macrocyclic peptides

Abstract

This disclosure relates to the field of unconventional neurotrophic factors and to the treatment of degenerative, chronic, or progressive diseases and conditions, as well as monogenic hereditary diseases with ER stress as a pathogenic compound. More specifically, this disclosure relates to modified peptides, particularly macrocyclic peptides. This disclosure also relates to pharmaceutical compositions comprising said peptides. Furthermore, this disclosure relates to said peptides and pharmaceutical compositions used as pharmaceuticals and for treating degenerative, chronic, or progressive diseases and conditions, as well as monogenic hereditary diseases with ER stress as a pathogenic compound, and methods for treating said diseases and conditions.

Description

Technical Field

[0001] This disclosure relates to the field of unconventional neurotrophic factors and proteins located in the endoplasmic reticulum (ER), and specifically to the field of treating degenerative, chronic, or progressive diseases and conditions, as well as monogenic hereditary diseases with ER stress as a pathogenic compound. More specifically, this disclosure relates to macrocyclic peptides. This disclosure also relates to pharmaceutical compositions comprising said peptides. Furthermore, this disclosure relates to said peptides and pharmaceutical compositions used as pharmaceuticals and for treating degenerative, chronic, or progressive diseases and conditions, as well as monogenic hereditary diseases with ER stress as a pathogenic compound, and methods for treating said diseases and conditions. Background Technology

[0002] Neurotrophic factors (NTFs) are a subgroup of growth factors that promote neuronal survival and differentiation and possess neuroprotective and neuroregenerative properties (Hefti, 1994). NTFs are small proteins that support the growth, survival, and differentiation of developing and maturing neurons, protecting them from damage and toxins. Brain dopamine neurotrophic factor (CDNF) and its more recent counterpart, midbrain astrocyte-derived neurotrophic factor (MANF), form a new, unconventional family of NTFs, differing structurally and mechanistically from other growth factors (Lindholm and Saarma, 2010; Huttunen and Saarma, 2019). CDNF and MANF are small monomeric proteins with a molecular weight of approximately 18 kDa, consisting of mature proteins of 161 and 158 amino acids, respectively, expressed in the central nervous system and non-neuronal tissues. CDNF and MANF are primarily located in the endoplasmic reticulum (ER) lumen. They contain N-terminal signal peptides that guide them to the ER. Both CDNF and MANF contain a C-terminal KDEL (SEQ ID NO:37)-like ER retention signal, which is typically absent in growth factors intended for secretion. They interact with ER proteins such as BiP / GRP78, regulating unfolded protein response (UPR) signaling and protecting cells from ER stress-induced cell death. Both CDNF and MANF accumulate in the ER lumen of healthy cells, and disruption of the C-terminal ER retention signal leads to their secretion. Detectable levels of CDNF and MANF have been found in normal human serum, and MANF has also been found in cerebrospinal fluid (CSF). Based on these characteristics, CDNF and MANF are considered general stress-protective proteins rather than highly specific neurotrophic factors (Huttunen and Saarma, 2019). MANF has also been described as a cardiac factor (Glembotski, 2011).

[0003] CDNF and MANF are currently the most effective proteins used to treat degenerated dopaminergic neurons in a rat model of Parkinson's disease 6-OHDA (Lindholm and Saarma, 2010). When applied before the toxin, both factors effectively prevented 6-OHDA-induced dopaminergic neuronal loss and Parkinson's-like motor symptoms (Lindholm et al., 2007; Voutilainen et al., 2009). More importantly, when applied at a stage where 6-OHDA-induced Parkinson's symptoms were already very severe, post-injury administration of either factor effectively restored normal striatal motor behavior and dopaminergic innervation (Lindholm et al., 2007; Voutilainen et al., 2011). CDNF also protects and repairs dopaminergic neurons in a mouse MPTP model of Parkinson's disease (Airavaara et al., 2012), and in severe 6-OHDA models, it is more effective than glial cell line-derived neurotrophic factor (GDNF) (Airavaara et al., 2012; Voutilainen et al., 2011). The mechanisms underlying the neuroprotective effects of these factors are not fully understood, but they have been shown to activate pathways designed to mitigate oxidative and ER stress and inhibit apoptotic cell death. Many pathophysiological conditions, including diabetes and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS), are associated with ER stress. Therefore, the roles of CDNF and MANF have been demonstrated in various central nervous system disorders (WO 2009133247; WO 2007068803; and Airavaara et al., 2009). Non-cellular autonomous mechanisms, including the regulation of immune and glial cell responses, have been shown to contribute to the cytoprotective effects of CDNF and MANF (Sousa-Victor et al., 2018). Specifically, CDNF and MANF have been shown to suppress neuroinflammation, which is involved in the pathophysiology of most (if not all) CNS diseases and injuries (Nadella et al., 2014; Zhao et al., 2013).

[0004] CDNF and MANF share approximately 60% amino acid sequence homology, but they have highly similar three-dimensional structures. Both CDNF and MANF consist of two independently folded domains connected by a flexible loop region (Lindholm and Saarma, 2010). The secondary structure is primarily α-helices, with five α-helices in the N-terminal domain and three α-helices in the C-terminal domain. Three disulfide bridges stabilize the N-terminal domain, while the C-terminal CRAC (SEQ ID NO: 38) sequence in CDNF and the CKGC (SEQ ID NO: 39) sequence in MANF form internal disulfide bridges. These CXXC (SEQ ID NO: 40) disulfide bridges are present in both CDNF and MANF and play a central role in the cytoprotective activity of these proteins.

[0005] CDNF is expressed in the brain, but also in many other tissues, including, for example, skeletal muscle, liver, heart, lungs, pancreas, testes, salivary glands, and the enteric nervous system (Lindholm et al., 2007). MANF is expressed in the brain, but also in peripheral tissues such as the pancreas and heart.

[0006] Natural peptides (such as those disclosed in publications WO2013 / 3034805 and WO2018 / 202957) are rarely used as pharmaceutical products.

[0007] Document WO2013 / 3034805A1 discloses MANF and CDNF fragments of 4-40 amino acids in length, containing the sequence CKGC (SEQ IN NO:39) or CRAC (SEQ ID NO:38). Document WO2018 / 202957A1 discloses CDNF fragments of at least 50 amino acids in length. Hellmann et al. (2011) disclosed an active C-terminal fragment construct of MANF containing residues 96-158. Fletcher and Hughes (2006) disclosed a brain-derived neurotrophic factor (BDNF)-derived peptide containing CRAC (SEQ ID NO:38) with engineered cysteine ​​residues for cyclization. Therefore, there remains a need in the art for therapeutic agents with improved metabolic stability and distribution properties. Summary of the Invention

[0008] One object of this disclosure is to provide novel modified macrocyclic peptides. Another object of this disclosure is to provide uses of the novel peptides.

[0009] This disclosure provides tools with these aforementioned properties by using macrocyclic peptides, in particular, in a novel and inventive manner.

[0010] The inventors have discovered that linear natural CDNF and MANF peptides are poor drug molecules because they are rapidly metabolized and poorly distributed when administered to humans or animals, particularly parenterally. Therefore, unmodified natural peptides (such as those disclosed in the prior art) are rarely used as pharmaceutical products. The inventors have developed novel stable peptides derived from CDNF and MANF that encapsulate the cytoprotective effects of CDNF and MANF but are well-suited for non-invasive peripheral administration. The inventors have found that macrocyclization of CDNF and MANF peptides significantly improves their metabolic stability and distribution characteristics without sacrificing their cytoprotective activity, as shown in the data of this disclosure. Furthermore, the modified peptides of this invention are shorter than those disclosed in the prior art.

[0011] The biological activity of CDNF / MANF resides in the C-terminal domain of the protein. This disclosure describes 8-32 amino acid peptides derived from the C-terminal domain of CDNF and MANF (particularly in macrocyclic form). Short linear octapeptides surrounding the CXXC (SEQ ID NO:40) motif exhibit cytoprotective activity comparable to the full-length protein and the ability to penetrate cell membranes and the experimental blood-brain barrier in in vitro models as disclosed herein.

[0012] The inventors have for the first time demonstrated that short macrocyclic peptides, such as head-to-tail cyclic CDNF / MANF macrocyclic peptides with a CXXC (SEQ ID NO:40) motif or a specific type of CXXXC (SEQ ID NO:27) motif, exhibit significantly improved pharmaceutical properties, such as metabolic stability, blood-brain barrier (BBB) ​​penetration, and in vivo pharmacokinetics. These short and semi-short macrocyclic peptides can be used to develop drugs for degenerative, chronic, and / or progressive diseases and conditions, or monogenic hereditary diseases with ER stress as a pathogenic component.

[0013] This disclosure provides a macrocyclic peptide of 8-32 amino acids in length or a pharmaceutically acceptable salt thereof, comprising the amino acid sequence C-X1-X2-X3-C (SEQ ID NO:27).

[0014] in

[0015] X1 can be selected from the groups composed of R, K, I, G, A, and S;

[0016] X2 either does not exist or is selected from the group consisting of G, A, R, K, I, and S; and

[0017] X3 can be selected from the groups consisting of A, G, and S.

[0018] In some embodiments, a macrocyclic peptide is a peptide in which the N-terminus of the peptide is linked to the C-terminus of the peptide (i.e., a "head-tail bond"). In some cases, the peptide is a pseudopeptide. In some cases, the peptide has at least one (e.g., one, two, three, four, five, six, or seven) of the following properties: (i) the peptide can dose-dependently protect TH-positive neurons from MPP. + Toxicity; (ii) The peptide reduces the number of α-synuclein inclusion bodies in TH-positive neurons; (iii) The peptide has improved stability in plasma compared to its linear counterpart; (iv) The peptide has improved stability in hepatocytes compared to its linear counterpart; or (v) The peptide has improved ability to cross the blood-brain barrier compared to its linear counterpart.

[0019] This disclosure also provides the macrocyclic peptide, which is used as a pharmaceutical agent.

[0020] This disclosure also provides the macrocyclic peptide for the treatment of degenerative, chronic and / or progressive diseases and conditions, such as neurodegenerative diseases or conditions, or monogenic hereditary diseases with ER stress as a pathogenic component.

[0021] This disclosure also provides a pharmaceutical composition comprising the macrocyclic peptide and at least one of the following: a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a preservative, a stabilizer, and / or a diluent.

[0022] This disclosure further provides pharmaceutical compositions for use as medicines.

[0023] This disclosure also provides a pharmaceutical composition for treating degenerative, chronic and / or progressive diseases and conditions, such as neurodegenerative diseases or conditions, or monogenic hereditary diseases with ER stress as a pathogenic component.

[0024] This disclosure also provides a method for treating a subject with a degenerative, chronic, or progressive disease or condition, such as a neurodegenerative disease or condition, or a monogenic hereditary disease with ER stress as a pathogenic component, the method comprising administering to the subject a pharmaceutical composition comprising the aforementioned macrocyclic peptide.

[0025] The present invention will now be described in more detail through preferred embodiments. Attached Figure Description

[0026] Figure 1A list of compounds studied is presented. The compound number, SEQ ID NO, amino acid sequence indicating the cyclization scheme and Cys-Cys disulfide bond, sequence length, modification description, and monoisotopic mass (Da) are shown. Column 5 shows the detailed charged mass peaks observed in the MS spectra, and column 6 displays the monoisotopic mass of the compounds. The difference in monoisotopic mass between the linear and cyclic forms of the same peptide, approximately 18 Da (the mass of a water molecule), confirms successful head- and tail-cyclization.

[0027] Figure 2A-2P The biophysical characteristics of macrocyclic compounds 2 (SEQ ID NO:2), 4 (SEQ ID NO:4), 6 (SEQ ID NO:6), 8 (SEQ ID NO:8), 10 (SEQ ID NO:10), 12 (SEQ ID NO:12), 14 (SEQ ID NO:14), 24 (SEQ ID NO:24), and 26 (SEQ ID NO:26) are presented.

[0028] Figure 2A Compound 2 (SEQ ID NO:2) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (head; Val1) and carboxyl-terminal (tail; Glu27) residues. The disulfide bridge forming cysteine ​​is shown in gray with bars and correspondingly labeled.

[0029] Figure 2B The 2D diagram of compound 2 (SEQ ID NO:2) is shown. 1 A contour plot of the number of amide protons observed in the H NMR TOCSY amide fingerprint. The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 27-amino acid-long cyclic peptides, i.e., the Val1 amide proton was observed, consistent with the expected N-terminal amidation, unlike its linear analogues, in which no N-terminal amide proton was observed. The Val1 and Val8 protons were identified, and they are respectively derived from H NMR protons. N Linear indications of 7.9 and 8.05 ppm.

[0030] Figure 2C Compound 4 (SEQ ID NO:4) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (Val1) and carboxyl-terminal (Glu27) residues. The disulfide bridge forming cysteine ​​is shown in gray with bars and correspondingly labeled.

[0031] Figure 2DThe 2D diagram of compound 4 (SEQ ID NO:4) is shown. 1 A contour plot of the number of amide protons observed in the 1H NMR TOCSY amide fingerprint (left panel). The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 27-amino acid-long cyclic peptides, i.e., the Val1 amide proton was observed, consistent with the expected N-terminal amidation, unlike its linear analogues, in which no N-terminal amide proton was observed. The first (Val1) and last (Glu27) residues were identified. 1 H chemical shifts and they are respectively determined by H corresponding to H N Linear indications of 8.0 and 8.35 ppm.

[0032] For these two residues, a strong ROE was observed between the amide proton and the α proton of the preceding amino acid residue, HN(i)-Hα(i-1), as in 2D. 1 The expansion of the H ROESY fingerprint region is shown in the right figure. The establishment of the Val1:HN-Glu27:Hα correlation at 8.02 / 4.25 ppm strongly indicates that the peptide is in its cyclized form and the proton spacing is less than [missing value].

[0033] Figure 2E Compound 6 (SEQ ID NO:6) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (Met1) and carboxyl-terminal (Lys23) residues. The disulfide bridge forming cysteine ​​is shown in gray with bars and correspondingly labeled.

[0034] Figure 2F The 2D diagram of compound 6 (SEQ ID NO:6) is shown. 1 A contour plot of the number of amide protons observed in the H NMR TOCSY amide fingerprint (left panel). The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 23-amino acid-long cyclic peptides; that is, Met1 amide protons were observed, consistent with the expected N-terminal amidation, unlike its linear analogues, in which N-terminal amide protons were not observed. The Met1 amide protons were identified and their composition corresponds to H NMR. N A linear indicator of 7.98 ppm.

[0035] Figure 2GCompound 8 (SEQ ID NO:8) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (Leu1) and carboxyl-terminal (Lys23) residues. The disulfide bridge forming cysteine ​​is shown in gray with bars and correspondingly labeled.

[0036] Figure 2H The 2D diagram of compound 8 (SEQ ID NO:8) is shown. 1 A contour plot of the number of amide protons observed in the 1H NMR TOCSY amide fingerprint (left panel). The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 23-amino acid-long cyclic peptides, i.e., five lysine amide protons were observed, consistent with the expected amidation at the N-terminus, unlike its linear analogues, in which the N-terminal Lys1 amide proton was not observed. All Lys amide protons were identified. 1 H chemical shifts and they are respectively determined by H corresponding to H N Linear indications of 7.51, 8.05, 8.09, 8.11 and 8.21 ppm.

[0037] Figure 2I Compound 10 (SEQ ID NO: 10) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (Lys1) and carboxyl-terminal (Glu16) residues. The disulfide bridge forming cysteine ​​is shown in gray with bars and correspondingly labeled.

[0038] Figure 2J The 2D diagram of compound 10 (SEQ ID NO: 10) is shown. 1 A contour plot of the number of amide protons observed in the 1H NMR TOCSY amide fingerprint (left panel). The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 16-amino acid-long cyclic peptides, i.e., all amino acids in the sequence have one amide proton, in contrast to its linear analogues, in which no one acid proton (peptide acid) or two amide protons (peptide amide) were observed at the N-terminus. The first (Lys1) and last (Glu16) residues were identified. 1 H chemical shift, which is determined by the corresponding H... N 8.18 and 8.3 ppm. For these two residues, a strong ROE was observed between the amide proton and the α proton of the preceding amino acid residue, HN(i)-Hα(i-1), as in 2D. 1The expansion of the H ROESY fingerprint region is shown in the right figure. The establishment of the Lys1:HN–Glu16:Hα correlation at 8.19 / 4.24 ppm strongly indicates that the peptide is in its cyclized form and the proton spacing is less than [missing information].

[0039] Figure 2K Compound 12 (SEQ ID NO:12) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (Trp1) and carboxyl-terminal (Thr12) residues. The disulfide bridge forming cysteine ​​is shown in gray with bars and correspondingly labeled.

[0040] Figure 2L The 2D diagram of compound 12 (SEQ ID NO:12) is shown. 1 A contour plot of the number of amide protons observed in the 1H NMR TOCSY amide fingerprint (left panel). The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 12-amino acid-long cyclic peptides, i.e., the Trp1 amide proton was observed, consistent with the expected N-terminal amidation, unlike its linear analogues, in which no N-terminal amide proton was observed. The first (Trp1) and last (Thr12) residues were identified. 1 H chemical shifts and they are respectively determined by H corresponding to H N Linear indications at 8.21 and 8.06 ppm. For these two residues, a strong ROE, HN(i)-Hα(i-1), as shown in 2D, was observed between the amide proton and the α proton of the preceding amino acid residue. 1 The expansion of the H ROESY fingerprint region is shown in the right figure. The establishment of the Trp1:HN-Thr12:Hα correlation at 8.21 / 4.30 ppm strongly indicates that the peptide is in its cyclized form and the proton spacing is less than [missing value].

[0041] Figure 2M Compound 14 (SEQ ID NO:14) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (Trp1) and carboxyl-terminal (Ser12) residues. The disulfide bridge forming cysteine ​​is shown in gray with bars and correspondingly labeled.

[0042] Figure 2N The 2D diagram of compound 14 (SEQ ID NO:14) is shown. 1A contour plot of the number of amide protons observed in the 1H NMR TOCSY amide fingerprint (left panel). The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 12-amino acid-long cyclic peptides, i.e., the Trp1 amide proton was observed, consistent with the expected N-terminal amidation, unlike its linear analogues, in which no N-terminal amide proton was observed. The first (Trp1) and last (Ser12) residues were identified. 1 H chemical shifts and they are respectively determined by H corresponding to H N Linear indications of 7.98 and 8.24 ppm. For these two residues, a strong ROE, HN(i)-Hα(i-1), as shown in 2D, was observed between the amide proton and the α proton of the preceding amino acid residue. 1 The expansion of the H ROESY fingerprint region is shown in the right figure. The establishment of the Trp1:HN-Ser12:Hα correlation at 7.99 / 4.4 ppm strongly indicates that the peptide is in its cyclized form and the proton spacing is less than [missing information].

[0043] Figure 2O Compound 24 (SEQ ID NO:24) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (Trp1) and carboxyl-terminal (Thr13) residues. The disulfide bridge forming cysteine ​​is shown and correspondingly labeled in gray bars. The inserted glycine residues between the Cys residues of the CXXC motif in compound 24 (SEQ ID NO:24) are also shown as gray bars.

[0044] Figure 2P The 2D diagram of compound 24 (SEQ ID NO:24) is shown. 1 A contour plot of the number of amide protons observed in the 1H NMR TOCSY amide fingerprint (left panel). The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 13-amino acid-long cyclic peptides, i.e., the Trp1 amide proton was observed, consistent with the expected N-terminal amidation, unlike its linear analogues, in which no N-terminal amide proton was observed. The first (Trp1) and last (Thr13) residues were identified. 1 H chemical shifts and they are respectively determined by H corresponding to H N Linear indications of 7.96 and 8.26 ppm. For these two residues, a strong ROE was observed between the amide proton and the α proton of the preceding amino acid residue, HN(i)-Hα(i-1), as in 2D.1 The expansion of the H ROESY fingerprint region is shown in the right figure. The establishment of the Trp1:HN-Thr13:Hα correlation at 8.26 / 4.35 ppm strongly indicates that the peptide is in its cyclized form and the proton spacing is less than [missing information].

[0045] Figure 2Q Compound 26 (SEQ ID NO:26) is shown, with arrows indicating the closed-loop region between the corresponding amino-terminal (Trp1) and carboxyl-terminal (Ser13) residues. The disulfide bridge forming cysteine ​​is shown and correspondingly labeled in gray bars. The inserted glycine residues in compound 26 (SEQ ID NO:26) are also shown in gray bars.

[0046] Figure 2R The 2D diagram of compound 26 (SEQ ID NO:26) is shown. 1 A contour plot of the number of amide protons observed in the 1H NMR TOCSY amide fingerprint (left panel). The horizontal axis is the direct dimension (F2, presented in ppm), and the vertical axis is the indirect dimension (F1, presented in ppm). The number of amide protons observed in the TOCSY data is consistent with 13-amino acid-long cyclic peptides, i.e., the Trp1 amide proton was observed, consistent with the expected N-terminal amidation, unlike its linear analogues, in which no N-terminal amide proton was observed. The first (Trp1) and last (Ser13) residues were identified. 1 H chemical shifts and they are respectively determined by H corresponding to H N Linear indications at 8.13 and 8.1 ppm. For these two residues, a strong ROE, HN(i)-Hα(i-1), as shown in 2D, was observed between the amide proton and the α proton of the preceding amino acid residue. 1 The expansion of the H ROESY fingerprint region is shown in the right figure. The establishment of the Trp1:HN-Ser13:Hα correlation at 8.13 / 4.45 ppm strongly indicates that the peptide is in its cyclized form and the proton spacing is less than [missing information].

[0047] Figure 3A-3LThis study demonstrates the neuroprotective effects of rhCDNF with linear and macrocyclic compounds (compounds 1-10, each having SEQ ID NO: 1-10) on dopaminergic TH (tyrosine hydroxylase)-positive neurons damaged by MPP+ (1-methyl-4-phenylpyridinium), and its effect on α-synuclein aggregation in TH-positive neurons. Data are presented as percentages of the control undamaged condition, i.e., mean + / - SEM (n = 4-6; MPP+ negative controls collected from multiple studies n = 122-127). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Brown-Forsythe and Welch ANOVA tests, as well as post-hoc unpaired t-tests and Welch-related paired comparisons, compared to MPP+ negative controls. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, Brown-Forsythe and Welch ANOVA tests with post-hoc unpaired t-tests were used to compare the linearity with the corresponding macrocyclic compound at the same concentration.

[0048] Figure 3A The figures show the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells following MPP+ injury with increased rhCDNF concentration. The upper dashed line represents the control level (100%) of parameters obtained from uninjured cells; the lower dashed line represents the negative control level of parameters obtained from MPP+-injured cells that were not further treated with the compound.

[0049] Figure 3B This diagram illustrates the aggregation of α-synuclein in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of increasing concentrations of rhCDNF. The upper dashed line represents the negative control level of α-synuclein aggregation in MPP+-injured cells without additional treatment with the test compound; the lower dashed line represents the negative control level (100%) of the parameter obtained from uninjured cells.

[0050] Figure 3C The results show the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury in the presence of compound 1 (SEQ ID NO:1) and compound 2 (SEQ ID NO:2).

[0051] Figure 3DThe study demonstrates the aggregation of α-synuclein in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 1 (SEQ ID NO:1) and 2 (SEQ ID NO:2).

[0052] Figure 3E The results show the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury in the presence of compounds 3 (SEQ ID NO:3) and 4 (SEQ ID NO:4).

[0053] Figure 3F This study demonstrates the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 3 (SEQ ID NO:3) and 4 (SEQ ID NO:4).

[0054] Figure 3G The results show the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury in the presence of compounds 5 (SEQ ID NO:5) and 6 (SEQ ID NO:6).

[0055] Figure 3H This study demonstrates the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 5 (SEQ ID NO:5) and 6 (SEQ ID NO:6).

[0056] Figure 3I The diagram shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury in the presence of compounds 7 (SEQ ID NO:7) and 8 (SEQ ID NO:8).

[0057] Figure 3J This study demonstrates the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 7 (SEQ ID NO:7) and 8 (SEQ ID NO:8).

[0058] Figure 3KThe results show the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury in the presence of compounds 9 (SEQ ID NO:9) and 10 (SEQ ID NO:10).

[0059] Figure 3L This study demonstrates the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 9 (SEQ ID NO:9) and 10 (SEQ ID NO:10).

[0060] Figure 4A-4L This study demonstrates the neuroprotective effects of linear and macrocyclic compounds (compounds 11-26, SEQ ID NO: 11-26, respectively) on dopaminergic TH-positive neurons damaged by MPP+, and their influence on α-synuclein aggregation in TH-positive neurons. Data are presented as a percentage of the control undamaged condition, i.e., mean + / - SEM (n = 4-6; MPP+ negative controls collected from multiple studies, n = 122-127). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Brown-Forsythe and Welch ANOVA tests, as well as post-hoc unpaired t-tests and Welch-related paired comparisons, compared to MPP+ negative controls. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, Brown-Forsythe and Welch ANOVA tests with post-hoc unpaired t-tests were used to compare the linearity with the corresponding macrocyclic compound at the same concentration.

[0061] Figure 4A The figures show the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury in the presence of compounds 11 (SEQ ID NO:11) and 12 (SEQ ID NO:12). The upper dashed line represents the control level (100%) of parameters obtained from undamaged cells; the lower dashed line represents the negative control level of parameters obtained from cells injured with MPP+ without additional treatment with the compounds.

[0062] Figure 4BThe diagram shows the aggregation of α-synuclein (aSyn) in primary cultures of midbrain cells following MPP+ injury in the presence of compounds 11 (SEQ ID NO:11) and 12 (SEQ ID NO:12). The upper dashed line represents the negative control level of α-synuclein aggregation in cells injured with MPP+ without additional treatment with the test compounds; the lower dashed line represents the negative control level (100%) of the parameter obtained from uninjured cells.

[0063] Figure 4C The number of TH neurons in primary cultures of midbrain cells after MPP+ injury is shown in the presence of compounds 13 (SEQ ID NO:13) and 14 (SEQ ID NO:14).

[0064] Figure 4D This study demonstrates the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 13 (SEQ ID NO:13) and 14 (SEQ ID NO:14).

[0065] Figure 4E The number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury are shown in the presence of compounds 15 (SEQ ID NO:15), 16 (SEQ ID NO:16), 17 (SEQ ID NO:17), and 18 (SEQ ID NO:18).

[0066] Figure 4F The study demonstrates the aggregation of α-synuclein in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 15 (SEQ ID NO:15), 16 (SEQ ID NO:16), 17 (SEQ ID NO:17), and 18 (SEQ ID NO:18).

[0067] Figure 4G The number of TH neurons in primary cultures of midbrain cells after MPP+ injury is shown in the presence of compounds 19 (SEQ ID NO:19), 20 (SEQ ID NO:20), 21 (SEQ ID NO:21), and 22 (SEQ ID NO:22).

[0068] Figure 4HThe study demonstrates the aggregation of α-synuclein in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 19 (SEQ ID NO:19), 20 (SEQ ID NO:20), 21 (SEQ ID NO:21), and 22 (SEQ ID NO:22).

[0069] Figure 4I The results show the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury in the presence of compounds 23 (SEQ ID NO:23) and 24 (SEQ ID NO:24).

[0070] Figure 4J This study demonstrates the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 23 (SEQ ID NO:23) and 24 (SEQ ID NO:24).

[0071] Figure 4K The results show the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurons in primary cultures of midbrain cells after MPP+ injury in the presence of compounds 25 (SEQ ID NO:25) and 26 (SEQ ID NO:26).

[0072] Figure 4L This study demonstrates the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells following MPP+ injury in the presence of compounds 25 (SEQ ID NO:25) and 26 (SEQ ID NO:26).

[0073] Figure 5A A computational molecular model of the nucleotide-binding domain of GRP78 (GRP78-NBD) complexed with compound 12 is shown. GRP78-NBD is presented as a translucent surface model and its cartoon trace. The Cys-Cys bond in compound 12 (SEQ ID NO:12) is shown as a bar.

[0074] Figure 5B The binding affinity (Kd, in μM) of a group of representative compounds to GRP78-NBD is presented in tabular form. Binding affinity was obtained by cell-free assay based on micro-thermophoresis.

[0075] Figure 5CThe neuroprotective activities of compounds 10 (SED ID NO: 10) and 14 (SEQ ID NO: 14) are shown to be dependent on the activity of unfolded protein response (UPR) pathway signaling. GSK2606414 was used to inhibit PERK signaling, and KIRA6 was used to inhibit IRE1α signaling. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 One-way ANOVA and post-hoc Fisher's LSD tests were used for pairwise comparisons against the MPP+ negative control. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 One-way ANOVA and post-hoc Fisher's LSD tests were used for pairwise comparisons between the effects of the compounds alone and in combination with UPR signaling inhibitors.

[0076] Figures 6A-6B The in vitro metabolic stability of linear and macrocyclic compounds is demonstrated in rats (compounds 1-14 and 23-26, respectively, having SEQ ID NO: 1-14 and 23-26) and human plasma (compounds 1-8, 11-14 and 23-26, respectively, having SEQ ID NO: 1-8, 11-14 and 23-26).

[0077] Figure 6A The calculated half-life is shown based on the disappearance of the compound in rat plasma.

[0078] Figure 6B This displays the calculated half-life based on the disappearance of compounds in human plasma. The square tops (arks) and numbers above each column reflect the variation in plasma half-life for macrocyclic compounds that exhibit linearity. The maximum calculated half-life at 789 min reflects the experimental cutoff time constraint.

[0079] Figures 7A-7B The in vitro metabolic stability of linear and macrocyclic compounds is demonstrated in rats (compounds 1-14 and 23-26, respectively, with SEQ ID NO: 1-14 and 23-26) and human hepatocytes (compounds 1-8, 11-14 and 23-26, respectively, with SEQ ID NO: 1-8, 11-14 and 23-26).

[0080] Figure 7A The calculated half-life is shown based on the disappearance of the compound in rat hepatocytes.

[0081] Figure 7BThis diagram illustrates the calculated half-life based on the disappearance of compounds in human hepatocytes. The squares and numbers above each column reflect the changes in the half-life of macrocyclic compounds in hepatocytes for the corresponding linear compounds. The maximum calculated half-life at 395 min reflects the experimental cutoff time limit.

[0082] Figure 8 This study demonstrates the penetration of linear and macrocyclic compounds (compounds 1-8, 11-14, and 23-26, respectively, with SEQ ID NO: 1-8-11-14 and 23-26) across a 3D in vitro blood-brain barrier model. The amount of compound that crossed the artificial blood-brain barrier is expressed as a percentage of the original applied concentration of the compound. Data are presented as mean + / - SEM (n = 3-4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. Paired comparisons between linear and corresponding macrocyclic compounds were performed using Welch correlation with post-hoc unpaired t-tests, Brown-Forsythe and Welch ANOVA tests.

[0083] Figure 9A Plasma concentrations of macrocyclic compounds 2, 10, and 14 (SEQ ID NO: 2, 10, and 14) and linear compounds 3 and 9 (SEQ ID NO: 3 and 9) measured at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h after intravenous administration of 5 mg / kg to rats are shown. Data are presented as mean ± SEM (n = 3).

[0084] Figure 9B The distribution kinetics of compound 14 (SEQ ID NO: 14) in the interstitial fluid (ISF; striatum) following an intravenous bolus injection of 10 mg / kg are illustrated. ISF concentrations were normalized by recovery rate (% as determined by in vitro experiments) via a microdialysis filter. The compound was detected from ISF and plasma using LC-MS / MS.

[0085] Figure 10 Pairwise alignments of the C-terminal domains (61-63aa) of human CDNF and MANF are shown. Alignments were performed using sequences retrieved from Genbank: human CDNF accession number NP_001025125.2 and human MANF accession number NP_006001.5. The CXXC motif is shown against a gray background, and the positions of the three α-helices are indicated.

[0086] Figure 11ClustalW multiple sequence alignments of the C-terminal domains of CDNF and MANF (61-63aa) from 10 different species (SEQ ID NO: 53-72, respectively) are shown. Genbank accession numbers are shown in the sequence alignments. The CXXC motif is shown against a gray background, and the positions of the three α-helices are indicated. Conserved residues between these representative sequences (in both CDNF and MANF) are shown in bold. Below the sequence alignments, natural variants found in the 10 representative species at each position are shown. The presented list of sequences and species can be used to identify conserved and variable positions and demonstrates that only limited variation is possible for most non-essential amino acid residues.

[0087] sequence list

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] Detailed Implementation

[0094] The term "modified peptide" refers to a peptide or polypeptide that has been modified. Peptide modification or synthetic options include, for example, macrocyclic peptides, peptide mimics, N-terminal modifications, C-terminal modifications, isotope-labeled peptides, biotinylated and labeled peptides, fluorescent dye-labeled peptides, peptide dimers, post-translational modifications, internal quenching / FRET peptides, linkers / spacers / PEGylation, peptide aggregation, protein conjugation, immunogenic peptides, and incorporation of non-naturally encoded amino acids. "Non-naturally encoded amino acids" refers to an amino acid that is not one of the 20 common amino acids or pyrrolidone or selenocysteine. Other terms that may be used synonymously with "non-naturally encoded amino acids" are "non-natural amino acid," "non-naturally occurring amino acid," "non-naturally present amino acid," and their various hyphenated and unhyphenated forms. The term "non-naturally encoded amino acids" also includes, but is not limited to, amino acids that appear through modification (e.g., post-translational modifications) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolidone and selenocysteine), but which are not naturally incorporated into the growing polypeptide chain by the translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosyl-L-serine, N-acetylglucosyl-L-threonine, and O-phosphotyrosine.

[0095] The term "macrocyclic peptide" refers to a polypeptide chain having a cyclic ring structure. As used herein, a macrocyclic peptide comprises a ring structure in addition to the ring structure formed by the disulfide bridge if the two cysteine ​​residues in the CX1X2X3C motif are present in the form of a disulfide bridge. In some embodiments, macrocyclic peptides comprise a ring structure formed by more than five amino acid residues. The ring structure may involve linkages, including N-terminal-C-terminal (head-tail), head-side chain, side chain-tail, and side chain-side chain linkages. In one embodiment, this application provides a peptide having head-tail linkages. Linkages can be formed by connecting one amino acid of the peptide to another amino acid of the peptide (e.g., end-to-end, side chain-to-end) using an amide bond or other chemically stable bond (such as a lactone, ether, thioether, disulfide, etc.). In one embodiment, bicyclic and monocyclic peptides can be cyclized via a disulfide bond between two cysteine ​​residues. Cyclization options include, for example, Cys-Cys (up to 4 disulfide bonds in a peptide, site-specific or thermodynamic cyclization), cyclized peptides via amides (head-tail or side-chain-side-chain), and thioethers (Cys-bromoacetate).

[0096] The term "pseudopeptide" refers to an amide of an amino acid not present in natural peptides or proteins, particularly an amide introduced into the polypeptide chain. Pseudopeptide or amino bond substitute is one of many terms that can be used to describe skeletally modified peptides. These synthetic analogs of peptides have a variety of potential uses, but the broadest interest in these areas focuses on their potential to develop metabolically stable and potentially orally active peptide hormone analogs or enzyme inhibitors with enhanced bioefficacy. Specifically, the term includes peptide skeletal modifications (i.e., amide bond mimics) known to those skilled in the art. Such modifications include modifications of the amide nitrogen, α-carbon, or amide carbonyl group, complete substitution, extension, deletion, or skeletal crosslinking of the amide bond. Several peptide skeletal modifications are known, including ψ[CH2S], ψ[CH2NH], ψ[CSNH2], ψ[NHCO], ψ[COCH2], and ψ[(E) or (Z)CH=CH]. In the terms used above, ψ indicates the absence of an amide bond. The structure of the substituted amide group is described in parentheses.

[0097] As used herein, when two entities are “conjugated” to each other, they are connected by direct or indirect covalent or non-covalent interactions. In some respects, association is covalent. In other respects, said association is non-covalent. Non-covalent interactions include hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. Indirect covalent interactions are, optionally, through a linker group when two entities are covalently connected. “Conjugation” herein means a peptide conjugated or coupled to a detectable chemical or biochemical moiety or PEG or other moiety used to prolong plasma half-life. In some cases, one or more peptides disclosed herein may be conjugated, for example, to a carrier protein. Such conjugated compositions may be monovalent or polyvalent. For example, a conjugated composition may include one peptide disclosed herein conjugated to a carrier protein. Alternatively, a conjugated composition may include two or more peptides disclosed herein conjugated to a carrier.

[0098] The blood-brain barrier (BBB) ​​is a highly selective semipermeable membrane barrier that separates circulating blood from the brain and extracellular fluid in the central nervous system. The BBB is formed by the endothelial cells of the capillary walls, the astrocytes that surround the capillaries, and pericytes embedded in the capillary basement membrane. This system allows some molecules to pass through by passive diffusion, as well as the selective transport of molecules essential for neural function, such as glucose, water, and amino acids. Large molecules, such as proteins, typically cannot cross the BBB. However, some peptides can cross the BBB through various mechanisms, and some proteins containing transporter-specific recognition motifs located on the surface of brain vascular endothelial cells can also cross the BBB.

[0099] As used herein, "pharmaceutically acceptable carriers" can include one or more solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption delay agents that are acceptable for the formulation of pharmaceuticals (such as those suitable for human administration). The use of such media and reagents for pharmaceutically active substances is well known in the art. Complementary active ingredients may also be incorporated into the composition.

[0100] CDNF and MANF share approximately 60% amino acid sequence homology. Figure 10 and 11While both CDNF and MANF are highly similar in their three-dimensional structures, they are composed of two independently folded domains connected by a flexible loop region. The secondary structure is primarily α-helices, with five α-helices in the N-terminal domain and three α-helices in the C-terminal domain. Three disulfide bridges stabilize the N-terminal domain, while the C-terminal CRAC (SEQ ID NO:38) sequence in CDNF and the CKGC (SEQ ID NO:39) sequence in MANF form internal disulfide bridges. Such CXXC (SEQ ID NO:40) disulfide bridges are present in both CDNF and MANF. The CXXC (SEQ ID NO:40) motif is beneficial for the neuroprotective activity of both MANF and CDNF. However, the data presented here show that the CXXC (SEQ ID NO:40) motif can be adapted to some modifications, such as the addition of small amino acids (e.g., glycine and serine), i.e., specific types of CXXXC motifs can also be used. In some respects, CDNF has a sequence derived from NP_001025125.2 (SEQ ID NO:43). In some respects, MANF has a sequence derived from NP_006001.5 (SEQ ID NO:44).

[0101] In addition to naturally occurring allelic variants of the MANF and CDNF peptides, alterations can be introduced into the MANF / CDNF sequence through mutation, resulting in changes to the amino acid sequence of the encoded MANF / CDNF peptide. Nucleotide substitutions that result in the substitution of amino acids at "non-essential" amino acid residues can be made within the sequence of the MANF / CDNF peptide. MANF / CDNF peptides containing one or more "non-essential" substitutions, or functional fragments thereof, can be considered equivalent to the wild-type MANF / CDNF peptides disclosed herein.

[0102] Each amino acid can be either natural or non-natural. The term "non-natural amino acid" refers to an organic compound that is an analogue of a natural amino acid because it has a similar structure to the natural amino acid, thus mimicking its structure and reactivity. Non-natural amino acids can be modified amino acids and / or amino acid analogues that are not one of the 20 common naturally occurring amino acids or one of the rare natural amino acids selenocysteine ​​or pyrrolidone.

[0103] Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, asparagine, aspartic acid, cysteine, cyclohexylalanine, 2,3-diaminopropionic acid, 4-fluorophenylalanine, glutamine, glutamic acid, glycine, histidine, homoproline, isoleucine, leucine, lysine, methionine, naphthylalanine, ortholeucine, phenylalanine, phenylglycine, hexahydropyridinecarboxylic acid, proline, pyroglutamic acid, sarcosine, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, derivatives, or combinations thereof.

[0104] The term “pharmaceutically acceptable” is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio.

[0105] "Pharmaceutically acceptable salt" is intended to mean a salt of the free acid or base of the compound represented herein that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. See also SMBerge et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of a subject without excessive toxicity, irritation, or allergic reactions. The compounds described herein may have sufficiently acidic groups, sufficiently basic groups, two types of functional groups, or more than one of each type, and thus react with many inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts.

[0106] For the compounds containing basic groups (such as amines) described herein, pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, nitric acid, boric acid, phosphoric acid, etc.) or with organic acids (such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, oxaloacetic acid, ethanesulfonic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, pyranoside (such as glucuronic acid)). Any compatible mixture of acids (such as taruronic acid or galacturonic acid), α-hydroxy acids (such as mandelic acid, citric acid or tartaric acid), amino acids (such as aspartic acid or glutamic acid), aromatic acids (such as benzoic acid, 2-acetoxybenzoic acid, naphtholic acid or cinnamic acid), sulfonic acids (such as lauryl sulfonic acid, p-toluenesulfonic acid, methanesulfonic acid or ethanesulfonic acid), or acids (such as those given as examples herein), and any other acids and mixtures thereof considered equivalents or acceptable substitutes according to the ordinary level of the art to treat free bases.

[0107] For compounds containing acidic groups (such as carboxylic acid groups) as described herein, base addition salts can be prepared by any suitable method available in the art, such as treating the compound with a sufficient amount of the desired base (soda ash or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include, but are not limited to, lithium, sodium, potassium, calcium, ammonium, zinc, or magnesium salts, or other metal salts; organic amino salts, such as alkyl, dialkyl, trialkyl, or tetraalkylammonium salts.

[0108] Other pharmaceutically acceptable examples of salts include, but are not limited to, camphor sulfonates, sulfates, pyrosulfates, bisulfates, sulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, hexanoates, heptarates, propionates, oxalates, malonates, succinates, octanoates, sebacic acid, fumarates, and maleic acid. Salts, butyn-1,4-diacidates, hexyn-1,6-diacidates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methanesulfonates, propanesulfonates, benzenesulfonates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, and mandelates. A list of other suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., 1985.

[0109] The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and separating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in some physical properties (such as solubility in polar solvents), but in other respects, the salt is equivalent to the parent form of the compound for the purposes of this application.

[0110] In embodiments of this disclosure, the macrocyclic peptide or fragment is in the range of 8-32 amino acids in length, wherein the macrocyclic peptide or fragment thereof comprises CX1X2X3C (SEQ ID NO:27) as described herein. In some embodiments, preferred peptides or fragments may consist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids. In some embodiments, the length of the peptide or fragment is in the range of 8-31, 8-29, 8-27, 8-25, 8-23, 8-21, 8-19, 8-17, 8-15, 11-27, 11-25, 11-23, 11-21, 11-19, 11-17, 11-15, 13-25, 13-23, 13-21, 13-19, 13-17, 13-15, 23-27, 24-27, or 25-27 amino acids. In some embodiments, the length of the peptide or fragment is in the range of 12-27, 13-27, 12-23, 13-23, or 23-27 amino acids. Peptides or fragments thereof may contain any naturally occurring amino acids, such as alanine [Ala(A)], arginine [Arg(R)], asparagine [Asn(N)], aspartic acid [Asp(D)], cysteine ​​[Cys(C)], glutamine [Gln(Q)], glutamic acid [Glu(E)], glycine [Gly(G)], histidine [His(H)], isoleucine [Ile(I)], leucine [Leu(L)], lysine [Lys(K)], methionine [Met(M)], phenylalanine [Phe(F)], proline [Pro(P)], serine [Ser(S)], threonine [Thr(T)], tryptophan [Trp(W)], tyrosine [Tyr(Y)], and valine [Val(V)], as well as non-natural or modified amino acids.

[0111] Cyclotides are small, disulfide-rich peptides isolated from plants. They typically contain 28-37 amino acids, with a head- and tail-cyclic peptide backbone and three interlocking disulfide bonds. Although the plant cyclotide family may contain macrocyclic peptides with potential CXXC and CXXXC motifs, their cytoprotective properties in mammalian cells are not similar to those of CDNF and MANF (i.e., protection against ER stress-induced cell dysfunction or cell death, such as apoptosis).

[0112] In one embodiment, the macrocyclic peptides claimed in this disclosure are not related to plant cyclic peptides or the plant cyclic peptide family.

[0113] Preferably, the peptides disclosed in this disclosure are not from the protein disulfide isomerase family of thioredoxin and / or proteins.

[0114] This disclosure provides a macrocyclic peptide or a pharmaceutically acceptable salt thereof composed of 8-32 amino acids of length, comprising the amino acid sequence C-X1-X2-X3-C (SEQ ID NO:27).

[0115] in

[0116] X1 can be selected from the groups composed of R, K, I, G, A, and S;

[0117] X2 either does not exist or is selected from the group consisting of G, A, R, K, I, and S; and

[0118] X3 can be selected from the groups consisting of A, G, and S.

[0119] In one embodiment, a macrocyclic peptide is a peptide having a bond between its N-terminus and C-terminus.

[0120] In a preferred embodiment, the macrocyclic peptide comprises the amino acid sequence E-X4-C-X1-X2-X3-CAE (SEQ ID NO:28).

[0121] in

[0122] X1 can be selected from the groups composed of R, K, I, G, A, and S;

[0123] X2 does not exist or can be freely selected from the group consisting of G, A, R, K, I, and S;

[0124] X3 can be selected from the groups composed of A, G, and S; and

[0125] X4 can be selected from the groups consisting of E, T, V, D, M, and G.

[0126] In one embodiment, a macrocyclic peptide is a peptide having a bond between its N-terminus and C-terminus.

[0127] Based on different species (humans, horses, bison, pigs, dogs, mice, hamsters, alligators, dolphins, and zebrafish), CDNF and MANF are used. Figure 10 Natural variations in the CDNF and MANF sequences in the example sequences (in the example sequences) allow for adaptation of the peptide sequences with limited changes to the X-group without loss of biological activity, compared to human sequences.

[0128] In another preferred embodiment, the macrocyclic peptide comprises the amino sequence X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO:29),

[0129] in

[0130] X1 can be selected from the groups composed of R, K, I, G, A, and S;

[0131] X2 does not exist or can be freely selected from the group consisting of G, A, R, K, I, and S;

[0132] X3 can be selected from the groups composed of A, G, and S;

[0133] X4 can be selected from the groups consisting of E, T, V, D, M, and G;

[0134] X5 does not exist or can be selected from the group consisting of H, D, Q, R, Y, N and S;

[0135] X6 does not exist or can be selected from the group consisting of S, D, G, N and R;

[0136] X7 does not exist or is W;

[0137] X8 does not exist or is G;

[0138] X9 does not exist or is K;

[0139] X 10 There is no group that can be selected from T, S, A, I, and N; and

[0140] X 11 It does not exist or is selected from D and E.

[0141] In one embodiment, a macrocyclic peptide is a peptide having a bond between its N-terminus and C-terminus.

[0142] In another preferred embodiment, the macrocyclic peptide comprises an amino acid sequence, which in X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -VX 24 -ELKX 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 Within the amino acid sequence of (SEQ ID NO:30),

[0143] in

[0144] X1 can be selected from the groups composed of R, K, I, G, A, and S;

[0145] X2 does not exist or can be freely selected from the group consisting of G, A, R, K, I, and S;

[0146] X3 can be selected from the groups composed of A, G, and S;

[0147] X4 can be selected from the groups consisting of E, T, V, D, M, and G;

[0148] X5 does not exist or can be selected from the group consisting of H, D, Q, R, Y, N and S;

[0149] X6 does not exist or can be selected from the group consisting of S, D, G, N and R;

[0150] X7 does not exist or is W;

[0151] X8 does not exist or is G;

[0152] X9 does not exist or is K;

[0153] X 10 There is no group, or the group consisting of T, S, A, I, and N is selected.

[0154] X 11 It does not exist or is selected from D and E.

[0155] X 12 There is no group that can be selected from L, I, and V;

[0156] X 13 It does not exist or is D;

[0157] X 14 It does not exist or is selected from L and W;

[0158] X 15 There is no group, or the group consisting of A, S, T, E, and N is selected.

[0159] X 16 It does not exist or is selected from S and T;

[0160] X 17 It does not exist or is selected from V and D;

[0161] X 18 It does not exist or is selected from D and A;

[0162] X 19 It does not exist or is L;

[0163] X 20 There is no group that can be formed by R, K, S, and W;

[0164] X 21 It either does not exist or is K;

[0165] X22 There is no group consisting of M, L, I, and V;

[0166] X 23 It does not exist or is R;

[0167] X 24 Choose the group consisting of A, K, T, L, and V;

[0168] X 25 Choose a group consisting of Q, K, and R; and

[0169] X 26 Selected from I and V.

[0170] In one embodiment, a macrocyclic peptide is a peptide having a bond between its N-terminus and C-terminus.

[0171] In some embodiments, the macrocyclic peptide comprises an 8-27 amino acid-long peptide within SEQ ID NO:28, wherein the macrocyclic peptide includes the CX1X2X3C (SEQ ID NO:27) motif.

[0172] In another preferred embodiment, the macrocyclic peptide comprises an amino acid sequence selected from the group consisting of:

[0173] X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -VX 24 -ELKX 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO:33),

[0174] X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -VX 24 -ELKX 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X10 (SEQ ID NO:34),

[0175] X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -VX 24 -ELKX 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9 (SEQ ID NO: 35), and

[0176] X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -VX 24 -ELKX 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE (SEQ ID NO: 36).

[0177] In some embodiments, the macrocyclic peptide comprises a peptide of 8-27 amino acids in length from any one of SEQ ID NO:33-36, wherein the macrocyclic peptide includes the CX1X2X3C (SEQ ID NO:27) motif.

[0178] In some cases, macrocyclic peptides contain amino acid sequences that may be within the amino acid sequence SEQ ID NO:45 or within the amino acid sequence SEQ ID NO:49.

[0179] Preferably, the macrocyclic peptide comprises or consists of a sequence selected from the group consisting of: VDLRKMRVAELKQILHSWGEECRACAE (SEQ ID NO:2), VDLKKLRVKELKKILDDWGETCKGCAE (SEQ ID NO:4), MRVAELKQILHSWGEECRACAEK (SEQ ID NO:6), LRVKELKKILDDWGETCKGCAEK (SEQ ID NO:8), KSILDDWGETCKGCAE (SEQ ID NO:10), WGEECRACAEKT (SEQ ID NO:12), WGETCKGCAEKS (SEQ ID NO:14), WGEECRGACAEKT (SEQ ID NO:24), and WGETCKGGCAEKS (SEQ ID NO:26).

[0180] In one implementation, the macrocyclic peptide protects against endoplasmic reticulum (ER) stress-induced cell dysfunction or cell death, such as apoptosis.

[0181] In one embodiment, a macrocyclic peptide is a peptide having a bond between its N-terminus and C-terminus.

[0182] In some cases, the macrocyclic peptide is 11-32 amino acids long. In some cases, the peptide is 12-32 amino acids long. In some cases, the peptide is 12-27 amino acids long. In some cases, the peptide is 8-27 amino acids long. In some cases, the peptide is 8-13 amino acids long. In some cases, the peptide is 8-12 amino acids long.

[0183] In a preferred embodiment, in the macrocyclic peptide, cysteine ​​(C) is in its reduced form or disulfide-bridged form.

[0184] In some embodiments, the macrocyclic peptides described herein are bound to GRP78.

[0185] In some respects, the macrocyclic peptides described herein are at least 1.5 times more stable than their linear counterparts. In one embodiment, the peptides described herein are at least 2, 3, or 4 times more stable than their linear counterparts.

[0186] In some respects, the macrocyclic peptide has a half-life at least 1.5 times longer than its linear counterpart. In one embodiment, the peptide described herein has a half-life at least 2, 3, or 4 times longer than its linear counterpart.

[0187] In some respects, macrocyclic peptides may include a linker that connects the N-terminus of the peptide to the C-terminus.

[0188] In some respects, the N-terminus of the peptide may be acetylated.

[0189] In some respects, the C-terminus of the peptide is amidated.

[0190] In some respects, the N-terminus of the peptide may be acetylated, and the C-terminus of the peptide may be amidated.

[0191] In another preferred embodiment, the macrocyclic peptide is conjugated with a detectable moiety, a chemical moiety, a biochemical moiety, or polyethylene glycol (PEG).

[0192] Macrocyclic peptides can be conjugated to detectable chemical or biochemical moieties such as fluorophores (e.g., fluorescein or rhodamine). Radiolabeling of the peptides can be used, for example, for SPECT or PET imaging. As used herein, “detectable chemical or biochemical moieties” refers to chemical tags exhibiting an amino acid sequence or a detectable chemical or biochemical moieties for the purpose of facilitating peptide detection; such as detectable molecules selected from: visible, fluorescent, chemiluminescent, or other detectable chemical tags; enzymes detectable in the presence of a substrate, such as alkaline phosphatases with NBT+BCIP or peroxidases with a suitable substrate; and detectable proteins, such as green fluorescent protein. Preferably, the tag does not prevent or hinder the fragment from penetrating into target cells or otherwise alter the biological activity of the compound.

[0193] It is also preferred to modify the C-terminal CDNF fragment or C-terminal MANF fragment with N and / or C-terminus to further increase the stability and / or cell permeability of the peptide or fragment. Acetylation-amidation (i.e. N-terminal acetylation and C-terminal amidation) of the CDNF fragment or MANF fragment is one of the known options in the art (see, for example, Marino et al. 2015, ACS Chem. Biol. [ACS Chemical Biology] 10:1754-1764).

[0194] For side-chain-side-chain cyclized peptides, acetylation-amidation at the peptide terminus (i.e., N-terminal acetylation and C-terminal amidation) increases peptide stability and cell permeability.

[0195] In some cases, macrocyclic peptides possess at least one of the following properties (e.g., 1, 2, 3, 4, 5, 6, or 7): (i) the peptide can dose-dependently protect TH-positive neurons from MPP. + Toxicity; (ii) The peptide reduces the number of α-synuclein inclusion bodies in TH-positive neurons; (iii) The peptide has improved stability in plasma compared to its linear counterpart; (iv) The peptide has improved stability in hepatocytes compared to its linear counterpart; or (v) The peptide has improved ability to cross the blood-brain barrier compared to its linear counterpart.

[0196] One implementation provides a macrocyclic peptide as described herein, which is used as a pharmaceutical agent.

[0197] Because CDNF / MANF peptides effectively protect dopamine neurons from death, existing technologies such as WO2009133247 and EP 1969003 show that the peptides can be used to treat central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease (PD), multiple system atrophy, amyotrophic lateral sclerosis (ALS), frontotemporal degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease (HD), traumatic brain injury, drug addiction, and stroke.

[0198] CDNF and MANF regulate signaling in the unfolded protein response (UPR) pathway and protect cells from ER stress-related cell death. ER stress is known to play a crucial pathophysiological role in a variety of chronic diseases, such as neurodegenerative and metabolic disorders and acute injuries (Wang and Kaufman, 2016). GRP78 (also known as BiP and HSPA5) is a major ER cavity chaperone and a key regulator of UPR (Bertolotti et al., 2000; Wang and Kaufman, 2016). The dynamic association and dissociation of GRP78 with UPR receptors IRE1α, PERK, and ATF6 are key steps in regulating UPR receptor signaling activity under ER stress. The interaction between MANF and GRP78 regulates its cellular activity (Yan et al., 2019).

[0199] Therefore, this disclosure relates to a method for treating degenerative, chronic, or progressive diseases or conditions (such as CNS diseases or conditions) or single-gene inherited diseases (which have ER stress as a pathogenic component), wherein a pharmaceutically effective amount of a macrocyclic peptide of length 8-32 amino acids or a pharmaceutically acceptable salt thereof is administered to a patient, said macrocyclic peptide or pharmaceutically acceptable salt thereof comprising the sequence C-X1-X2-X3-C (SEQ ID NO:27), E-X4-C-X1-X2-X3-CAE (SEQ ID NO:28), X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO:29) or X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22-X 23 -VX 24 -ELKX 25 -X 26 -L-X5 X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO:30).

[0200] Another implementation provides macrocyclic peptides for treating degenerative, chronic, or progressive diseases or conditions, such as neurodegenerative diseases or conditions.

[0201] The neurodegenerative diseases or disorders are preferably central nervous system diseases selected from the group consisting of: Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease, traumatic brain injury, traumatic spinal cord injury, progressive supranuclear palsy, Pick's disease, simple autonomic failure, corticobasal degeneration, chronic traumatic encephalopathy, spinocerebellar ataxia, bipolar disorder, and peripheral neuropathy, and the scope of their diseases and symptoms.

[0202] Neurodegenerative diseases can have partially overlapping, dynamic, and non-linear progressive "dimensions" present in a wide range of neuroproteopathies. The expression of several combinations of various proteases within the central nervous system can be variable. Therefore, the coexistence of mixed neuropathologies may be observed in patients. The genetic spectrum of neurodegenerative diseases can differ; for example, different diseases may manifest in identical twins with the same genotype.

[0203] Another implementation provides a macrocyclic peptide for treating monogenic hereditary diseases selected from the group consisting of: Wolcott-Rallison syndrome, Wolfram syndrome, Marinesco- Syndromes, Machado-Joseph disease, and degenerative retinal diseases such as retinitis pigmentosa, as well as inherited nephrotic syndromes such as primary nephrotic syndrome and autosomal dominant polycystic kidney disease. These monogenic inherited diseases are those with ER stress as a pathogenic component.

[0204] One embodiment provides a macrocyclic peptide for use according to the present disclosure, wherein the peptide is administered by: peripheral administration, such as intravenous, intra-arterial, subcutaneous, intranasal, intraocular, intratympanic, or local administration; enteric, parenteral, or local routes, including oral, rectal, sublingual, or buccal administration; intraperitoneal, intramuscular, intra-articular, transdermal, intracochlear, ocular, or inhalation administration; or intracranial, intrathecal, epidural, or intralesional administration.

[0205] In one implementation, the macrocyclic peptide is administered subcutaneously.

[0206] Pharmaceutical Composition

[0207] One or more macrocyclic peptides disclosed herein can be formulated for use in pharmaceutical compositions or for use in pharmaceutical compositions. Such compositions can be formulated or modified for administration to a subject via any route, such as any route approved by the appropriate authority.

[0208] One embodiment provides a pharmaceutical composition comprising the macrocyclic peptide described herein and at least one of the following: a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a preservative, a stabilizer, and / or a diluent.

[0209] In one embodiment, this disclosure also relates to a pharmaceutical composition comprising a macrocyclic peptide of length 8-32 amino acids or a pharmaceutically acceptable salt thereof, said macrocyclic peptide or pharmaceutically acceptable salt thereof comprising the sequence C-X1-X2-X3-C (SEQ ID NO:27), E-X4-C-X1-X2-X3-CAE (SEQ ID NO:28), X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO:29) or X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -VX 24 -ELKX 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO:30).

[0210] In some cases, a pharmaceutical composition may contain an effective amount of one or more macrocyclic peptides. As used herein, the terms “effective amount” and “effective treatment” refer to the amount or concentration of one or more compounds or pharmaceutical compositions described herein used over a period of time (including acute or chronic administration and periodic or continuous administration) that is effective in producing the intended effect or physiological outcome when administered.

[0211] In one embodiment of this disclosure, macrocyclic peptides may be incorporated into pharmaceutical compositions. Such compositions of this disclosure are prepared for storage by mixing peptides of desired purity with optional physiologically acceptable carriers (such as nanocarriers), excipients, buffers, or stabilizers (Remington's Pharmaceutical Sciences, 22nd edition, edited by Allen, Loyd V., Jr., (2012)). The peptides are in the form of lyophilized cakes or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronic, polyethylene glycol (PEG), or excipients for enhancing naso-to-brain delivery such as chitosan, methylated pectin, alkyl sugar-based mucosal absorption enhancers, and hydroxy fatty acyl esters of PEG.

[0212] The actual dose (e.g., effective amount) of the peptide administered to a patient can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, prior or concurrent therapeutic interventions, the patient's spontaneous disease, and the route of administration. The physician responsible for administration can determine the concentration of one or more active ingredients in the composition and one or more appropriate doses for the individual subject.

[0213] The peptides can also be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by coarse-grained techniques or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in coarse-drop emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 22nd edition, edited by Allen, Loyd V., Jr., (2012). Controlled-release gel formulations can also be used.

[0214] In one embodiment, the pharmaceutical composition may comprise, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may comprise, for example, about 2% to about 75% per unit weight, or about 25% to about 60%, and any range thereof.

[0215] In other non-limiting examples, the dosage of the pharmaceutical composition or formulation may include, per administration, about 1 ng / kg / body weight of macrocyclic peptide, about 5 ng / kg / body weight, about 10 ng / kg / body weight, about 50 ng / kg / body weight, about 100 ng / kg / body weight, about 200 ng / kg / body weight, about 350 ng / kg / body weight, about 500 ng / kg / body weight, 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 1 ... Peptides in the range of 0 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight, to about 1000 mg / kg / body weight, or more, and any ranges derived therefrom. In a non-limiting example of the ranges from which the figures listed herein can be derived, peptides in the range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, or about 5 μg / kg / body weight to about 500 mg / kg / body weight, can be administered based on the figures described above.

[0216] The methods described herein consider administering an effective amount of a compound or compound composition to achieve a desired or stated effect. Typically, the pharmaceutical compositions of this disclosure will be administered about 1 to about 6 times daily, such as 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, or 2-5 times daily, or alternatively, as a continuous infusion. The pharmaceutical compositions may be administered, for example, 1, 2, 3, 4, 5, or 6 times daily. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration. Typical formulations will contain about 5% to about 95% (w / w) of the active compound. Alternatively, such formulations contain about 20% to about 80% of the active compound.

[0217] Various techniques can be used to determine the dosage. The selected dosage level can depend on a variety of factors, including, for example, the activity of the specific compound used, the route of administration, the time of administration, the rate of excretion or metabolism of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific compound used, the age, sex, weight, condition, general health status and / or medical history of the patient being treated, and similar factors well known in the medical field. Dosage values ​​can also be varied depending on the severity of the condition to be alleviated. For any particular subject, the specific dosage regimen can be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the composition.

[0218] In some respects, the appropriate daily dose of the compounds disclosed herein may be the amount of the lowest dose at which the compound effectively produces a therapeutic effect. Such an effective dose will generally depend on the factors mentioned above. The precise timing and amount of administration of any particular compound that will produce the most effective treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of the particular compound, the patient's physiological condition (including age, sex, disease type and stage, general physical condition, response to a given dose and type of drug), route of administration, etc.

[0219] A doctor or veterinarian can prescribe an effective amount of the desired pharmaceutical composition. For example, a doctor or veterinarian can start with a dose of the disclosed compound used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0220] The pharmaceutical compositions described herein can be unit dosage forms suitable for precise single-dose administration. In a unit dosage form, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit dose can be in the form of packaging containing discrete amounts of the formulation. A non-limiting example is a liquid in a vial or ampoule. Aqueous suspension compositions can be packaged in single-dose, non-reclosed containers. For example, multi-dose, reclosed containers can be used in combination with preservatives. Formulations for parenteral injection can be presented in unit dosage forms, such as in ampoules or multi-dose containers containing preservatives.

[0221] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with the compounds of the present invention, and that is non-toxic and does not impair the pharmacological activity of the compound when administered at a dose sufficient to deliver a therapeutic amount.

[0222] Pharmaceutically acceptable carriers, adjuvants, and mediators that may be used in the pharmaceutical compositions disclosed herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as D-α-tocopherol polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tween or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, saturated mixtures of partial glycerides of vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0223] The pharmaceutical compositions disclosed herein may contain any conventionally non-toxic, pharmaceutically acceptable carrier, adjuvant, or mediator. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form. As used herein, the term parenteral includes parenteral, epidural, subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques.

[0224] The effective amount of the compounds disclosed herein can be administered by any acceptable route of administration, in single or multiple doses (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any suitable amount). The number of doses can be within the range defined by any two of the values ​​above. Regardless of the chosen route of administration, the compounds and / or pharmaceutical compositions of this disclosure are formulated into pharmaceutically acceptable dosage forms. The compounds according to this disclosure can be formulated by analogy to other pharmaceuticals for administration in any convenient manner for use in humans or veterinary medicine.

[0225] In one aspect, this disclosure provides pharmaceutical formulations comprising a therapeutically effective dose of one or more of the aforementioned compounds formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. In another aspect, one or more of the compounds described herein are formulated for parenteral administration. For parenteral administration, one or more of the compounds disclosed herein may be formulated as aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, or sterile powders, which may be reformed into sterile injectable solutions or dispersions prior to use. Such formulations may contain sugars, alcohols, antioxidants, buffers, antibacterial agents, solutes or suspending or thickening agents that make the formulation isotonic with the blood of the intended recipient. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action on the subject compound can be ensured by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin. If desired, the formulation can be diluted with, for example, an isotonic saline solution or a dextran solution prior to use. In some embodiments, the compound is formulated as an aqueous solution and administered intravenously.

[0226] Pharmaceutical compositions may be in the form of injectable solutions or powders. Such compositions may be formulated using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents, according to techniques known in the art. Sterile injectable formulations may also be sterile injectable solutions or suspensions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable media and solvents that may be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild fixed oil may be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glycerol derivatives may be used in the preparation of injectable formulations, as may natural, pharmaceutically acceptable oils such as olive oil or castor oil (especially their polyoxyethylene forms). These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethyl cellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants such as Tween or Spans and / or other similar emulsifiers or bioavailability enhancers (typically used to manufacture pharmaceutically acceptable solid, liquid or other dosage forms) may also be used for formulation purposes.

[0227] Pharmaceutical compositions can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. In the case of tablets for oral administration, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When administered orally in aqueous suspensions and / or emulsions, the active ingredient can be suspended or dissolved in an oil phase combined with an emulsifier and / or suspending agent. Sweeteners and / or flavoring agents and / or coloring agents may be added if desired.

[0228] The pharmaceutical compositions disclosed herein can also be administered in the form of suppositories for rectal use. These compositions can be prepared by mixing the compositions of this disclosure with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0229] Alternatively or additionally, the pharmaceutical composition may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the field of pharmaceutical formulation and may be prepared as saline solutions using benzyl alcohol or other suitable preservatives, absorption enhancers (to improve bioavailability), fluorocarbons, and / or other solubilizers or dispersants known in the art.

[0230] In some cases, one or more peptides disclosed herein may be conjugated, for example, to a carrier protein. Such conjugated compositions may be monovalent or polyvalent. For example, a conjugated composition may include one peptide disclosed herein conjugated to a carrier protein. Alternatively, a conjugated composition may include two or more peptides disclosed herein conjugated to a carrier.

[0231] This document provides methods for using the peptides described herein. For example, the methods provided herein may include administering the peptides as described herein to a patient. Patients may include both mammals and non-mammals.

[0232] Pharmaceutically acceptable carriers can be selected based on the chosen route of administration and standard pharmaceutical practices. For example, compositions can be formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release formulations, or elixirs for oral administration, or sterile solutions or suspensions for parenteral and intraperitoneal injection, as well as transdermal patch formulations, dry powder inhalers, and ointments (see, for example, Ansel, Introduction to Pharmaceutical Dosage Forms, 4th edition, 1985, p. 126). Peptides and / or immunoglobulins can be formulated into dosage forms according to standard practices in the pharmaceutical formulation field. See Alphonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th edition (1990), Mack Publishing Co., Easton, PA.

[0233] For parenteral administration, pharmaceutical compositions may contain suitable carriers or diluents such as water, oils (especially vegetable oils), ethanol, salt solutions, aqueous dextran (glucose) and related sugar solutions, glycerol, or glycols such as propylene glycol or polyethylene glycol. Solutions for parenteral administration preferably contain water-soluble salts of peptides and / or active agents. Stabilizers, antioxidants, and preservatives may also be added. Suitable antioxidants include sulfites, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. Compositions for parenteral administration may be in the form of aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions.

[0234] For oral administration, the pharmaceutical composition may contain one or more solid inactive ingredients for use in the preparation of tablets, capsules, pills, powders, granules, or other suitable oral dosage forms. For example, the pharmaceutical composition may contain at least one excipient, such as a filler, binder, wetting agent, disintegrant, solution barrier, absorption enhancer, humectant, absorbent, or lubricant.

[0235] A further feature of this disclosure is a pharmaceutical composition that may further comprise nerve cells. The nerve cells may be, for example, neurons, neural stem cells, or neuronal precursor cells.

[0236] This disclosure relates to pharmaceutical compositions used as pharmaceutical agents, comprising a macrocyclic peptide as described herein and at least one of the following: a pharmaceutically acceptable carrier, excipient, preservative, stabilizer, and / or diluent.

[0237] In one treatment method, a pharmaceutically effective amount of a macrocyclic peptide as defined herein is administered to a patient. In other words, the macrocyclic peptide according to this disclosure is used to treat degenerative, chronic, or progressive diseases or conditions (such as CNS diseases or conditions) or single-gene inherited diseases (which have ER stress as a pathogenic component).

[0238] The pharmaceutical composition is used to treat degenerative, chronic, or progressive diseases or disorders, such as neurodegenerative diseases or disorders.

[0239] The neurodegenerative diseases or conditions referred to are central nervous system diseases selected from the group consisting of: Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease, traumatic brain injury, traumatic spinal cord injury, progressive supranuclear palsy, Pick's disease, pure autonomic failure, corticobasal degeneration, chronic traumatic encephalopathy, spinocerebellar ataxia, bipolar disorder, and peripheral neuropathy.

[0240] In one embodiment, the pharmaceutical composition is administered subcutaneously.

[0241] The macrocyclic peptides are administered via the same routes as known methods and are generally administered by injection or infusion, via the following routes: intravenous, intra-arterial, subcutaneous, intranasal, intraocular, intratympanic, or local administration; enteric, parenteral, or local routes, including oral, rectal, sublingual, or buccal administration; intracranial, intrathecal, or epidural administration; intraperitoneal, intramuscular, intra-articular, transdermal, intracochlear, ocular, intralesional, or inhalation administration; or via sustained-release systems as described below.

[0242] Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing the peptide, said matrix being in the form of a molded article (e.g., a membrane) or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels, or polyvinyl alcohol, polylactic acid (US Patent No. 3,773,919, EP 58,481), or non-degradable ethylene-vinyl acetate (Langer et al., see above) as described in Langer et al., J. Biomed. Mater. Res. [Research on Biomedical Materials], 15:167-277 (1981) and Langer, Chem. Tech. [Chemical Techniques], 12:98-105 (1982).

[0243] This disclosure also relates to a method for treating degenerative, chronic, or progressive diseases or conditions (such as CNS diseases or conditions) or single-gene inherited diseases (which have ER stress as a pathogenic component), wherein a pharmaceutically effective amount of a macrocyclic peptide as defined herein is administered to a patient. Preferably, the fragment is administered peripherally.

[0244] This disclosure also relates to the use of macrocyclic peptides as defined herein in the preparation of medicaments for the treatment of degenerative, chronic or progressive diseases or conditions (such as CNS diseases or conditions) or monogenic inherited diseases (which have ER stress as a pathogenic component).

[0245] This disclosure relates to a method for treating a subject with a degenerative, chronic, or progressive disease or condition (such as a neurodegenerative disease or condition), the method comprising administering to the subject a pharmaceutical composition comprising a macrocyclic peptide as described herein.

[0246] In one embodiment, a method for treating neurodegenerative diseases or conditions (such as central nervous system diseases) includes administering to a subject a pharmaceutical composition comprising a macrocyclic peptide as described herein, wherein the central nervous system disease is selected from the group consisting of: Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease, traumatic brain injury, traumatic spinal cord injury, progressive supranuclear palsy, Pick's disease, pure autonomic failure, corticobasal degeneration, chronic traumatic encephalopathy, spinocerebellar ataxia, bipolar disorder, and peripheral neuropathy.

[0247] This disclosure relates to a method for treating a single-gene hereditary disease selected from the group consisting of: Wolcott-Rallison syndrome, Wolfram syndrome, Marinesco- Syndromes, Machado-Joseph disease, and degenerative retinal diseases such as retinitis pigmentosa, as well as inherited nephrotic syndromes such as primary nephrotic syndrome and autosomal dominant polycystic kidney disease, the method comprising administering to a subject a pharmaceutical composition containing a macrocyclic peptide as defined herein. The monogenic inherited diseases have ER stress as a pathogenic component.

[0248] The subjects who need to be tested can be human.

[0249] The macrocyclic peptides or pharmaceutical compositions comprising said peptides of this disclosure can be administered continuously by infusion or bolus injection. Generally, where barriers permit, said fragments should be formulated and administered for site-specific delivery. Administration can be continuous or periodic. Administration can be accomplished by an implantable pump with a constant or programmable flow or by periodic injection. Peripheral or systemic administration is preferred because this disclosure demonstrates that macrocyclic peptides can effectively penetrate across neuronal cell membranes and cross the blood-brain barrier in vitro and in vivo (respectively). Figure 8 and 9B Other preferred routes of administration are subcutaneous, intrathecal, intraventricular, intranasal, or transdermal.

[0250] In another embodiment, this disclosure provides a method for promoting the survival of dopaminergic neurons, comprising contacting the dopaminergic neurons with a macrocyclic peptide comprising 8-32 amino acids of the sequence SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. Preferably, the method is performed in vitro, as shown in the Experimental Section below. The dopaminergic neurons are preferably cultured non-human neurons, such as mouse or rat sympathetic neurons, or human neurons derived from induced pluripotent cells (iPSCs).

[0251] Based on the results provided in this disclosure, this disclosure also relates to a macrocyclic peptide of 8-32 amino acids in length comprising the peptides of sequences SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30, for the treatment of degenerative, chronic or progressive diseases or conditions (such as CNS diseases or conditions) or single-gene hereditary diseases (which have ER stress as a pathogenic component).

[0252] Methods for preparing macrocyclic peptides

[0253] Methods for synthesizing the compounds disclosed herein are known in the art. The following exemplary methods can be used. It should be understood that the steps can be performed in an alternating order or sequence to obtain the desired compound. Synthetic chemical transformations and protecting group methods (protection and deprotection) for synthesizing the compounds described herein are known in the art and include, for example, methods described in the following literature: R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G. M. Tauts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0254] The peptides disclosed herein can be prepared by chemical synthesis methods well known to those skilled in the art. See, for example, Fields et al., Synthetic Peptides: A User's Guide, Chapter 3, edited by Grant, WH Freeman & Co., New York, 1992, p. 77. One method for preparing the peptides described herein is using solid-phase peptide synthesis (SPPS). The C-terminal amino acid is linked to a cross-linked polystyrene resin via an acid-labile bond with a linker molecule. This resin is insoluble in the solvent used for synthesis, making the washing away of excess reagents and byproducts relatively simple and rapid. The N-terminus is protected by an Fmoc group, which is stable in acid but can be removed by a base. Any side chain functional groups are protected by base-stable, acid-labile groups.

[0255] The publications and other materials used herein illustrate the background of the invention, and in particular provide additional details regarding its implementation, which are incorporated herein by reference.

[0256] It will be apparent to those skilled in the art that the basic ideas of this disclosure can be implemented in various ways as technology advances. Therefore, this disclosure and its embodiments are not limited to the following examples, but can vary within the scope of the claims.

[0257] Example

[0258] Example 1

[0259] Neuroprotective effects of linear and macrocyclic compounds on dopaminergic TH-positive neurons damaged by MPP+

[0260] The neuroprotective effects of compound 1-26 (SEQ ID NO: 1-26) were tested in an in vitro model in which primary cultures of rat embryonic brain neurons were stressed with MPP+ (an active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)). MPP+ kills dopaminergic (TH-positive) neurons via a variety of toxic mechanisms, including mitochondrial dysfunction, production of peroxynitrite, oxidative stress, ER stress, and apoptosis induction. Because peptides derived from the C-terminal domains of CDNF and MANF were modified into pseudopeptides, their neuroprotective activity was tested in a model in which full-length CDNF protein demonstrated neuroprotective protection against MPP+-induced damage to TH+ dopaminergic neurons, their synapses, and neurite networks. Figure 3A And reduce the accumulation of α-synuclein aggregates in TH+ neurons. Figure 3B ).

[0261] Materials and methods for the synthesis and characterization of head- and tail-cyclic derivatives of CDNF and MANF, as well as linear peptides.

[0262] General Scheme - Resin Loading

[0263] Resins with auxiliary moieties (cryptothioester Gly-Cys(Hnb)-NH) were prepared for all syntheses. For this purpose, Fmoc-Gly-OH and Fmoc-Cys(StBu)-OH were coupled to TentaGel R RAM resin (0.19–0.26 mmol / g) using 4 equivalents of the corresponding amino acid and equimolar amounts of DIC (0.5 M, in DMF) and OxymaPure (0.5 M, in DMF) as coupling agents. After removing the Fmoc group by treatment with 20% piperidine in DMF, reductive amination was performed using 2-hydroxy-5-nitrobenzaldehyde and sodium cyanoborohydride according to the original procedure (Terrier et al., 2016). The first amino acid (Ala or Gly) is then coupled using 10 equivalents of Fmoc-AA-OH, 9.5 equivalents of HCTU or 10 equivalents of PyBOP / HOBt and 20 equivalents of DIPEA, followed by capping via acetic anhydride / DIPEA. This assisted resin is used for further solid-phase peptide synthesis.

[0264] General Solution - SPPS

[0265] Solid-phase peptide synthesis was performed on an automated peptide synthesizer (Biotage Initiator + Alstra or Activotec Activo-P11). Standard Fmoc-protected amino acids were used for peptide elongation: Ala, Arg (Pbf), Asp (tBu), Gln (Trt), Glu (OtBu), Gly, His (Trt), Ile, Lys (Boc), Leu, Met, Ser (tBu), Thr (tBu), Trp (Boc), Val, Cys (Trt), and Cys (StBu). Fmoc groups were removed using 20% ​​piperidine in DMF, and coupling was performed using 4 equivalents of the corresponding amino acid, 3.9 equivalents of HBTU, 4 equivalents of HOBt, and 8 equivalents of DIPEA under microwave irradiation or at room temperature. The crude peptides were deprotected and cleaved from the resin by treatment with TFA / H2O / iPr3SiH for 2 h, followed by precipitation in cold Et2O and lyophilization.

[0266] General approach - Natural chemical linkage (NCL) and SS oxidation

[0267] Head-to-tail cyclization was performed using an NCL between the N-terminal Cys and the C-terminal cryptothioester Gly-Cys (Hnb). An NCL buffer (0.2 M phosphate buffer, pH 6.4, containing 6 M Gn*HCl, 25 mM MPAA, and 50 mM TCEP*HCl) was prepared according to a published protocol (Terrier et al., 2016). The linear peptide was dissolved in the buffer to a concentration of 1 mM, and the reaction mixture was stirred overnight at 38 °C. The cyclic peptide was separated by RP-HPLC using an appropriate gradient. The purified peptide was lyophilized, then dissolved in acetic acid and treated with 20 mM iodine in MeOH until a disulfide bridge was formed. The target peptide was then purified by RP-HPLC.

[0268] The purity of the peptide was assessed using standard HPLC methods, and its identity was confirmed using standard LC-MS procedures. The mass of charged ions obtained from the peptide during the process is visible in [the following data is missing from the original text]. Figure 1 .

[0269] Materials and methods for synthesizing linear derivatives of CDNF and MANF.

[0270] As previously described, linear peptides were synthesized using a standard SPPS protocol. Purity and identity were determined in the same manner. The amount of peptide was confirmed using an LC-MS procedure. The mass of the charged ions obtained during the process is visible in [the data]. Figure 1 .

[0271] The cyclization of the head and tail cyclic derivatives of CDNF and MANF was confirmed using NMR.

[0272] All head and tail cyclic peptides were dissolved at 25°C in a buffer solution containing 20 mM sodium phosphate (pH 6.0), 10 v / v D₂O, 1 mM EDTA, 100 mM NaCl, 3 μM NaN₃, and 0.5 mM DSS. Their concentrations were recorded on a 500 MHz spectrometer. 1 H / 13 C1D and 2DNMR spectra.

[0273] Head-tail cyclic peptide compounds mainly use 1D 1 H, 2D 1 H TOCSY, 2D 1 H ROESY and 2D 1 H- 13 C HSQC NMR analysis ( Figure 2A-2P Record the 1D of compounds 2, 4, 6, 8, 10, 12, 14, 24, and 26. 1 H and 2D 1H TOCSY NMR data were used to confirm head- and tail-cyclization. All main-chain amides should each have one proton, which is incorrect for the corresponding linear peptides, where the N-terminus has one acid proton (peptide acid) or two amide protons (peptide amide). Peak profiles of the N-terminal amide protons for compounds 2, 4, 6, 8, 10, 12, 14, 24, and 26 were determined and analyzed at [data missing]. Figure 2B , 2D The numbers 2F, 2H, 2J, 2L, 2N, 2P, and 2R indicate this. In all cases, the number of amide protons observed is consistent with the expected number of amide protons in the head-tail cyclized peptide of the specific amino acid (unlike the corresponding linear analogs, which do not include the N-terminus). This in itself indicates that the compound is cyclized. However, when based on the LC-MS data of the compound (e.g., ... Figure 1 The presented data, when interpreted, doublely confirmed that the compound is a cyclized compound.

[0274] Due to spectral overlap between NH and Hα, some i1-i23 / 27 ROE correlations could not be definitively verified (for compounds 2, 6, and 8). For the remaining peptides (compounds 4, 10, 12, 14, 24, and 26), 2D ROEs were also collected. 1 H ROESY data are sufficient to independently verify the through-space ROE correlation between i1:HN and i(12 / 13 / 23):_Hα, i.e., the sequential HN(i)-H_α(i_-1) correlation, to support the cyclic structure (and these coupled protons are less than [missing information]). ). 2D of these compounds (compounds 4, 10, 12, 14, 24, and 26) 1 The expansion of the H ROESY amide fingerprint region is respectively shown in Figure 2D , 2J In 2L, 2N, 2P, and 2R, the critical sequence ROE cross-peak is marked at the corresponding H. N frequency.

[0275] Materials and methods related to the testing of the neuroprotective activity of compounds

[0276] Culture of midbrain neurons. Rat dopaminergic neurons were cultured as described by Visanji et al., 2008. Briefly, the midbrain was dissected from 15-day-old rat embryos (Janvier, France), and the ventral portion of the midbrain convexity (a region of the developing brain rich in dopaminergic neurons) was used for cell preparation. Midbrain cells were dissociated by trypsin digestion for 20 min at 37°C (final solution concentrations of 0.05% trypsin and 0.02% EDTA). The reaction was terminated by adding Dürbeco Modified Eagle's Medium (DMEM) containing DNase I-II (0.5 mg / mL) and 10% fetal bovine serum (FCS). Cells were then mechanically dissociated by passing through a 10 mL pipette three times. Cells were then centrifuged at 180 x g for 10 min at +4°C on a layer of BSA (3.5%) in L15 medium. The cell pellet was resuspended in serum-free culture medium supplemented with Neurobasal (Invitrogen) containing B27 (2%), L-glutamine (2 mM), and 2% PS solution, along with 10 ng / mL brain-derived neurotrophic factor (BDNF) and 1 ng / mL glial-derived neurotrophic factor (GDNF). Viable cells were counted using a Neubauer hematology counter using a trypan blue exclusion test. Cells were seeded at a density of 40,000 cells / well in 96-well plates (pre-coated with poly-L-lysine) and incubated in a humidified incubator at 37°C under 5% CO2 / 95% air atmosphere. Half of the medium was replaced with fresh medium every 2 days. Only 60 wells were used in each 96-well plate. To avoid any edge effects, the first and last lines, as well as the column, were not used for culture and were filled with sterile water.

[0277] Test compounds and MPP+ exposure.

[0278] CDNF, as well as linear and macrocyclic compounds 1-24 (SEQ ID NO: 1-26), were tested. On day 6 of culture, the compounds were dissolved in the culture medium and then pre-incubated with midbrain neurons for 4 hours, followed by the application of MPP+. Four hours after the pre-incubation of the compounds, MPP+ was added to a final concentration of 4 μM and diluted for 48 hours in control medium still containing the compounds.

[0279] Immunostaining: Survival neurite network of TH neurons and α-syn aggregation in TH neurons.

[0280] Forty-eight hours post-poisoning, cells were fixed for 20 minutes at room temperature in PBS (pH 7.3) with 4% paraformaldehyde. Cells were washed twice in PBS and then blocked at permeabilized and nonspecific sites for 15 minutes at room temperature with PBS containing 0.1% saponin and 1% FCS. Next, cells were incubated for 2 hours at room temperature in PBS containing 1% FCS and 0.1% saponin with: (a) a monoclonal antibody against tyrosine hydroxylase (TH) produced in mice, diluted 1:10000; and (b) a polyclonal antibody against α-synuclein (α-syn) produced in rabbits, diluted 1:400. After 1 hour at room temperature in PBS containing 1% FCS and 0.1% saponin, these antibodies were observed with a 1:800 dilution of the secondary antibody Alexa Fluor 488 conjugated goat anti-mouse IgG and a 1:400 dilution of Alexa 568 conjugated goat anti-rabbit IgG.

[0281] Synaptic immunostaining: TH neurons and PSD-95 (overlap between TH / PSD-95 neurons).

[0282] Forty-eight hours after poisoning, the cell culture supernatant was removed, and the cells were fixed for 20 min at room temperature in PBS (pH 7.3) with 4% paraformaldehyde. The cells were washed twice in PBS and then blocked at permeabilized and nonspecific sites for 15 min at room temperature with PBS containing 0.1% saponin and 1% FCS. The cells were then incubated with: a) a mouse monoclonal anti-tyrosine hydroxylase (TH) antibody at a 1:10000 dilution in PBS containing 1% FCS and 0.1% saponin for 2 h at room temperature; and b) a rabbit polyclonal anti-postsynaptic density protein-95 (PSD-95) antibody at a 1:200 dilution in PBS containing 1% FCS and 0.1% saponin for 2 h at room temperature. This antibody specifically stains synapses. At room temperature, in PBS containing 1% FCS and 0.1% saponins for 1 hour, these antibodies were observed with Alexa Fluor 488 conjugated goat anti-mouse IgG diluted 1:800 and with Alexa Fluor 568 conjugated goat anti-rabbit IgG diluted 1:400.

[0283] For each condition, ImageXpress (Molecular Devices) is used to automatically acquire images representing the entire aperture area at 10x magnification (20 images for TH and α-syn) or 40x magnification (60 images for TH and PSD-95). The following readouts are automatically determined using the Custom Module Editor (Molecular Devices):

[0284] Analysis of the total number of -TH neurons (TH-positive neurons)

[0285] Total neurite network of -TH positive neurons (in μm)

[0286] The number of α-syn inclusion bodies in TH-positive neurons (overlap between TH and α-syn staining)

[0287] The number of synapses in TH-positive neurons (overlap of TH and PSD-95, in μm) 2 (Unit: )

[0288] The total number of TH+ neurons, the total neurite network of TH+ neurons, and the number of synapses of TH+ neurons are presented in... Figure 3A (CDNF) Figure 3C (Compounds 1 and 2) Figure 3D (Compounds 3 and 4) Figure 3G (Compounds 5 and 6) Figure 3I (Compounds 7 and 8) Figure 3K (Compounds 9 and 10) Figure 4A (Compounds 11 and 12) Figure 4C (Compounds 13 and 14) Figure 4I (Compounds 23 and 24) and Figure 4K (Compounds 25 and 26)

[0289] The total number of TH+ neurons is presented in Figure 4E (Compounds 15, 16, 17 and 18) Figure 4G (Among compounds 19, 20, 21 and 22).

[0290] Following MPP+ injury, α-synuclein aggregation in TH-positive neurons of primary cultures of midbrain cells was observed. Figure 3B (CDNF) Figure 3D (Compounds 1 and 2) Figure 3F (Compounds 3 and 4) Figure 3H (Compounds 5 and 6) Figure 3J (Compounds 7 and 8) Figure 3L (Compounds 9 and 10) Figure 4B (Compounds 11 and 12) Figure 4D (Compounds 13 and 14), Figure F (Compounds 15, 16, 17 and 18), Figure 4H (Compounds 19, 20, 21 and 22) Figure 4J (Compounds 23 and 24) and Figure 4L (Compounds 25 and 26)

[0291] Data show that many macrocyclic peptides dose-dependently protect TH-positive neurons, their neurites, and synapses from MPP+ toxicity. Furthermore, these macrocyclic compounds effectively reduce the number of α-Syn inclusion bodies in TH-positive neurons, the aggregation of which is strongly induced by MPP+. In most cases, the potency of the macrocyclic peptides (compounds 2, 6, 8, 10, 12, 14, 24, and 26) is comparable to that of their linear counterparts (compounds 1, 5, 7, 9, 11, 13, 23, and 25, respectively).

[0292] Example 2

[0293] Interaction with the ER stress response pathway

[0294] CDNF and MANF protect cells from ER-induced cellular dysfunction or death, such as apoptosis, by modulating cellular responses to ER stress. Yan et al. (2019) showed that the C-terminus of MANF binds to the nucleotide-binding domain (NBD) of GRP78 and modulates its cellular activity. This data suggests that MANF (and CDNF) have a regulatory interaction with GRP78 (the most abundant chaperone protein in the ER cavity), rather than a substrate-like interaction. The binding of compounds to GRP78-NBD was assessed in a cell-free binding assay using purified recombinant GRP78-NBD and synthetic peptides. Since GRP78 also acts as a key ligand for three receptors in the unfolded protein response (UPR) pathway—IRE1α, PERK, and ATF6—the neuroprotective effects of the compounds were also tested for dependence on UPR signaling. Figure 5A Molecular modeling of compound 12 in the MANF binding pocket of GRP78-NBD is shown. Figure 5B The binding affinity of the selected compound to GRP78-NBD in the cell-free binding assay is shown. Figure 5C The neuroprotective effects of compounds 10 and 14 were eliminated in the presence of pharmacological inhibitors of PERK (GSK2606414) and IRE1α (KIRA6).

[0295] Materials and Methods

[0296] Molecular modeling.

[0297] Based on the previously resolved structure of the GRP78-NBD:MANF complex, using Schrodinger suite version 2018-4 ( The PRIME module of LLC, USA, via the MAESTRO interface, was used to analyze GRP78-NBD (complexed with different compounds mentioned herein) Figure 2A-2PModeling was performed using a template structure. The generated model was manually reviewed (PDB:6HAB, Yan et al., 2019). The model was also validated by examining its Ramachandran plot.

[0298] Cell-free binding assay.

[0299] His-tagged GRP78-NBD was recombinantly overexpressed and purified from *E. coli* cells and labeled with NHS-red dye (Nanotemper Technologies, Inc.). The His tag was cleaved from the protein using a TEV protease. Binding of different peptides (serial dilutions) to the labeled, untagged GRP78-NBD was measured in a Monolith NT standard capillary at high power in a PBS environment using a Monolith NA device (Nanotemper Technologies, Inc.). Figure 5B List data obtained from the MST combined experiment is shown.

[0300] Culture of midbrain neurons.

[0301] Neuronal cell culture, MPP+ poisoning, immunostaining, and neuroprotective effects analysis of the compounds were performed as in Example 1. One hour before adding the test compounds, either the PERK inhibitor GSK2606414 (2 μM, Sigma) or the IRE1α inhibitor KIRA6 (2 μM, Sigma) was added to the culture.

[0302] This data shows that macrocyclic compounds bind to GRP78, a key target molecule for ER stress regulation, and that the neuroprotective effects of macrocyclic compounds depend on UPR signaling activity.

[0303] Example 3

[0304] In vitro metabolic stability of linear and macrocyclic compounds in rat plasma.

[0305] In this embodiment, the metabolic stability of the macrocyclic compound was tested. Since metabolic stability is relevant to compounds planned for parenteral administration in vivo, rat plasma stability studies were conducted using both macrocyclic and linear compounds.

[0306] Materials and Methods

[0307] Linear and macrocyclic compounds 1-14 and 23-26 (SEQ ID NO: 1-14 and 23-26) were incubated at 1 μM with rat plasma (Sprague-Dawley, male; 400 μl) at 37 °C for different time points (0, 20, 40, 60, or 120 min). Incubation was terminated with acetonitrile. The collected samples were centrifuged at 2272 × g for 20 min and analyzed. Stock solutions were prepared using 50% DMSO, and the compounds were incorporated into the incubation at a 1 / 100 ratio to have a final DMSO content of 0.5%. The disappearance of the compounds was monitored by UHPLC / PDA analysis of the samples using high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode). Enalapril 1 μM was used as a disappearance rate control. For the analysis of compounds 9, 10, 13, and 14, stock solutions were prepared with PBS, and the samples were analyzed by UHPLC-ToF mass spectrometry. Optimal chromatographic characteristics (peak shape and retention time) and mass spectrometric ionization were obtained by optimizing the analytical method using parent compounds. Ion chromatograms were extracted from the total ion chromatogram using calculated single isotope precise masses in a 5 mDa window. Disappearance was based on LC / MS peak area, with 0 min marked as 100%. The first-order rate constant k(min⁻¹) of metabolism was obtained from the slope of the time-to-logarithm (% of remaining compound) plot using Excel software. The in vitro half-life (t) of the compounds was investigated. 1 / 2 ) is defined as: t 1 / 2 = ln2 / k. The calculated half-life is based on the disappearance of the compound from rat plasma.

[0308] exist Figure 6A In the figures, each pair of bars shows data for the unmodified peptide (linear) and its corresponding modified peptide (cyclic). In vitro plasma metabolic stability increased from <30 min to >789 min. Many macrocyclic compounds (compounds 2, 4, 8, 10, 14, and 26) showed improved stability in rat plasma compared to their linear counterparts (compounds 1, 3, 7, 9, 13, and 25, respectively).

[0309] Example 4

[0310] In vitro metabolic stability of linear and macrocyclic compounds in human plasma.

[0311] Metabolic stability was studied using human plasma over a 120-minute period, with an initial test concentration of 1 μM. Samples were analyzed using LC / QE-orbitrap-MS. The calculated half-life was based on the disappearance of the compound from human plasma.

[0312] Materials and Methods

[0313] Linear or macrocyclic compounds 1-8, 11-14, and 23-24 (SEQ ID NO: 1-8, 11-14, and 23-26) were incubated at 1 μM with human plasma (mixed sex, 400 μl) at 37 °C for different time points (0, 20, 40, 60, or 120 min). Incubation was terminated with acetonitrile. The collected samples were centrifuged at 2272 × g for 20 min and analyzed. The samples were analyzed by UHPLC / PDA and high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) to monitor compound disappearance. Propanthelin bromide 1 μM was used as a disappearance rate control. The analytical method was optimized using the parent compound to obtain optimal chromatographic characteristics (peak shape and retention time) and mass spectrometric ionization. Ion chromatograms were extracted from the total ion chromatogram using calculated single isotope precision mass with a 5 mDa window. Disappearance was based on LC / MS peak area, with 0 min marked as 100%. Using Excel software, the first-order rate constant k (min⁻¹) of metabolism was obtained from the slope of the time-to-logarithmic (%) graph of the remaining compounds. The in vitro half-life (t) of the compounds was studied. 1 / 2 ) is defined as: t 1 / 2 = ln2 / k. In Figure 6B In the image, each pair of bars displays data for the unmodified peptide (linear) and its corresponding modified peptide (cyclic).

[0314] Compared to their stability in rat plasma, the linear and macrocyclic compounds exhibited better peptide stability in human plasma, as evidenced by the maximum reported half-life in human plasma (a maximum value specific to the half-life test was 795 min). The two macrocyclic compounds (compounds 2 and 26) showed improved stability in human plasma compared to their linear counterparts (compounds 1 and 25, respectively).

[0315] Example 5

[0316] In vitro metabolic stability of linear and macrocyclic compounds in rat hepatocytes

[0317] Since liver metabolism plays a central role in the clearance of compounds, the metabolic stability of macrocyclic and linear compounds was tested in cultured rat hepatocytes.

[0318] Materials and methods

[0319] Linear or macrocyclic compounds 1-14 and 23-26 (SEQ ID NO: 1-14 and 21-26) were incubated at 1 μM with pooled cryopreserved rat hepatocytes (Spraque-Dawley, male; 400 μl, 1 million viable cells / mL (compounds 1-8, 11-12, and 23-26) or 100 μl, 100,000 viable cells / mL (compounds 9-10 and 13-14)) at 37 °C for different time points (0, 10, 20, 40, or 60 min). Cell density and viability were determined by trypan blue exclusion assay. Incubation was terminated with acetonitrile. The collected samples were centrifuged at 2272 × g for 20 min and analyzed. Samples were analyzed by UHPLC / PDA and high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) (compounds 1-8, 11-12, and 23-26) or HHPLC-ToF mass spectrometry (compounds 9-10 and 13-14) to monitor compound disappearance. Verapamil 1 μM was used as a disappearance rate control. Analytical methods were optimized using parent compounds to achieve optimal chromatographic characteristics (peak shape and retention) and mass spectrometric ionization. Ion chromatograms were extracted from the total ion chromatogram using calculated single isotope precision mass in a 5 mDa window. Disappearance was based on LC / MS peak area, with 0 min marked as 100%. The first-order rate constant k(min⁻¹) of metabolism was obtained from the slope of the time-relative logarithm (percentage of remaining compound) curve using Excel software. The in vitro half-life (t) of the compounds was investigated. 1 / 2 ) is defined as: t 1 / 2 =ln2 / k. The calculated half-life is based on the disappearance of the compound in rat hepatocytes.

[0320] exist Figure 7A In the figure, each pair of bars shows data for unmodified peptides (linear) and their corresponding modified peptides (cyclic). In vitro hepatocyte metabolic stability increased from >10 minutes to >395 minutes.

[0321] Compared with their linear counterparts (compounds 1, 3, 5, 7, 9, 11, 13, 23, and 25, respectively), all macrocyclic compounds (compounds 2, 4, 6, 8, 10, 12, 14, 24, and 26) showed improved stability in rat hepatocytes.

[0322] Example 6

[0323] In vitro metabolic stability of linear and macrocyclic compounds in human hepatocytes

[0324] Materials and methods

[0325] Linear or macrocyclic compounds 1-8, 11-14, and 23-26 (SEQ ID NO: 1-8, 11-14, and 23-26) were incubated at 1 μM with pooled cryopreserved human hepatocytes (mixed sex; 400 μl, 1 million viable cells / mL) at 37 °C for different time points (0, 10, 20, 40, or 60 min). Cell density and viability were determined by trypan blue exclusion. Incubation was terminated with acetonitrile. The collected samples were centrifuged at 2272 × g for 20 min and analyzed. Samples were analyzed by UPLC / PDA and high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) to monitor compound disappearance. Verapamil 1 μM was used as a disappearance rate control. The analytical method was optimized using parent compounds to achieve optimal chromatographic characteristics (peak shape and retention) and mass spectrometric ionization. Ion chromatograms were extracted from the total ion chromatogram using calculated single isotope precision mass with a 5 mDa window. The disappearance was determined based on LC / MS peak area, with 0 minutes marked as 100%. The first-order rate constant k (min⁻¹) of metabolism was obtained from the slope of the time-to-logarithmic (%) plot using Excel software. The in vitro half-life (t) of the compounds was investigated. 1 / 2 ) is defined as: t 1 / 2 =ln2 / k. The calculated half-life is based on the disappearance of the compound in human liver cells.

[0326] exist Figure 7B In the image, each pair of bars displays data for the unmodified peptide (linear) and its corresponding modified peptide (cyclic).

[0327] Compared with their linear counterparts (compounds 1, 3, 5, 11, 23, and 25, respectively), almost all macrocyclic compounds (compounds 2, 4, 6, 12, 14, 24, and 26) showed significantly improved stability in human hepatocytes.

[0328] Example 7

[0329] Permeation properties of linear and macrocyclic compounds in a 3D in vitro blood-brain barrier model

[0330] Due to interest in developing CDNF and MANF-derived peptides for peripheral administration in the treatment of CNS diseases, and the fact that macrocyclization can improve the cell penetration and distribution properties of peptides, the ability of compounds to cross the blood-brain barrier was tested in an established in vitro blood-brain barrier model. For this purpose, macrocyclic and linear compounds were incubated at 500 nM for 2 h in a two-compartment in vitro blood-brain barrier model (n=4), followed by sample collection and LC-MS / MS analysis.

[0331] Figure 8Results are presented; the amount of compounds crossing the artificial blood-brain barrier is expressed as a percentage of the original applied concentration of the compound. Macrocyclic compounds were observed to have better BBB channel penetration compared to linear compounds. BBB penetration increased from <3% (unmodified >30aa peptide) to >15% (modified).

[0332] Compared with the linear counterparts (compounds 1, 3, 5, 7, 11, 13, 23 and 25, respectively), all macrocyclic compounds (compounds 2, 4, 6, 8, 12, 14, 24 and 26) showed improved stability across the in vitro blood-brain barrier.

[0333] Materials and Methods

[0334] Primary culture of astrocytes. Rat astrocytes were prepared from E15 embryos. Briefly, pregnant female rats (Wistar, Janvier laboratory) at 15 days of gestation were deeply anesthetized in a CO2 chamber and then euthanized by cervical dislocation. Fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium containing 2% penicillin (10000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). The whole brain was treated with trypsin-EDTA solution at 37°C for 20 min at a final concentration of 0.05% trypsin and 0.02% EDTA. The dissociated cells were cultured in DMEM 10% fetal bovine serum. Purified astrocytes were used at passage 4 (P4).

[0335] Culture of human endothelial cells. A vial of HBMEC (primary human brain microvascular endothelial cells, ACBRI 376) was used at a specific 8th passage (P8).

[0336] Primary culture of cortical neurons. Rat cortical neurons were cultured as described by Callizot et al., 2013, but with modifications. Briefly, pregnant female rats (Wistar, Janvier laboratory) at 15 days of gestation were deeply anesthetized in a CO2 chamber and then euthanized by cervical dislocation. Fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium containing 2% penicillin (10000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). The cortex was treated with trypsin-EDTA solution at 37°C for 20 min at a final concentration of 0.05% trypsin and 0.02% EDTA. Dissociation was terminated by adding Dürbeco Modified Eagle's Medium (DMEM) with 4.5 g / L glucose containing DNase I grade II (final concentration 0.5 mg / mL) and 10% fetal bovine serum (FCS). Cells were mechanically dissociated by forcing the flow through the tip of a 10-ml pipette three times. Cells were centrifuged at 515x g for 10 minutes at 4°C. The pellet was resuspended in a defined medium consisting of a neural basal medium containing 2% B27 supplementation solution, 2 mmol / L L-glutamine, 2% PS solution, and 10 ng / mL brain-derived neurotrophic factor (BDNF). Viable cells were counted using a Neubauer hematology counter using a trypan blue exclusion test. Cortical neurons were seeded at a density of 255,000 cells / well in 24-well plates pre-coated with poly-L-lysine at the bottom of the wells and cultured at 37°C in an air (95%)-CO2 (5%) incubator. The medium was changed every other day.

[0337] Co-culture of endothelial cells, astrocytes, and primary cortical neurons. The procedure was performed as previously reported (Xue et al., 2013, but modified; Callizot et al., 2017). Briefly, on day 0, purified astrocytes (P4) were rapidly thawed in a 37°C water bath. The cells were immediately placed in DMEM containing 10% FCS. The cell suspension was centrifuged at 515 x g for 5 min at 4°C, and the pellet was resuspended in DMEM F12 containing 10% FCS. Cells were seeded at a density of 45,000 cells / insertion on the outside of an insert membrane (PET, 1 μm) and cultured at 37°C in an air (95%)-CO2 (5%) incubator. Thirty-six (36) hours after astrocyte seeding, HBMECs (P8) were rapidly thawed in a 37°C water bath and immediately placed in DMEM containing 10% FCS. The cell suspension was centrifuged at 515x g for 5 minutes at 4°C, and the pellet was resuspended in an EGM-2 bullet kit containing 5% FCS, 1% PS solution, 1.4 μM hydrocortisone, 5 μg / mL acidic ascorbic acid, 1% lipid mixture, 10 mM HEPES, and 1 ng / mL bFGF. Cells were seeded at a density of 50,000 cells / insertion on the inside of an insert membrane (PET, 1 μm) and cultured at 37°C in an air (95%)-CO2 (5%) incubator. Thirty-six (36) hours after HBMEC seeding (72 hours after astrocyte seeding), cortical neurons were seeded at a density of 170,000 cells / well at the bottom of poly-L-lysine-pre-coated wells and cultured at 37°C in an air (95%)-CO2 (5%) incubator.

[0338] Application of linear or macrocyclic compounds. Five (5) days after HBMEC inoculation, following the first test of endothelial cell layer integrity, test compounds (1-8, 11-14 and 23-26, SEQ ID NO: 1-8, 11-14 and 21-26) were added to the chamber and incubated at a concentration of 500 nM for 2 hours.

[0339] Quantification of test compounds. Further detection and quantification of each compound in the near-chamber supernatant were performed by mass spectrometry (MS). After sample thawing, each cell culture sample in 100 μL aliquots was analyzed by peptide quantification using mass spectrometry. Calculated percentages represent the percentage of compounds applied to the near-chamber, measured in the near-chamber at the end of application.

[0340] Example 8

[0341] In vivo pharmacokinetic characteristics of macrocyclic compounds after peripheral administration to rats

[0342] The clearance and elimination of peptides can be mediated by multiple in vivo processes, including metabolism and renal elimination. Since in vitro studies have shown improved metabolic stability of macrocyclic peptides, preliminary pharmacokinetic properties were tested by administering the compound at single-dose levels peripherally (subcutaneously and intravenously) and then determining the presence of the compound in plasma at different time points following peripheral administration.

[0343] Figure 9A Plasma concentrations of macrocyclic compounds 2, 10, and 14 (SEQ ID NO: 2, 10, and 14) and linear compounds 3 and 9 (SEQ ID NO: 3 and 9) at different time points following intravenous administration are presented. Compound 14 exhibited improved blood retention behavior and was detected in plasma at least 2 hours after administration.

[0344] Table 1 presents the in vivo pharmacokinetic characteristics calculated based on plasma concentrations of linear and cyclic compounds measured at different time points after peripheral administration to rats.

[0345] Table 1. In vivo pharmacokinetic characteristics of linear and macrocyclic compounds after intravenous bolus injection of 5 mg / kg into male Sprague-Dawley rats.

[0346]

[0347] Figure 9B The brain distribution dynamics of compound 14 in male Sprague-Dawley rats are illustrated. This was achieved by implanting a guiding cannula... microdialysis A probe was inserted into the ventral striatum of rats and perfused with aCSF. Compound 14 was administered as a single intravenous bolus injection of 10 mg / kg, and microdialysis samples were collected at 20-minute intervals over 4 hours. The concentration of the compound in the interstitial fluid (ISF) was determined by LC-MS / MS and normalized to the recovery rate of the microdialysis membrane (% as determined in vitro).

[0348] Low molecular weight natural linear peptides typically have short circulating lifetimes because they are eliminated from plasma within minutes due to metabolic degradation and clearance mechanisms (Li et al., 2015; Lin et al., 2009). Plasma concentrations of selected linear and macrocyclic compounds were measured at different time points following intravenous administration to rats. Macrocyclic compound 14 (SEQ ID NOs: 14) showed improved plasma half-life, volume of distribution, and mean residence time. Furthermore, brain microdialysis studies showed that compound 14 penetrated into the brain parenchyma after a single intravenous bolus injection.

[0349] Materials and Methods

[0350] Male Spraque-Dawley rats (approximately 6 weeks old, n=3 for each compound) were administered test compounds intravenously (iv) at a dose of 5 mg / kg. Blood samples were collected from the jugular vein at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h after compound administration by inserting an indwelling catheter (250 μl of blood) into a labeled polypropylene tube containing an anticoagulant (heparin) and incubating on wet ice for up to 30 min. The blood samples were centrifuged to achieve plasma separation (4 °C, 21100 G, 5 min). Tolbutamide at 500 ng / ml containing 10% TFA in acetonitrile or MeCN was used as an internal standard solution. Standards were prepared as rat plasma by incorporating the matrix into analytes at concentrations of 2–10000 ng / ml, or otherwise treated as samples. 200 μl of the internal standard was added to 50 μl aliquots of the sample plasma. The samples were mixed (150 rpm, 15 min) and centrifuged (3000 rpm, 15 min). The analytical method was optimized for reaction monitoring chromatographic (peak shape and retention time) shift and mass spectrometric characteristics (ionization efficiency, MS / MS detection). The supernatant was analyzed by UHPLC-TOF mass spectrometry using electrospray ionization.

[0351] Brain microdialysis was studied in a separate group of conscious animals treated intravenously with compound 14. One week prior to the microdialysis experiment, a guide cannula was implanted in the striatum of Sprague-Dawley rats at the following coordinates: AP +0.6 mm; L -3.0 mm; V -2.8 mm, thus providing a final V -6.8 mm for the tip of the microdialysis probe. On the day of the experiment, the microdialysis probe (EicomA-I: 0.22 mm OD, 4 mm membrane length, cutoff 50 kDa) was inserted into the guide cannula and perfused with artificial cerebrospinal fluid (aCSF) at a constant flow rate of 0.1 μL / min. After a stabilization period of 120–150 minutes, compound 18 was administered intravenously at a single bolus dose of 10 mg / kg, and samples were collected at 20-minute intervals over 4 hours. The intracranial fluid concentration of the compound was analyzed by UHPLC-MS / MS. When perfused with aCSF under conditions similar to in vivo testing, additional in vitro tests were performed to determine the recovery of the test compound from the tubing, connector, and microdialysis probe. The determined recovery percentage (29.3%) was used to correct for data obtained in the microdialysis studies.

[0352] Reference List

[0353] Patent documents

[0354] EP 58,481;

[0355] US 3,773,919;

[0356] WO 2007068803;

[0357] WO 2009133247;

[0358] WO 2013 / 3034805;

[0359] WO 2018 / 202957

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[0362] Airavaara M, Harvey BK, Voutilainen MH, Shen H, Chou J, Lindholm P, Lindahl M, Tuominen RK, Saarma M, Hoffer B, Wang Y. 2012. CDNF protects the nigrostriatal dopamine system and promotes recovery after MPTP treatment in mice. Cell Transplant. 21(6):1213-23.

[0363] Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. 2000 Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2(6):326-32.

[0364] Callizot N, Combes M, Steinschneider R, Poindron P. 2013. Operational dissection of beta-amyloid cytophathic effects on cultured neurons. J Neurosci Res. 91(5):706-16

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[0368] Glembotski CC. 2011. Functions for the cardiomyokine, MANF, in cardioprotection, hypertrophy and heart failure. J. Mol. Cell. Cardiol. 51:512–517.

[0369] Hefti F. Neurotrophic factor therapy for nervous system degenerative diseases. 1994. J Neurobiol., 25:1418-1435.

[0370] Hellman M, Arumae U, Yu LY, Lindholm P, Peranen J, Saarma M and Permi P. 2011. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons. J. Biol. Chem. 286:2675-2680.

[0371] Huttunen, HJ and Saarma M. 2019. CDNF protein therapy in Parkinson’s disease. Cell Transplantation 1-18.

[0372] Langer R, Brem H, Tapper D. 1981. Biocompatibility of polymeric delivery systems for macromolecules. J. Biomed. Mater. Res. 15:167-277

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[0374] Lindholm P., Voutilainen M.H., Lauren J., Peranen J., Leppanen V.M., Andressoo J.O., Lindahl M., Janhunen S., Kalkkinen N., Timmusk T., Tuominen R.K. and Saarma M. 2007. Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo. Nature. 448:73-77.

[0375] Lindholm P and Saarma M. 2010. Novel CDNF / MANF family of neurotrophic factors. Dev. Neurobiol. 70:360-371.

[0376] Lin JH. 2009. Pharmacokinetics of biotech drugs: peptides, proteins and monoclonal antibodies. Curr Drug Metab. 10(7): 661 - 91.

[0377] Li Y, Wang Y, Wei Q, Zheng X, Tang L, Kong D, Gong M. 2015. Variant fatty acid - like molecules Conjugation, novel approaches for extending the stability of therapeutic peptides. Sci Rep. 5: 18039.

[0378] Nadella R, Voutilainen MH, Saarma M, Gonzalez - Barrios JA, Leon - Chavez BA, Jiménez JM, Jiménez SH, Escobedo L, Martinez - Fong DJ. 2014. Transient transfection of human CDNF gene reduces the 6 - hydroxydopamine - induced neuroinflammation in the rat substantia nigra. Neuroinflammation. 16; 11: 209.

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[0383] Visanji NP, Orsi A, Johnston TH, Howson PA, Dixon K, Callizot N, Brotchie JM and Rees DD. 2008. PYM50028, a novel, orally active, nonpeptide neurotrophic factor inducer, prevents and reverses neuronal damage induced by MPP+ in mesencephalic neurons and by MPTP in a mouse model of Parkinson's disease. FASEB J., 22(7):2488 - 97.

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[0385] Voutilainen MH, Back S, Porsti E, Toppinen L, Lindgren L, Lindholm P, Peranen J, Saarma M and Tuominen RK. 2009. Mesencephalic astrocyte - derived neurotrophic factor is neurorestorative in rat model of Parkinson's disease. J. Neurosci. 29:9651 - 9659.

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[0389] Zhao H, Liu Y, Cheng L, Liu B, Zhang W, Guo YJ, Nie L. 2013. Mesencephalic astrocyte-derived neurotrophic factor inhibits oxygen-glucose deprivation-induced cell damage and inflammation by suppressing endoplasmic reticulum stress in rat primary astrocytes. J Mol Neurosci. 51(3):671-8. Sequence Listing <110> Herantis Pharma Oy <120> Macrocyclic Peptide <130> P4701EP00 <150> EP19218576.7 <151> 2019-12-20 <160> 72 <170> BiSSAP 1.3.6 <210> 1 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connect (22..25) <400> 1 Val Asp Leu Arg Lys Met Arg Val Ala Glu Leu Lys Gln Ile Leu His 1 5 10 15 Ser Trp Gly Glu Glu Cys Arg Ala Cys Ala Glu 20 25 <210> 2 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connect (22..25) <400> 2 Val Asp Leu Arg Lys Met Arg Val Ala Glu Leu Lys Gln Ile Leu His 1 5 10 15 Ser Trp Gly Glu Glu Cys Arg Ala Cys Ala Glu 20 25 <210> 3 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connect (22..25) <400> 3 Val Asp Leu Lys Lys Leu Arg Val Lys Glu Leu Lys Lys Ile Leu Asp 1 5 10 15 Asp Trp Gly Glu Thr Cys Lys Gly Cys Ala Glu 20 25 <210> 4 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connect (22..25) <400> 4 Val Asp Leu Lys Lys Leu Arg Val Lys Glu Leu Lys Lys Ile Leu Asp 1 5 10 15 Asp Trp Gly Glu Thr Cys Lys Gly Cys Ala Glu 20 25 <210> 5 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connect (17..20) <400> 5 Met Arg Val Ala Glu Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu 1 5 10 15 Cys Arg Ala Cys Ala Glu Lys 20 <210> 6 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connect (17..20) <400> 6 Met Arg Val Ala Glu Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu 1 5 10 15 Cys Arg Ala Cys Ala Glu Lys 20 <210> 7 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connect (17..20) <400> 7 Leu Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr 1 5 10 15 Cys Lys Gly Cys Ala Glu Lys 20 <210> 8 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connect (17..20) <400> 8 Leu Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr 1 5 10 15 Cys Lys Gly Cys Ala Glu Lys 20 <210> 9 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connection (11..14) <400> 9 Lys Ser Ile Leu Asp Asp Trp Gly Glu Thr Cys Lys Gly Cys Ala Glu 1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connection (11..14) <400> 10 Lys Ser Ile Leu Asp Asp Trp Gly Glu Thr Cys Lys Gly Cys Ala Glu 1 5 10 15 <210> 11 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connect (5..8) <400> 11 Trp Gly Glu Glu Cys Arg Ala Cys Ala Glu Lys Thr 1 5 10 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connect (5..8) <400> 12 Trp Gly Glu Glu Cys Arg Ala Cys Ala Glu Lys Thr 1 5 10 <210> 13 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connect (5..8) <400> 13 Trp Gly Glu Thr Cys Lys Gly Cys Ala Glu Lys Ser 1 5 10 <210> 14 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connect (5..8) <400> 14 Trp Gly Glu Thr Cys Lys Gly Cys Ala Glu Lys Ser 1 5 10 <210> 15 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connection (4..7) <400> 15 Gly Glu Glu Cys Arg Ala Cys Ala Glu Lys Thr 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connections (4..8) <400> 16 Gly Glu Glu Cys Arg Gly Ala Cys Ala Glu Lys Thr 1 5 10 <210> 17 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connections (4..8) <400> 17 Gly Glu Glu Cys Arg Ala Ala Cys Ala Glu Lys Thr 1 5 10 <210> 18 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connections (4..8) <400> 18 Gly Glu Glu Cys Arg Ser Ala Cys Ala Glu Lys Thr 1 5 10 <210> 19 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connection (4..7) <400> 19 Gly Glu Thr Cys Lys Gly Cys Ala Glu Lys Ser 1 5 10 <210> 20 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connections (4..8) <400> 20 Gly Glu Thr Cys Lys Gly Gly Cys Ala Glu Lys Ser 1 5 10 <210> twenty one <211> 12 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connections (4..8) <400> twenty one Gly Glu Thr Cys Lys Ala Gly Cys Ala Glu Lys Ser 1 5 10 <210> twenty two <211> 12 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connections (4..8) <400> twenty two Gly Glu Thr Cys Lys Ser Gly Cys Ala Glu Lys Ser 1 5 10 <210> twenty three <211> 13 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connect (5..9) <400> twenty three Trp Gly Glu Glu Cys Arg Gly Ala Cys Ala Glu Lys Thr 1 5 10 <210> twenty four <211> 13 <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connect (5..9) <400> twenty four Trp Gly Glu Glu Cys Arg Gly Ala Cys Ala Glu Lys Thr 1 5 10 <210> 25 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Disulfide bridge <220> <221> DISULFID <222> Connect (5..9) <400> 25 Trp Gly Glu Thr Cys Lys Gly Gly Cys Ala Glu Lys Ser 1 5 10 <210> 26 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Head and tail ring-shaped, disulfide bridge <220> <221> DISULFID <222> Connect (5..9) <400> 26 Trp Gly Glu Thr Cys Lys Gly Gly Cys Ala Glu Lys Ser 1 5 10 <210> 27 <211> 5 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 2 <223> X1 = Not present, R, K, I, G, A, or S <220> <221> variants <222> 3 <223> X2 = G, A, R, K, I, or S <220> <221> variants <222> 4 <223> X3 = A, G, or S <400> 27 Cys Xaa Xaa Xaa Cys 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 2 <223> X4 = E, T, V, D, M, or G <220> <221> variants <222> 4 <223> X1 = R, K, I, G, A, or S <220> <221> variants <222> 5 <223> X2 = Not present, G, A, R, K, I, or S <220> <221> variants <222> 6 <223> X3 = A, G, or S <400> 28 Glu Xaa Cys Xaa Xaa Xaa Cys Ala Glu 1 5 <210> 29 <211> 16 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 1 <223> X5 = Not present, H, D, Q, R, Y, N, or S <220> <221> variants <222> 2 <223> X6 = Not present, S, D, G, N, or R <220> <221> variants <222> 3 <223> X7 = Not present or W <220> <221> variants <222> 4 <223> X8 = Not present or G <220> <221> variants <222> 6 <223> X4 = E, T, V, D, M, or G <220> <221> variants <222> 8 <223> X1 = R, K, I, G, A, or S <220> <221> variants <222> 9 <223> X2 = Not present, G, A, R, K, I, or S <220> <221> variants <222> 10 <223> X3 = A, G, or S <220> <221> variants <222> 14 <223> X9 = Not present or K <220> <221> variants <222> 15 <223> X10 = Not present, T, S, A, I, or N <220> <221> variants <222> 16 <223> X11 = Not present, D or E <400> 29 Xaa Xaa Xaa Xaa Glu Xaa Cys Xaa Xaa Xaa Cys Ala Glu Xaa Xaa Xaa 1 5 10 15 <210> 30 <211> 36 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 1 <223> X12 = Not present, L, I, or V <220> <221> variants <222> 2 <223> X13 = Not present or D <220> <221> variants <222> 3 <223> X14 = Not present, L or W <220> <221> variants <222> 4 <223> X15 = Not present, A, S, T, E, or N <220> <221> variants <222> 5 <223> X16 = Not present, S or T <220> <221> variants <222> 6 <223> X17 = Not present, V or D <220> <221> variants <222> 7 <223> X18 = Not present, D or A <220> <221> variants <222> 8 <223> X19 = Not present or L <220> <221> variants <222> 9 <223> X20 = ​​Not present, R, K, S, or W <220> <221> variants <222> 10 <223> X21 = Not present or K <220> <221> variants <222> 11 <223> X22 = Not present, M, L, I, or V <220> <221> variants <222> 12 <223> X23 = Not present or R <220> <221> variants <222> 14 <223> X24 = A, K, T, L, or V <220> <221> variants <222> 18 <223> X25 = Q, K, or R <220> <221> variants <222> 19 <223> X26 = I or V <220> <221> variants <222> twenty one <223> X5 = Not present, H, D, Q, R, Y, N, or S <220> <221> variants <222> twenty two <223> X6 = Not present, S, D, G, N, or R <220> <221> variants <222> twenty three <223> X7 = Not present or W <220> <221> variants <222> twenty four <223> X8 = Not present or G <220> <221> variants <222> 26 <223> X4 = E, T, V, D, M, or G <220> <221> variants <222> 28 <223> X1 = R, K, I, G, A or S <220> <221> Variant <222> 29 <223> X2 = Absent, G, A, R, K, I or S <220> <221> Variant <222> 30 <223> X3 = A, G or S <220> <221> Variant <222> 34 <223> X9 = Absent or K <220> <221> Variant <222> 35 <223> X10 = Absent, T, S, A, I or N <220> <221> Variant <222> 36 <223> X11 = Absent, D or E <400> 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Glu Leu 1 5 10 15 Lys Xaa Xaa Leu Xaa Xaa Xaa Xaa Glu Xaa Cys Xaa Xaa Xaa Cys Ala 20 25 30 Glu Xaa Xaa Xaa 35 <210> 31 <211> 37 <212> PRT <213> Homo sapiens <220> <223> Native human CDNF peptide <400> 31 Arg Val Ala Glu Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu Cys 1 5 10 15 Arg Ala Cys Ala Glu Lys Thr Asp Tyr Val Asn Leu Ile Gln Glu Leu 20 25 30 Ala Pro Lys Tyr Ala 35 <210> 32 <211> 37 <212> PRT <213> Homo sapiens <220> <223> Natural human MANAF peptide <400> 32 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 1 5 10 15 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 20 25 30 Met Pro Lys Tyr Ala 35 <210> 33 <211> 32 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 1 <223> X16 = Not present, S or T <220> <221> variants <222> 2 <223> X17 = Not present, V or D <220> <221> variants <222> 3 <223> X18 = Not present, D or A <220> <221> variants <222> 4 <223> X19 = Not present or L <220> <221> variants <222> 5 <223> X20 = ​​Not present, R, K, S, or W <220> <221> variants <222> 6 <223> X21 = Not present or K <220> <221> variants <222> 7 <223> X22 = Not present, M, L, I, or V <220> <221> variants <222> 8 <223> X23 = Not present or R <220> <221> variants <222> 10 <223> X24 = A, K, T, L, or V <220> <221> variants <222> 14 <223> X25 = Q, K, or R <220> <221> variants <222> 15 <223> X26 = I or V <220> <221> variants <222> 17 <223> X5 = Not present, H, D, Q, R, Y, N, or S <220> <221> variants <222> 18 <223> X6 = Not present, S, D, G, N, or R <220> <221> variants <222> 19 <223> X7 = Not present or W <220> <221> variants <222> 20 <223> X8 = Not present or G <220> <221> variants <222> twenty two <223> X4 = E, T, V, D, M, or G <220> <221> variants <222> twenty four <223> X1 = R, K, I, G, A, or S <220> <221> variants <222> 25 <223> X2 = Not present, G, A, R, K, I, or S <220> <221> variants <222> 26 <223> X3 = A, G, or S <220> <221> variants <222> 30 <223> X9 = Not present or K <220> <221> variants <222> 31 <223> X10 = Not present, T, S, A, I, or N <220> <221> variants <222> 32 <223> X11 = Not present, D or E <400> 33 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Glu Leu Lys Xaa Xaa Leu 1 5 10 15 Xaa Xaa Xaa Xaa Glu Xaa Cys Xaa Xaa Xaa Cys Ala Glu Xaa Xaa Xaa 20 25 30 <210> 34 <211> 32 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 1 <223> X15 = Not present, A, S, T, E, or N <220> <221> variants <222> 2 <223> X16 = Not present, S or T <220> <221> variants <222> 3 <223> X17 = Not present, V or D <220> <221> variants <222> 4 <223> X18 = Not present, D or A <220> <221> variants <222> 5 <223> X19 = Not present or L <220> <221> variants <222> 6 <223> X20 = ​​Not present, R, K, S, or W <220> <221> variants <222> 7 <223> X21 = Not present or K <220> <221> variants <222> 8 <223> X22 = Not present, M, L, I, or V <220> <221> variants <222> 9 <223> X23 = Not present or R <220> <221> variants <222> 9 <223> X23 = Not present or R <220> <221> variants <222> 11 <223> X24 = A, K, T, L, or V <220> <221> variants <222> 15 <223> X25 = Q, K, or R <220> <221> variants <222> 16 <223> X26 = I or V <220> <221> variants <222> 18 <223> X5 = Not present, H, D, Q, R, Y, N, or S <220> <221> variants <222> 19 <223> X6 = Not present, S, D, G, N, or R <220> <221> variants <222> 20 <223> X7 = Not present or W <220> <221> variants <222> twenty one <223> X8 = Not present or G <220> <221> variants <222> twenty three <223> X4 = E, T, V, D, M, or G <220> <221> variants <222> 25 <223> X1 = R, K, I, G, A, or S <220> <221> variants <222> 26 <223> X2 = Not present, G, A, R, K, I, or S <220> <221> variants <222> 27 <223> X3 = A, G, or S <220> <221> variants <222> 31 <223> X9 = Not present or K <400> 34 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Glu Leu Lys Xaa Xaa 1 5 10 15 Leu Xaa Xaa Xaa Xaa Glu Xaa Cys Xaa Xaa Xaa Cys Ala Glu Xaa Xaa 20 25 30 <210> 35 <211> 32 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 1 <223> X14 = Not present, L or W <220> <221> variants <222> 2 <223> X15 = Not present, A, S, T, E, or N <220> <221> variants <222> 3 <223> X16 = Not present, S or T <220> <221> variants <222> 4 <223> X17 = Not present, V or D <220> <221> variants <222> 5 <223> X18 = Not present, D or A <220> <221> variants <222> 6 <223> X19 = Not present or L <220> <221> variants <222> 7 <223> X20 = ​​Not present, R, K, S, or W <220> <221> variants <222> 8 <223> X21 = Not present or K <220> <221> variants <222> 9 <223> X22 = Not present, M, L, I, or V <220> <221> variants <222> 10 <223> X23 = Not present or R <220> <221> variants <222> 12 <223> X24 = A, K, T, L, or V <220> <221> variants <222> 16 <223> X25 = Q, K, or R <220> <221> variants <222> 17 <223> X26 = I or V <220> <221> variants <222> 19 <223> X5 = Not present, H, D, Q, R, Y, N, or S <220> <221> variants <222> 20 <223> X6 = Not present, S, D, G, N, or R <220> <221> variants <222> twenty one <223> X7 = Not present or W <220> <221> variants <222> twenty two <223> X8 = Not present or G <220> <221> variants <222> twenty four <223> X4 = E, T, V, D, M, or G <220> <221> variants <222> 26 <223> X1 = R, K, I, G, A, or S <220> <221> variants <222> 27 <223> X2 = Not present, G, A, R, K, I, or S <220> <221> variants <222> 28 <223> X3 = A, G, or S <220> <221> variants <222> 32 <223> X9 = Not present or K <400> 35 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Glu Leu Lys Xaa 1 5 10 15 Xaa Leu Xaa Xaa Xaa Xaa Glu Xaa Cys Xaa Xaa Xaa Cys Ala Glu Xaa 20 25 30 <210> 36 <211> 32 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 1 <223> X13 = Not present or D <220> <221> variants <222> 2 <223> X14 = Not present, L or W <220> <221> variants <222> 3 <223> X15 = Not present, A, S, T, E, or N <220> <221> variants <222> 4 <223> X16 = Not present, S or T <220> <221> variants <222> 5 <223> X17 = Not present, V or D <220> <221> variants <222> 6 <223> X18 = Not present, D or A <220> <221> variants <222> 7 <223> X19 = Not present or L <220> <221> variants <222> 8 <223> X20 = ​​Not present, R, K, S, or W <220> <221> variants <222> 9 <223> X21 = Not present or K <220> <221> variants <222> 10 <223> X22 = Not present, M, L, I, or V <220> <221> variants <222> 11 <223> X23 = Not present or R <220> <221> variants <222> 13 <223> X24 = A, K, T, L, or V <220> <221> variants <222> 17 <223> X25 = Q, K, or R <220> <221> variants <222> 18 <223> X26 = I or V <220> <221> variants <222> 20 <223> X5 = Not present, H, D, Q, R, Y, N, or S <220> <221> variants <222> twenty one <223> X6 = Not present, S, D, G, N, or R <220> <221> variants <222> twenty two <223> X7 = Not present or W <220> <221> variants <222> twenty three <223> X8 = Not present or G <220> <221> variants <222> 25 <223> X4 = E, T, V, D, M, or G <220> <221> variants <222> 27 <223> X1 = R, K, I, G, A, or S <220> <221> variants <222> 28 <223> X2 = Not present, G, A, R, K, I, or S <220> <221> variants <222> 29 <223> X3 = A, G, or S <400> 36 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Glu Leu Lys 1 5 10 15 Xaa Xaa Leu Xaa Xaa Xaa Xaa Glu Xaa Cys Xaa Xaa Xaa Cys Ala Glu 20 25 30 <210> 37 <211> 4 <212> PRT <213> Artificial sequence <220> <223> peptides <400> 37 Lys Asp Glu Leu 1 <210> 38 <211> 4 <212> PRT <213> Artificial sequence <220> <223> peptides <400> 38 Cys Arg Ala Cys 1 <210> 39 <211> 4 <212> PRT <213> Artificial sequence <220> <223> peptides <400> 39 Cys Lys Gly Cys 1 <210> 40 <211> 4 <212> PRT <213> Artificial sequence <220> <223> peptides <220> <221> variants <222> 2 <223> X1 = K or R <220> <221> variants <222> 3 <223> X2 = G or A <400> 40 Cys Xaa Xaa Cys 1 <210> 41 <211> 8 <212> PRT <213> Artificial sequence <220> <223> peptides <400> 41 Glu Thr Cys Lys Gly Cys Ala Glu 1 5 <210> 42 <211> 6 <212> PRT <213> Artificial sequence <220> <223> peptides <400> 42 Thr Cys Lys Gly Cys Ala 1 5 <210> 43 <211> 187 <212> PRT <213> Homo sapiens <220> <223> NCBI Reference Sequence: NP_001025125.2 <400> 43 Met Trp Cys Ala Ser Pro Val Ala Val Val Ala Phe Cys Ala Gly Leu 1 5 10 15 Leu Val Ser His Pro Val Leu Thr Gln Gly Gln Glu Ala Gly Gly Arg 20 25 30 Pro Gly Ala Asp Cys Glu Val Cys Lys Glu Phe Leu Asn Arg Phe Tyr 35 40 45 Lys Ser Leu Ile Asp Arg Gly Val Asn Phe Ser Leu Asp Thr Ile Glu 50 55 60 Lys Glu Leu Ile Ser Phe Cys Leu Asp Thr Lys Gly Lys Glu Asn Arg 65 70 75 80 Leu Cys Tyr Tyr Leu Gly Ala Thr Lys Asp Ala Ala Thr Lys Ile Leu 85 90 95 Ser Glu Val Thr Arg Pro Met Ser Val His Met Pro Ala Met Lys Ile 100 105 110 Cys Glu Lys Leu Lys Lys Leu Asp Ser Gln Ile Cys Glu Leu Lys Tyr 115 120 125 Glu Lys Thr Leu Asp Leu Ala Ser Val Asp Leu Arg Lys Met Arg Val 130 135 140 Ala Glu Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu Cys Arg Ala 145 150 155 160 Cys Ala Glu Lys Thr Asp Tyr Val Asn Leu Ile Gln Glu Leu Ala Pro 165 170 175 Lys Tyr Ala Ala Thr His Pro Lys Thr Glu Leu 180 185 <210> 44 <211> 182 <212> PRT <213> Homo sapiens <220> <223> NCBI Reference Sequence: NP_006001.5 <400> 44 Met Arg Arg Met Trp Ala Thr Gln Gly Leu Ala Val Ala Leu Ala Leu 1 5 10 15 Ser Val Leu Pro Gly Ser Arg Ala Leu Arg Pro Gly Asp Cys Glu Val 20 25 30 Cys Ile Ser Tyr Leu Gly Arg Phe Tyr Gln Asp Leu Lys Asp Arg Asp 35 40 45 Val Thr Phe Ser Pro Ala Thr Ile Glu Asn Glu Leu Ile Lys Phe Cys 50 55 60 Arg Glu Ala Arg Gly Lys Glu Asn Arg Leu Cys Tyr Tyr Ile Gly Ala 65 70 75 80 Thr Asp Asp Ala Ala Thr Lys Ile Ile Asn Glu Val Ser Lys Pro Leu 85 90 95 Ala His His Ile Pro Val Glu Lys Ile Cys Glu Lys Leu Lys Lys Lys 100 105 110 Asp Ser Gln Ile Cys Glu Leu Lys Tyr Asp Lys Gln Ile Asp Leu Ser 115 120 125 Thr Val Asp Leu Lys Lys Leu Arg Val Lys Glu Leu Lys Lys Ile Leu 130 135 140 Asp Asp Trp Gly Glu Thr Cys Lys Gly Cys Ala Glu Lys Ser Asp Tyr 145 150 155 160 Ile Arg Lys Ile Asn Glu Leu Met Pro Lys Tyr Ala Pro Lys Ala Ala 165 170 175 Ser Ala Arg Thr Asp Leu 180 <210> 45 <211> 49 <212> PRT <213> Homo sapiens <400> 45 Thr Leu Asp Leu Ala Ser Val Asp Leu Arg Lys Met Arg Val Ala Glu 1 5 10 15 Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu Cys Arg Ala Cys Ala 20 25 30 Glu Lys Thr Asp Tyr Val Asn Leu Ile Gln Glu Leu Ala Pro Lys Tyr 35 40 45 Ala <210> 46 <211> 37 <212> PRT <213> Homo sapiens <400> 46 Arg Val Ala Glu Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu Cys 1 5 10 15 Arg Ala Cys Ala Glu Lys Thr Asp Tyr Val Asn Leu Ile Gln Glu Leu 20 25 30 Ala Pro Lys Tyr Ala 35 <210> 47 <211> 35 <212> PRT <213> Homo sapiens <400> 47 Thr Leu Asp Leu Ala Ser Val Asp Leu Arg Lys Met Arg Val Ala Glu 1 5 10 15 Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu Cys Arg Ala Cys Ala 20 25 30 Glu Lys Thr 35 <210> 48 <211> 32 <212> PRT <213> Homo sapiens <400> 48 Leu Ala Ser Val Asp Leu Arg Lys Met Arg Val Ala Glu Leu Lys Gln 1 5 10 15 Ile Leu His Ser Trp Gly Glu Glu Cys Arg Ala Cys Ala Glu Lys Thr 20 25 30 <210> 49 <211> 49 <212> PRT <213> Homo sapiens <400> 49 Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu Arg Val Lys Glu 1 5 10 15 Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys Lys Gly Cys Ala 20 25 30 Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu Met Pro Lys Tyr 35 40 45 Ala <210> 50 <211> 37 <212> PRT <213> Homo sapiens <400> 50 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 1 5 10 15 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 20 25 30 Met Pro Lys Tyr Ala 35 <210> 51 <211> 35 <212> PRT <213> Homo sapiens <400> 51 Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu Arg Val Lys Glu 1 5 10 15 Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys Lys Gly Cys Ala 20 25 30 Glu Lys Ser 35 <210> 52 <211> 32 <212> PRT <213> Homo sapiens <400> 52 Leu Ser Thr Val Asp Leu Lys Lys Leu Arg Val Lys Glu Leu Lys Lys 1 5 10 15 Ile Leu Asp Asp Trp Gly Glu Thr Cys Lys Gly Cys Ala Glu Lys Ser 20 25 30 <210> 53 <211> 61 <212> PRT <213> Homo sapiens <220> <223> Human CDNF (NP_001025125.2) <400> 53 Lys Tyr Glu Lys Thr Leu Asp Leu Ala Ser Val Asp Leu Arg Lys Met 1 5 10 15 Arg Val Ala Glu Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu Cys 20 25 30 Arg Ala Cys Ala Glu Lys Thr Asp Tyr Val Asn Leu Ile Gln Glu Leu 35 40 45 Ala Pro Lys Tyr Ala Ala Thr His Pro Lys Thr Glu Leu 50 55 60 <210> 54 <211> 61 <212> PRT <213> Equus <220> <223> Equine CDNF (XP_001498617.2) <400> 54 Lys Tyr Glu Lys Lys Leu Asp Leu Ala Ser Val Asp Leu Leu Lys Met 1 5 10 15 Arg Val Ala Glu Leu Lys Gln Ile Leu Asn Ser Trp Gly Glu Glu Cys 20 25 30 Arg Ala Cys Ala Glu Lys Ser Asp Tyr Val Asn Leu Ile Lys Glu Leu 35 40 45 Ala Pro Lys Tyr Ala Ala Met His Pro Lys Thr Glu Leu 50 55 60 <210> 55 <211> 61 <212> PRT <213> Bos <220> <223> Bison CDNF (XP_010858254.1) <400> 55 Lys Tyr Glu Lys Lys Leu Asp Leu Ala Ser Val Asp Leu Ser Lys Met 1 5 10 15 Arg Val Ala Glu Leu Lys Gln Ile Leu His Gly Trp Gly Glu Glu Cys 20 25 30 Arg Ala Cys Ala Glu Lys Thr Asp Tyr Val Asn Leu Ile Lys Glu Leu 35 40 45 Ala Pro Lys Tyr Ala Ala Thr His Pro Gln Thr Glu Leu 50 55 60 <210> 56 <211> 61 <212> PRT <213> Sus <220> <223> Porcine CDNF (XP_003130787.1) <400> 56 Lys Tyr Glu Lys Lys Leu Asp Leu Ala Ser Val Asp Leu Ser Lys Met 1 5 10 15 Arg Val Ala Glu Leu Lys Gln Ile Leu Tyr Ser Trp Gly Glu Glu Cys 20 25 30 Arg Ala Cys Ala Glu Lys Thr Asp Tyr Val Asn Leu Ile Lys Glu Leu 35 40 45 Ala Pro Lys Tyr Thr Glu Thr Pro Pro Gln Thr Glu Leu 50 55 60 <210> 57 <211> 61 <212> PRT <213> Canis <220> <223> Dog CDNF (XP_848954.2) <400> 57 Lys Tyr Glu Lys Lys Leu Asp Leu Ala Ser Val Asp Leu Ser Lys Met 1 5 10 15 Arg Val Ala Glu Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu Cys 20 25 30 Ile Ala Cys Ala Glu Lys Thr Asp Tyr Val Asn Leu Ile Thr Glu Leu 35 40 45 Ala Pro Lys Tyr Ala Ala Ala His Pro Lys Thr Glu Leu 50 55 60 <210> 58 <211> 61 <212> PRT <213> Musculus <220> <223> Mouse CDNF (NP_808315.1) <400> 58 Lys Tyr Gly Lys Lys Leu Asp Leu Ala Ser Val Asp Leu Trp Lys Met 1 5 10 15 Arg Val Ala Glu Leu Lys Gln Ile Leu Gln Arg Trp Gly Glu Glu Cys 20 25 30 Arg Ala Cys Ala Glu Lys Ser Asp Tyr Val Asn Leu Ile Arg Glu Leu 35 40 45 Ala Pro Lys Tyr Val Glu Ile Tyr Pro Gln Thr Glu Leu 50 55 60 <210> 59 <211> 61 <212> PRT <213> Cricetus <220> <223> Hamster CDNF (XP_027261009.1) <400> 59 Asn Tyr Glu Lys Lys Leu Asp Leu Ala Ser Val Asp Leu Trp Lys Met 1 5 10 15 Arg Asp Ala Glu Leu Lys Gln Ile Leu His Ser Trp Gly Glu Glu Cys 20 25 30 Arg Ala Cys Ala Glu Lys Asn Asp Tyr Val Asn Leu Ile Lys Glu Leu 35 40 45 Ala Pro Lys Tyr Val Glu Ile His Pro Gln Ile Glu Leu 50 55 60 <210> 60 <211> 61 <212> PRT <213> Alligator <220> <223> Alligator CDNF (XP_019343086.1) <400> 60 Lys Tyr Glu Arg Lys Leu Asp Leu Thr Ser Val Asp Leu Ser Lys Met 1 5 10 15 Arg Val Ala Glu Leu Arg Lys Ile Leu Asp Ser Trp Gly Glu Val Cys 20 25 30 Lys Ala Cys Ile Glu Lys Thr Glu Phe Val Asn Leu Ile Lys Glu Leu 35 40 45 Ala Pro Lys Tyr Ala Pro Pro Asn Ser Arg Ala Asp Leu 50 55 60 <210> 61 <211> 61 <212> PRT <213> Delphinus <220> <223> Dolphin CDNF (XP_026977721.1) <400> 61 Lys Tyr Glu Lys Lys Leu Asp Leu Ala Ser Val Asp Leu Ser Lys Met 1 5 10 15 Arg Val Ala Glu Leu Lys Gln Ile Leu Tyr Ser Trp Gly Glu Glu Cys 20 25 30 Arg Ala Cys Val Glu Lys Thr Asp Tyr Val Asn Leu Ile Lys Glu Leu 35 40 45 Ala Pro Lys Tyr Thr Ala Thr Tyr Pro Lys Thr Glu Leu 50 55 60 <210> 62 <211> 65 <212> PRT <213> Zebrafish (Danio rerio) <220> <223> Zebrafish CDNF (NP_001116753.1) <400> 62 Arg Tyr Glu Arg Leu Val Leu Asp Trp Ser Thr Asp Ala Leu Ser Lys 1 5 10 15 Met Arg Ala Leu Glu Leu Lys Arg Val Leu Ala Ser Trp Gly Glu Glu 20 25 30 Cys Arg Ala Cys Leu Glu Lys Ser Glu Phe Ile Ala Leu Ile Gln Glu 35 40 45 Val Ala Pro Lys His Ser Ala Ser Glu His Arg Ala His Thr Glu Glu 50 55 60 Phe 65 <210> 63 <211> 63 <212> PRT <213> Homo sapiens <220> <223> Human MANF (NP_006001.5) <400> 63 Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ala Arg Thr Asp Leu 50 55 60 <210> 64 <211> 63 <212> PRT <213> Equus <220> <223> Equus MANF (NP_001184244.1) <400> 64 Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ser Arg Thr Asp Leu 50 55 60 <210> 65 <211> 63 <212> PRT <213> Bison <220> <223> Bison MANF (XP_010850093.1) <400> 65 Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ser Arg Thr Asp Leu 50 55 60 <210> 66 <211> 63 <212> PRT <213> Sus <220> <223> Porcine MANF (NP_001231584.1) <400> 66 Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ser Arg Thr Asp Leu 50 55 60 <210> 67 <211> 63 <212> PRT <213> Canis <220> <223> Canine MANF (XP_003639808.2) <400> 67 Lys Tyr Asp Lys Gln Ile Asp Leu Arg Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Arg Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ser Arg Thr Asp Leu 50 55 60 <210> 68 <211> 63 <212> PRT <213> Muscle <220> <223> mouseMANF (NP_083379.2) <400> 68 Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Met Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ala Arg Thr Asp Leu 50 55 60 <210> 69 <211> 63 <212> PRT <213> Cricetus <220> <223> Hamster MANF (RLQ67668) <400> 69 Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Met Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ala Arg Thr Asp Leu 50 55 60 <210> 70 <211> 63 <212> PRT <213> Alligator <220> <223> Alligator MANF (XP_014455597.1) <400> 70 Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ser Arg Thr Asp Leu 50 55 60 <210> 71 <211> 63 <212> PRT <213> Delphinus <220> <223> Dolphin MANF (XP_026976745.1) <400> 71 Lys Tyr Asp Lys Gln Ile Asp Leu Ser Thr Val Asp Leu Lys Lys Leu 1 5 10 15 Arg Val Lys Glu Leu Lys Lys Ile Leu Asp Asp Trp Gly Glu Thr Cys 20 25 30 Lys Gly Cys Ala Glu Lys Ser Asp Tyr Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Lys Ala Ala Ser Ser Arg Thr Asp Leu 50 55 60 <210> 72 <211> 63 <212> PRT <213> Danio rerio <220> <223> Zebrafish MANF (NP_001070097.1) <400> 72 Lys Tyr Asp Lys Gln Val Asp Leu Ser Ser Val Asp Leu Lys Lys Leu 1 5 10 15 Lys Val Lys Asp Leu Lys Lys Ile Leu Glu Glu Trp Gly Glu Ser Cys 20 25 30 Gly Cys Val Glu Lys Ser Asp Phe Ile Arg Lys Ile Asn Glu Leu 35 40 45 Met Pro Lys Tyr Ala Pro Ser Ala Ala Lys Ala Arg Thr Asp Leu 50 55 60

Claims

1. A head- and tail-cyclic macrocyclic peptide or a pharmaceutically acceptable salt thereof, consisting of a length of 12-27 amino acids, wherein the amino acid sequence is selected from the group consisting of: VDLRKMRVAELKQILHSWGEECRACAE (SEQ ID NO: 2), VDLKKLRVKELKKILDDWGETCKGCAE (SEQ ID NO: 4), MRVAELKQILHSWGEECRACAEK (SEQ ID NO: 6), LRVKELKKILDDWGETCKGCAEK (SEQ ID NO: 8), KSILDDWGETCKGCAE (SEQ ID NO: 10), WGEECRACAEKT (SEQ ID NO: 12), WGETCKGCAEKS (SEQ ID NO: 14), WGEECRGACAEKT (SEQ ID NO: 24), and WGETCKGGCAEKS (SEQ ID NO: 26), in, The macrocyclic peptide or a pharmaceutically acceptable salt thereof has at least one of the following properties: (i) Improved stability in plasma compared to its linear counterpart; (ii) Improved stability in hepatocytes compared to its linear counterpart; or (iii) Improved ability to cross the blood-brain barrier compared to its linear counterpart.

2. The macrocyclic peptide of claim 1, wherein the macrocyclic peptide protects against endoplasmic reticulum (ER) stress-induced cell dysfunction or cell death.

3. The macrocyclic peptide according to claim 1 or 2, wherein the macrocyclic peptide is bound to GRP78.

4. The macrocyclic peptide according to claim 1, wherein the cysteine ​​is in a reduced form or a disulfide-bridged form.

5. The macrocyclic peptide according to claim 1, wherein the macrocyclic peptide is a pseudopeptide.

6. The macrocyclic peptide according to claim 1, wherein it is conjugated to a detectable chemical and / or biochemical moiety.

7. The macrocyclic peptide according to claim 1, wherein it is conjugated with polyethylene glycol (PEG).

8. The macrocyclic peptide according to claim 1, wherein the peptide has at least one of the following properties: (i) It can protect TH-positive neurons from MPP+ toxicity in a dose-dependent manner; (ii) Reduce the amount of α-synuclein contents in TH-positive neurons.

9. Use of the macrocyclic peptide according to any one of claims 1-8 in the preparation of a medicament for treating Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease, traumatic brain injury, or retinitis pigmentosa.

10. The use according to claim 9, wherein the peptide is administered by peripheral application, topical application, enteral application, or parenteral application.

11. The use according to claim 10, wherein the peripheral application is selected from subcutaneous and intranasal application.

12. The use according to claim 10, wherein the parenteral administration is selected from intravenous, intra-arterial, intraocular, intratympanic, intra-abdominal, intramuscular, intra-articular, intracranial, intrathecal, epidural, and intralesional administration.

13. The use according to claim 10, wherein the topical application is selected from transdermal, ocular, and inhalation applications.

14. The use according to claim 10, wherein the enteral administration is selected from oral, rectal, sublingual, and buccal administration.

15. The use according to claim 11, wherein the peptide is administered subcutaneously.

16. A pharmaceutical composition comprising a macrocyclic peptide according to any one of claims 1-8 and at least one of the following: a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutically acceptable carrier and the pharmaceutically acceptable excipient are selected from preservatives, stabilizers and / or diluents.

18. Use of the pharmaceutical composition of claim 16 in the preparation of a medicament for treating Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease, traumatic brain injury, or retinitis pigmentosa.

19. The use according to claim 18, wherein the peptide is administered by peripheral, topical, enteral, or parenteral administration.

20. The use according to claim 19, wherein the peripheral application is selected from subcutaneous and intranasal application.

21. The use according to claim 19, wherein the parenteral administration is selected from intravenous, intra-arterial, intraocular, intratympanic, intra-abdominal, intramuscular, intra-articular, intracranial, intrathecal, epidural, and intralesional administration.

22. The use according to claim 19, wherein the topical application is selected from transdermal, ocular, and inhalation applications.

23. The use according to claim 19, wherein the enteral administration is selected from oral, rectal, sublingual, and buccal administration.

24. The use according to claim 20, wherein the composition is administered subcutaneously.

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