependent decrease in relative body weight. In the group administered 30 nmol / kg, body weight decreased by approximately 20% of initial body weight.

[0255]

[0256] Data indicate a strong correlation between in vitro potency and in vivo effects. Figure 5SEQ ID NO: 215 is shown compared to GSEQ ID NO: 16, where SEQ ID NO: 215 provided a relatively higher weight loss at a dose of 10 nmol / kg. Significant weight loss after 4 weeks of treatment was associated with loss of adipose tissue mass, while absolute lean tissue mass was maintained. Consistent with MRI results, treatment with the selected peptide resulted in maintenance or increase in weight of the soleus and gastrocnemius muscles. Decreased blood HbA1c levels were observed in the group treated with the selected peptide, indicating improved glucose homeostasis.

[0257] In summary, treatment with the selected peptides resulted in a dose-dependent reduction in body weight and adipose tissue mass, while lean tissue mass was maintained.

[0258] Example 11: Remote sensing in mice

[0259] In mice treated with SEQ ID NO: 3, the results of blood pressure and heart rate assessments can be found in [the table / data]. Figure 6 and Table 18 and Figure 7 And in Table 19. Treatment with SEQ ID NO: 3 resulted in a transient, dose-dependent effect on mean arterial blood pressure and a permanent increase in heart rate.

[0260]

[0261] Example 12: Remote Sensing in Rats

[0262] The remote sensing results for rats are shown in Figure 8 And Tables 20 and 21. Treatment with SEQ ID NO: 3 resulted in a transient, dose-dependent decrease in blood pressure, accompanied by a transient, dose-dependent increase in heart rate (HR).

[0263]

[0264] Example 13: Remote Sensing of Pigs

[0265] Using an ascending dosing regimen (Study A, Figure 9A -F) or repeated single application (Study B, Figure 10A The hemodynamic effects of treatment with SEQ ID NO: 3 (-F) were assessed according to the general procedure described. Only animals exhibiting accurate phase signals were included in the analysis.

[0266] In Study A, treatment with SEQ ID NO: 3 resulted in a persistent positive inotropic effect, while left ventricular diastole was unaffected. Heart rate and mean arterial blood pressure were unaffected. A slight decrease in the PR interval was observed, but the QT interval was unaffected. No relevant effects on systemic or cardiac hemodynamics were observed by measuring left ventricular end-diastolic pressure, diastolic time constant, baroreceptor sensitivity, and heart rate variability. Similar results were observed in Study B. In summary, SEQ ID NO: 3 induced a persistent positive inotropic effect without safety-related findings.

[0267] Example 14: Acute effects of selected peptides on hemodynamics in anesthetized pigs

[0268] The acute effects of escalating doses of SEQ ID NO: 215 on hemodynamics in anesthetized pigs are shown in Figure 11A -D.

[0269] From the telemetry study, treatment with SEQ ID NO: 215 resulted in a positive inotropic effect ( Figure 11A ). This was associated with an increase in coronary blood flow ( Figure 11B ) and an increase in O2 consumption ( Figure 11C and 11D ), as seen with treatment with dobutamine.

[0270] In summary, SEQ ID NO: 215 demonstrated a strong positive inotropic effect.

[0271] Example 15: Rat myocardial ischemia model

[0272] The effect of SEQ ID NO: 3 on myocardial infarction was evaluated according to the general procedure. The results of 2 months of treatment are shown in Table 22.

[0273] Three months after MI, within 2 months after treatment induction, the MI-vehicle group showed left ventricular dysfunction with reduced ejection fraction and fractional shortening. In addition, stroke volume and cardiac output were significantly reduced in MI-vehicle animals compared to sham-operated animals. This was associated with a significant increase in the diastolic parameter - isovolumic relaxation time. Rats treated with SEQ ID NO: 3 showed improved cardiac function with significant increases in ejection fraction, fractional shortening, stroke volume, and cardiac output compared to MI animals treated with vehicle. In contrast, compared to MI animals treated with vehicle, the control treatment (A+L+S) only improved ejection fraction and fractional shortening.

[0274] Mean blood pressure and heart rate measured in anesthetized rats before termination were reduced in MI animals compared to sham-operated animals. Treatment did not affect these hemodynamic parameters.

[0275] At termination, the animals treated with SEQ ID NO: 3 were significantly heavier than the MI-carrier animals. This was associated with a significant increase in muscle mass.

[0276]

[0277] In summary, SEQ ID NO: 3 improved cardiac function in a rat model of myocardial infarction.

[0278] Example 16: Subchronic IRI in Mice

[0279] The results of treating the mouse uIRI model with SEQ ID NO: 3 are shown in Table 23. Compared with sham-operated animals, uIRI induction increased serum creatinine levels as well as renal tubular injury (KIM-1), inflammation (F4 / 80), and fibrosis (Col1a1). Compared with uIRI animals treated with the vector, treatment with SEQ ID NO: 3 (100 nmol / kg) reduced serum creatinine levels as well as renal fibrosis and inflammation.

[0280]

[0281] In summary, treatment with SEQ ID NO: 3 improved assessment parameters of renal function in the uIRI mouse model.

[0282] project

[0283] 1. A polypeptide or a pharmaceutically acceptable salt thereof, comprising the structure of formula (I):

[0284] in

[0285] X 4 Choose I, MeI, or P; X 6 Choose L or T; X 10 Choose Cle or V; X 11 Choose P or Hyp; X 12 Choose I or L; X 20 Choose from E, A, F, G, H, I, K, L, N, Q, R, S, T, D, P, W, Y, or V; X 21 Choose Q, E, or L; X 22 Choose A or E; X 24 Choose A or E; X 25 Choose R, E, or K; X 26 Choose A, E, or L; X 27 Choose A, E, or K; X 28 Choose R, E, or K; X 29 Choose E or K; X 30 Choose Q or R; X 32Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 36 Choose R or E; X 38 Choose L or Nle; X 39 Choose A or E; X 40 Choose R, E, or K; X 41 Choose either V or I; where X 20 X 25 X 27 X 28 X 29 X 32 X 33 or X 40 Only one of them is selected as K, and said K is lipid-modified, optionally lipid-modified via a linker / spacer; and wherein X 21 X 22 X 24 X 25 X 26 X 27 X 28 X 33 X 35 X 36 X 39 and X 40 At least two of them should be selected as E.

[0286] 2. The polypeptide or its pharmaceutically acceptable salt as described in Project 1, wherein X 33 Choose T, E, K, or Aib; X 35 Choose A, Aib, L, I, or V, and if X 33 If it is T, then X 35 Choose Aib, L, I, or V, or if X 35 If it is A, then X 33 Choose E, K, or Aib, where K is lipid-modified, optionally lipid-modified via a linker / spacer.

[0287] 3. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the foregoing items, wherein X 38 Selected as Nle.

[0288] 4. The polypeptide or its pharmaceutically acceptable salt as described in Project 3, wherein X 20 X 21 X 26 X 30 or X 41 One of the following options is: X20 Choose A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Selected as L; X 26 Selected as L; X 30 Choose R; or X 41 Select I.

[0289] 5. The polypeptide or its pharmaceutically acceptable salt as described in Project 3, wherein X 20 Choose from A, F, G, H, I, K, L, N, Q, R, S, T, or V, preferably X. 20 Choose from N, F, G, K, Q, S, or T, with X being the most preferred. 20 Choose S.

[0290] 6. The polypeptide or a pharmaceutically acceptable salt thereof as described in Project 5, wherein X 21 Choose Q; X 26 Choose A; X 30 Choose Q; and X 41 V is selected.

[0291] 7. A polypeptide or a pharmaceutically acceptable salt thereof according to any one of the foregoing items, wherein X 32 or X 33 Only one of them is selected as K, wherein K is lipid-modified, optionally lipid-modified via a linker / spacer; X 25 Choose R or E; X 27 Choose A or E; X 28 Choose R or E; X 29 Choose E; X 40 Choose R or E.

[0292] 8. A polypeptide or a pharmaceutically acceptable salt thereof according to any one of the foregoing items, wherein X 22 X 33 X 36 and X 40 At least two of them should be selected as E; X 21 Choose Q or L; X 24 Choose A; X 25 Select R; X 26 Choose A or L; X 27 Choose A; X 28 Choose R; and X 39 Choose A.

[0293] 9. A polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein X 36 and X 40 Choose E; X 21 Choose Q or L; X 22 Choose A; X 24 Choose A; X25 Select R; X 26 Choose A or L; X 27 Choose A; X 28 Choose R; and X 39 Choose A.

[0294] 10. A polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein X 4 Choose MeI or P; X 6 Selected as L; X 10 Selected as V; X 11 P was chosen as the first choice; and X was chosen as the second choice. 12 Select I.

[0295] 11. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein X29 is selected as E; and X30 is selected as Q.

[0296] 12. The polypeptide or a pharmaceutically acceptable salt thereof according to Project 1, wherein the polypeptide comprises the structure shown in formula (I): Where X 4 Choose MeI or P; X 20 Choose from N, F, G, K, Q, S, or T, with X being the most preferred. 20 Selected as S; X 33 Choose T or Aib; X 35 Choose A or Aib; and further, when X 33 When X is T, 35 Choose Aib, or when X 35 When X is A, 33 Selected as Aib; This indicates that the lipid is optionally covalently bound to the ε-amino group of the lysine side chain via a linker / spacer.

[0297] 13. The polypeptide or a pharmaceutically acceptable salt thereof according to Item 1, wherein the polypeptide comprises a sequence selected from the list of sequences comprising: [MeI]VLSLDVPIGLLQILLSQARARAAREQA[K ]TN[Aib]EI[Nle]AEV(NH2) (SEQ IDNO: 215), PVLSLDVPIGLLQILLSQARARAAREQA[K ][Aib]NAEI[Nle]AEV(NH2) (SEQ ID NO:238), [MeI]VLSLDVPIGLLQILLEQARARAAREQAT[K ]NAEILAEV(NH2) (SEQ ID NO: 6) or [MeI]VLSLDVPIGLLQILLEQARARAAREQA[K ]TN[Aib]EILAEV(NH2) (SEQ ID NO:16), wherein This indicates that the lipid is optionally covalently bonded to the ε-amino group of the lysine side chain via a linker / spacer.

[0298] 14. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein hCRHR1-EC 50 / hCRHR2-EC 50 The ratio must be at least 2000.

[0299] 15. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding items, used as a medicine.

[0300] 16. A polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence [MeI]VLSLDVPIGLLQILLSQARARAAREQA[ ]TN[Aib]EI[Nle]AEV(NH2) (SEQ ID NO: 215), wherein This indicates that the lipid is optionally covalently linked to the ε-amino group of the lysine side chain via a linker / spacer, or is a derivative having the sequence of SEQ ID NO: 215 and having one or two amino acid deviations.

[0301] 17. The polypeptide or a pharmaceutically acceptable salt thereof according to item 16, wherein the deviation is an amino acid substitution, preferably a conserved amino acid substitution.

[0302] 18. A polypeptide or a pharmaceutically acceptable salt thereof according to any one of items 16 to 17, wherein the deviation is not present in the group selected from X 32 (i.e. K) 32 ), X 35 (i.e., Aib) 35 ), X 36 (i.e. E) 36 ), X 38 (i.e. Nle) 38 ) and X 40 (i.e. E) 40 In any amino acid position in ).

[0303] 19. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein hCRHR1-EC 50 / hCRHR2-EC 50 The ratio must be at least 2000.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, comprising the structure shown in formula (I): in, X 4 Choose I, MeI, or P; X 20 Choose E, A, F, G, H, I, K, L, N, Q, R, S, T, or V; X 21 Choose Q or L; X 32 Choose T or K; X 33 Choose T, E, K, V, Y, W, S, P, F, L, I, H, G, Q, A, or Aib; X 35 Choose A, E, W, T, S, F, K, L, H, G, Q, D, N, R, Aib, L, I, or V; X 38 Choose L or Nle; X 41 It is V or I; And X 32 or X 33 Only one of them is selected as K, and K is lipid-modified, optionally lipid-modified via a linker / spacer.

2. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, wherein, X 33 Choose T, E, K, or Aib; X 35 Choose A, Aib, L, I, or V, and if X 33 If it is T, then X 35 Choose Aib, L, I, or V, or if X 35 If it is A, then X 33 Choose E, K, or Aib, where K is lipid-modified, optionally lipid-modified via a linker / spacer.

3. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein, X 38 Selected as Nle.

4. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 3, wherein, X 20 Choose A, F, G, H, I, K, L, N, Q, R, S, T, or V.

5. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 3, wherein, X 20 Choose N, F, G, K, Q, S, or T.

6. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 3, wherein, X 20 Choose S.

7. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein, X 20 Choose S or E.

8. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein, X 21 Choose Q.

9. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein, X 33 Choose T.

10. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein, X 41 V is selected.

11. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein X 4 Choose MeI or P.

12. The polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the structure shown in formula (I): Where X 4 Choose MeI or P; X 20 Choose from N, F, G, K, Q, S, or T, with X being the most preferred. 20 Selected as S; X 33 Choose T or Aib; X 35 Choose A or Aib; and further, when X 33 When X is T, 35 Choose Aib, or when X 35 When X is A, 33 Selected as Aib; This indicates that the lipid is optionally covalently bound to the ε-amino group of the lysine side chain via a linker / spacer.

13. The polypeptide or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The polypeptide comprises a sequence selected from the list of sequences consisting of: [MeI]VLSLDVPIGLLQILLSQARARAAREQA[K ]TN[Aib]EI[Nle]AEV(NH2) (SEQ ID NO: 215),PVLSLDVPIGLLQILLSQARARAAREQA[K ][Aib]NAEI[Nle]AEV(NH2)(SEQ ID NO: 238),[MeI]VLSLDVPIGLLQILLEQARARAAREQAT[K ]NAEILAEV(NH2) (SEQ IDNO: 6) or [MeI]VLSLDVPIGLLQILLEQARARAAREQA[K ]TN[Aib]EILAEV(NH2) (SEQ ID NO:16), wherein This indicates that the lipid is optionally covalently bonded to the ε-amino group of the lysine side chain via a linker / spacer.

14. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, wherein hCRHR1-EC 50 / hCRHR2-EC 50 The ratio must be at least 2000.

15. The polypeptide or a pharmaceutically acceptable salt thereof according to any one of the preceding claims, used as a medicament for treating metabolic disorders, preferably obesity.

16. A pharmaceutical composition comprising the polypeptide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-14.

Images