Macrocyclic peptides

Abstract

The present invention relates to the field of atypical neurotrophic factors and the field of treating degenerative, chronic or progressive diseases and disorders, and monogenic genetic disorders with ER stress as a pathogenic compound. More specifically, the present invention relates to modified peptides, particularly macrocyclic peptides. The present invention also relates to pharmaceutical compositions comprising the peptides. Furthermore, the present invention also relates to the peptides and pharmaceutical compositions used as medicines and in the treatment of degenerative, chronic or progressive diseases and disorders, and monogenic genetic disorders with ER stress as a pathogenic compound, and methods for treating the diseases and disorders.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the fields of atypical neurotrophic factors and proteins located in the endoplasmic reticulum (ER) and the treatment of degenerative, chronic, or progressive diseases and disorders, as well as monogenic inherited diseases with ER stress as a pathogenic factor. More specifically, the present invention relates to macrocyclic peptides. The present invention also relates to pharmaceutical compositions comprising the peptides. Furthermore, the present invention also relates to the peptides and pharmaceutical compositions for use as medicines and in the treatment of degenerative, chronic, or progressive diseases and disorders, as well as monogenic inherited diseases with ER stress as a pathogenic factor, as well as methods for treating the diseases and disorders. [Background technology]

[0002] Background of the Invention Neurotrophic factors (NTFs) are a subgroup of growth factors that promote neuronal survival and differentiation and possess neuroprotective and neurorestorative properties (Hefti, 1994). NTFs are small proteins that support the proliferation, survival, and differentiation of developing and mature neurons and protect them from injury and toxins. Brain dopamine neurotrophic factor (CDNF), along with its closest relative mesencephalic astrocyte-derived neurotrophic factor (MANF), form a novel family of atypical NTFs that differ both structurally and mechanistically from other growth factors (Lindholm and Saarma, 2010; Huttunen and Saarma, 2019). CDNF and MANF are small monomeric proteins with a mature molecular mass of approximately 18 kDa, consisting of 161 and 158 amino acids, respectively, that are expressed not only in the central nervous system but also in non-neuronal tissues. CDNF and MANF are primarily localized to the endoplasmic reticulum (ER) lumen. They contain an N-terminal signal peptide that targets them to the ER. Both CDNF and MANF also contain a C-terminal KDEL (SEQ ID NO: 37)-like ER retention signal, which is generally absent in secreted growth factors. They interact with ER proteins such as BiP / GRP78, regulate unfolded protein response (UPR) signaling, and protect against ER stress-induced cell death. Both CDNF and MANF accumulate in the ER lumen in healthy cells, and disruption of the C-terminal ER retention signal leads to secretion. Detectable levels of CDNF and MANF are found in normal human serum, and MANF is also 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 cardiomyokine (Glembotski, 2011).

[0003] CDNF and MANF inhibit degeneration in the rat 6-OHDA model of Parkinson's disease DopaThese are currently the most efficient proteins for the treatment of mitochondrial neurons (Lindholm and Saarma, 2010). Both factors, when applied before the toxin, inhibited 6-OHDA-induced Dopa CDNF strongly prevents dopaminergic neuron loss and Parkinson's disease-like motor symptoms (Lindholm et al., 2007; Voutilainen et al., 2009). More importantly, post-lesion administration of either factor efficiently restores normal motor behavior and dopaminergic innervation of the striatum when applied at a stage when 6-OHDA-induced symptoms of Parkinson's disease have already become widespread (Lindholm et al., 2007; Voutilainen et al., 2011). CDNF also effectively prevents dopaminergic neuron loss and Parkinson's disease-like motor symptoms (Lindholm et al., 2007; Voutilainen et al., 2011). Dopa CDNF protects and repairs endothelial cells (CNS) neurons (Airavaara et al., 2012) and is more effective than glial cell line-derived neurotrophic factor (GDNF) in severe 6-OHDA models (Airavaara et al., 2012; Voutilainen et al., 2011). The mechanisms by which these factors protect neuronally are not entirely clear, but they have been suggested to activate pathways aimed at reducing oxidative and ER stress and suppressing 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. Accordingly, the effects of CDNF and MANF have been demonstrated in various central nervous system disorders (WO2009133247; WO2007068803; and Airavaara et al., 2009). Non-cell-autonomous mechanisms, including modulation of immune and glial cell responses, have been shown to contribute to the cytoprotective effects of CDNF and MANF (Sousa-Victor et al., 2018).

[0004] 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).

[0005] CDNF and MANF not only share approximately 60% amino acid sequence identity, but also have very similar three-dimensional structures. Both CDNF and MANF consist of two independently folding domains connected by a flexible loop region (Lindholm and Saarma, 2010). The secondary structure is primarily α-helical, 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, and the C-terminal CRAC (SEQ ID NO: 38) sequence in CDNF and CKGC (SEQ ID NO: 39) sequence in MANF form an internal disulfide bridge. This CXXC (SEQ ID NO: 40) disulfide bridge is found in both CDNF and MANF and plays a central role in the cytoprotective activity of these proteins.

[0006] CDNF is expressed not only in the brain but also in many other tissues, including skeletal muscle, liver, heart, lung, pancreas, testis, salivary glands, and the enteric nervous system (Lindholm et al., 2007). MANF is expressed not only in the brain but also in peripheral tissues such as the pancreas and heart.

[0007] Natural peptides such as those disclosed in WO2013 / 3034805 and WO2018 / 202957 are largely unusable as pharmaceuticals.

[0008] WO2013 / 3034805A1 discloses MANF and CDNF fragments of 4 to 40 amino acids in length containing the sequence CKGC (SEQ ID NO: 39) or CRAC (SEQ ID NO: 38). WO2018 / 202957A1 discloses CDNF fragments of at least 50 amino acids in length. Hellmann et al., 2011, discloses an active C-terminal fragment of MANF containing residues 96 to 158. Fletcher and Hughes, 2006, discloses a brain-derived neurotrophic factor (BDNF)-derived peptide containing CRAC (SEQ ID NO: 38) in which cysteines have been engineered to create a loop. Therefore, there remains a need for therapeutic agents with improved metabolic stability and distribution properties. Summary of the Invention

[0009] Summary of the Invention An object of the present invention is to provide novel modified macrocyclic peptides. Another object of the present invention is to provide uses of the novel peptides.

[0010] The present invention provides tools with these aforementioned properties, particularly through the use of macrocyclic peptides in novel and innovative ways.

[0011] The present inventors have discovered that linear native CDNF and MANF peptides are unsuitable as drug molecules due to their rapid metabolism and poor distribution, especially when administered parenterally to humans or animals. Therefore, native, unmodified peptides, such as those disclosed in prior literature, are largely unusable as pharmaceuticals. The present inventors have developed novel stabilized peptides derived from CDNF and MANF that reproduce the cytoprotective effects of CDNF and MANF but are well suited for noninvasive peripheral administration. As demonstrated by the data herein, the present inventors have discovered that macrocyclization of CDNF and MANF peptides improves metabolic stability and distribution properties without loss of cytoprotective activity. Furthermore, the modified peptides are shorter than those disclosed in prior literature.

[0012] The biological activity of CDNF / MANF is localized to the C-terminal domain of the protein. The present invention discloses 8-32 amino acid peptides derived from the C-terminal domain of CDNF and MANF, particularly in macrocyclic form. A short linear octapeptide around the CXXC (SEQ ID NO: 40) motif, as disclosed herein, exhibited cytoprotective activity comparable to the full-length protein in in vitro models and the ability to cross cell membranes and experimental blood-brain barriers.

[0013] The present inventors have shown for the first time that short macrocyclic peptides, such as head-to-tail cyclized CDNF / MANF macrocyclic peptides with a CXXC (SEQ ID NO: 40) motif or a specific type of CXXXC (SEQ ID NO: 27) motif, have 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 in the development of pharmaceuticals for degenerative, chronic, and / or progressive diseases and disorders or monogenic inherited disorders with ER stress as a pathogenic factor.

[0014] The present invention relates to C-X1-X2-X3-C (SEQ ID NO: 27). [During the ceremony, X1 is selected from the group consisting of R, K, I, G, A and S; X2 is absent or selected from the group consisting of G, A, R, K, I, and S; and X3 is selected from the group consisting of A, G and S. or a pharmaceutically acceptable salt thereof.

[0015] In some embodiments, the macrocyclic peptide is a peptide in which the N-terminus of the peptide is linked to the C-terminus of the peptide (i.e., "head-to-tail linkage"). In some instances, the peptide is a pseudopeptide. In some instances, the peptide has at least one (e.g., one, two, three, four, five, six, or seven) of the following properties: (i) the peptide induces TH-positive neurons to become MPPs; + (ii) the peptide reduces the number of alpha-synuclein inclusions in TH-positive neurons; (iii) the peptide has improved plasma stability compared to its linear counterpart; (iv) the peptide has improved hepatocyte stability compared to its linear counterpart; or (v) the peptide has improved ability to cross the blood-brain barrier compared to its linear counterpart.

[0016] The present invention further provides said macrocyclic peptide for use as a pharmaceutical.

[0017] The present invention further provides said macrocyclic peptides for use in the treatment of degenerative, chronic and / or progressive diseases such as neurodegenerative diseases or disorders and disorders or monogenic inherited diseases having ER stress as a pathogenic factor.

[0018] The present invention further provides pharmaceutical compositions comprising the macrocyclic peptide and at least one of the following pharmaceutically acceptable carriers, pharmaceutically acceptable additives, preservatives, stabilizers and / or diluents:

[0019] The present invention further provides a pharmaceutical composition for use as a medicament.

[0020] The present invention further provides pharmaceutical compositions for use in treating degenerative, chronic and / or progressive diseases such as neurodegenerative diseases or disorders and disorders or monogenic inherited diseases having ER stress as a pathogenic factor.

[0021] The present invention further provides a method for treating a degenerative, chronic or progressive disease or disorder, such as a neurodegenerative disease or disorder, or a monogenic genetic disease having ER stress as a pathogenic factor, in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising said macrocyclic peptide.

[0022] The present invention will now be described in more detail by means of preferred embodiments. [Brief explanation of the drawings]

[0023] [Figure 1]The compounds tested are listed below. The compound number, sequence number, amino acid sequence showing the cyclization scheme and Cys-Cys disulfide bond, sequence length, description of modifications, and monoisotopic mass (Da) are shown. Column 5 details the charged mass peaks seen in the MS spectrum, and column 6 shows the monoisotopic mass of the compound. The difference in monoisotopic mass of approximately 18 Da (the mass of a water molecule) between the linear and cyclic forms of the same peptide confirms successful head-to-tail cyclization.

[0024] Figures 2A-2P show the biophysical characteristics of macrocyclic compound 2 (SEQ ID NO: 2), compound 4 (SEQ ID NO: 4), compound 6 (SEQ ID NO: 6), compound 8 (SEQ ID NO: 8), compound 10 (SEQ ID NO: 10), compound 12 (SEQ ID NO: 12), compound 14 (SEQ ID NO: 14), compound 24 (SEQ ID NO: 24), and compound 26 (SEQ ID NO: 26).

[0025] [Figure 2A] The ring-closure portion is shown as an arrow between the corresponding amino-terminal (head: Val1) and carboxyl-terminal (tail; Glu27) residues in compound 2 (SEQ ID NO: 2). Disulfide bridge-forming cysteines are shown as grey sticks and labeled accordingly.

[0026] [Figure 2B] For amide protons, a contour plot of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint is shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 27 amino acid long cyclic peptide; i.e., the Val1 amide proton is observed, consistent with expected amidation at the N-terminus, as opposed to the linear analog, where no N-terminal amide proton is observed. The Val1 and Val8 protons are assigned and are represented by lines corresponding to HN 7.9 ppm and 8.05 ppm, respectively.

[0027] [Figure 2C]The ring-closure moiety is shown as an arrow between the corresponding amino-terminal (Val1) and carboxyl-terminal (Glu27) residues, representing compound 4 (SEQ ID NO: 4). Disulfide bridge-forming cysteines are represented as grey sticks and labeled accordingly.

[0028] [Figure 2D] A contour plot of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint (left panel) is shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 27-amino acid-long cyclic peptide; i.e., the Val1 amide proton is observed, consistent with expected N-terminal amidation, as opposed to the linear analog, in which no N-terminal amide proton is observed. The 1H chemical shifts of the first (Val1) and last (Glu27) residues are assigned and shown as lines corresponding to HN 8.0 and 8.35 ppm, respectively. For both of these residues, a strong ROE between the amide proton of the preceding amino acid residue and the alpha proton, HN(i)-Hα(i_-1), is observed, as shown in a zoomed-in view of the 2D 1H ROESY fingerprint region (right panel). Establishment of the Val1:HN-Glu27:Hα correlation at 8.02 / 4.25 ppm strongly suggests that the peptide is in its cyclized form, with the protons less than 5 Å apart.

[0029] [Figure 2E] Compound 6 (SEQ ID NO: 6) is depicted, with the ring-closure moiety indicated by an arrow between the corresponding amino-terminal (Met1) and carboxyl-terminal (Lys23) residues. Disulfide bridge-forming cysteines are represented by grey sticks and labeled accordingly.

[0030] [Figure 2F]For amide protons, a contour plot of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint (left panel) is shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 23 amino acid long cyclic peptide; i.e., the Met1 amide proton is observed, consistent with expected amidation at the N-terminus, as opposed to the linear analog, where the N-terminal amide proton is not observed. The Met1 amide proton is assigned and is shown by the line corresponding to HN 7.98 ppm.

[0031] [Figure 2G] The ring-closure moiety is shown as an arrow between the corresponding amino-terminal (Leu1) and carboxyl-terminal (Lys23) residues, representing compound 8 (SEQ ID NO: 8). Disulfide bridge-forming cysteines are represented as grey sticks and labeled accordingly.

[0032] [Figure 2H] A contour plot of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint (left panel) is shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 23-amino acid-long cyclic peptide; five lysine amide protons are observed, consistent with expected N-terminal amidation, in contrast to the linear analog, in which the N-terminal Lys1 amide proton is not observed. 1H chemical shifts for all Lys amide protons have been assigned and are shown by lines corresponding to HN 7.51, 8.05, 8.09, 8.11, and 8.21 ppm, respectively.

[0033] [Figure 2I] The ring-closure moiety is shown as an arrow between the corresponding amino-terminal (Lys1) and carboxyl-terminal (Glu16) residues, representing compound 10 (SEQ ID NO: 10). Disulfide bridge-forming cysteines are represented as grey sticks and labeled accordingly.

[0034] [Figure 2J] Contour plots of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint (left panel) are shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 16-amino acid-long cyclic peptide; that is, all amino acids in the sequence have one amide proton, as opposed to linear analogs with one acid proton (peptide acid) or two amide protons (peptide amide) where the N-terminus is not observed. The 1H chemical shifts of the first (Lys1) and last (Glu16) residues can be assigned corresponding to HN 8.18 ppm and 8.3 ppm, respectively. For both of these residues, strong ROE between the amide proton of the preceding amino acid residue and the alpha proton, HN(i)-Hα(i_-1), is observed, as shown in the zoomed-in view of the 2D 1H ROESY fingerprint region (right panel). Establishment of the Lys1:HN-Glu16:Hα correlation at 8.19 / 4.24 ppm strongly suggests that the peptide is in its cyclized form, with the protons less than 5 Å apart.

[0035] [Figure 2K] Compound 12 (SEQ ID NO: 12) is depicted, with the ring-closure moiety indicated by an arrow between the corresponding amino-terminal (Trp1) and carboxyl-terminal (Thr12) residues. Disulfide bridge-forming cysteines are represented by grey sticks and labeled accordingly.

[0036] [Figure 2L]A contour plot of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint (left panel) is shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 12-amino acid-long cyclic peptide; i.e., the Trp1 amide proton is observed, consistent with expected N-terminal amidation, as opposed to the linear analog, in which no N-terminal amide proton is observed. The 1H chemical shifts of the first (Trp1) and last (Thr12) residues are assigned and shown as lines corresponding to HN 8.21 and 8.06 ppm, respectively. For both of these residues, a strong ROE between the amide proton of the preceding amino acid residue and the alpha proton, HN(i)-Hα(i_-1), is observed, as shown in a zoomed-in view of the 2D 1H ROESY fingerprint region (right panel). Establishment of the Trp1:HN-Thr12:Hα correlation at 8.21 / 4.30 ppm strongly suggests that the peptide is in its cyclized form, with the protons less than 5 Å apart.

[0037] [Figure 2M] The ring-closure moiety is shown as an arrow between the corresponding amino-terminal (Trp1) and carboxyl-terminal (Ser12) residues, representing compound 14 (SEQ ID NO: 14). Disulfide bridge-forming cysteines are represented as grey sticks and labeled accordingly.

[0038] [Figure 2N]A contour plot of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint (left panel) is shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 12-amino acid-long cyclic peptide; i.e., the Trp1 amide proton is observed, consistent with expected N-terminal amidation, as opposed to the linear analog, in which no N-terminal amide proton is observed. The 1H chemical shifts of the first (Trp1) and last (Ser12) residues are assigned and shown as lines corresponding to HN 7.98 ppm and 8.24 ppm, respectively. For both of these residues, a strong ROE between the amide proton of the preceding amino acid residue and the alpha proton, HN(i)-Hα(i_-1), is observed, as shown in a zoomed-in view of the 2D 1H ROESY fingerprint region (right panel). Establishment of the Trp1:HN-Ser12:Hα correlation at 7.99 / 4.4 ppm strongly suggests that the peptide is in its cyclized form, with the protons less than 5 Å apart.

[0039] [Figure 2O] Compound 24 (SEQ ID NO: 24) is shown with the ring-closure moiety indicated by an arrow between the corresponding amino-terminal (Trp1) and carboxyl-terminal (Thr13) residues. Disulfide bridge-forming cysteines are represented by gray bars and labeled accordingly. The glycine residues inserted between the Cys residues in compound 24 (SEQ ID NO: 24) are also represented by gray bars.

[0040] [Figure 2P]A contour plot of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint (left panel) is shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 13-amino acid-long cyclic peptide; i.e., the Trp1 amide proton is observed, consistent with expected N-terminal amidation, as opposed to the linear analog, in which no N-terminal amide proton is observed. The 1H chemical shifts of the first (Trp1) and last (Thr13) residues are assigned and shown as lines corresponding to HN 7.96 and 8.26 ppm, respectively. For both of these residues, a strong ROE between the amide proton of the preceding amino acid residue and the alpha proton, HN(i)-Hα(i_-1), is observed, as shown in a zoomed-in view of the 2D 1H ROESY fingerprint region (right panel). Establishment of the Trp1:HN-Thr13:Hα correlation at 8.26 / 4.35 ppm strongly suggests that the peptide is in its cyclized form, with the protons less than 5 Å apart.

[0041] [Figure 2Q] Figure 2 depicts compound 26 (SEQ ID NO: 26), with the ring-closure moiety indicated by an arrow between the corresponding amino-terminal (Trp1) and carboxyl-terminal (Ser13) residues. Disulfide bridge-forming cysteines are represented by gray bars and labeled accordingly. The inserted glycine residue in compound 26 (SEQ ID NO: 26) is also shown by a gray bar.

[0042] [Figure 2R]A contour plot of the number of amide protons observed in the 2D 1H NMR TOCSY amide fingerprint (left panel) is shown. The horizontal axis is the direct dimension (F2, expressed in ppm), and the vertical axis is the indirect dimension (F1, expressed in ppm). The number of amide protons observed in the TOCSY data is consistent with a 13-amino acid-long cyclic peptide; i.e., the Trp1 amide proton is observed, consistent with expected N-terminal amidation, as opposed to the linear analog, in which no N-terminal amide proton is observed. The 1H chemical shifts of the first (Trp1) and last (Ser13) residues are assigned and shown as lines corresponding to HN 8.13 and 8.1 ppm, respectively. For both of these residues, a strong ROE between the amide proton of the preceding amino acid residue and the alpha proton, HN(i)-Hα(i_-1), is observed, as shown in a zoomed-in view of the 2D 1H ROESY fingerprint region (right panel). Establishment of the Trp1:HN-Ser13:Hα correlation at 8.13 / 4.45 ppm strongly suggests that the peptide is in its cyclized form, with the protons less than 5 Å apart.

[0043] Figures 3A-3L show the neuroprotective effects of rhCDNF and linear and macrocyclic compounds (compounds 1-10, SEQ ID NOs: 1-10, respectively) on dopaminergic TH (tyrosine hydroxylase)-positive neurons injured with MPP + (1-methyl-4-phenylpyridinium) and on alpha-synuclein aggregation in TH-positive neurons. Data are expressed as mean ± SEM as a percentage of the control, non-injured state (n = 4-6; MPP + negative control n = 122-127, pooled across multiple studies). * p<0.05, ** p<0.01, *** p<0.001, *****p<0.0001 vs. MPP + negative control by Brown-Forsythe and Welch ANOVA test with post hoc independent t-test using Welch's correlation pairwise comparisons. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 between linear and corresponding macrocycle compounds at the same concentrations by Brown-Forsythe and Welch ANOVA test with post hoc independent t-test using Welch's correlation pairwise comparisons.

[0044] [Figure 3A] Figure 1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of increasing concentrations of rhCDNF. The upper dotted line indicates the control level (100%) of the parameter obtained from uninjured cells; the lower dotted line indicates the negative control level of the parameter obtained from MPP+ injured cells without further treatment with compound.

[0045] [Figure 3B] Figure 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of increasing concentrations of rhCDNF. The upper dotted line indicates the negative control level of alpha-synuclein aggregation in MPP+ injured cells without further treatment with test compound; the lower dotted line represents the negative control level of the parameter (100%) obtained from non-injured cells.

[0046] [Figure 3C] 1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 1 (SEQ ID NO: 1) and Compound 2 (SEQ ID NO: 2).

[0047] [Figure 3D] 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 1 (SEQ ID NO: 1) and Compound 2 (SEQ ID NO: 2).

[0048] [Figure 3E]1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 3 (SEQ ID NO: 3) and Compound 4 (SEQ ID NO: 4).

[0049] [Figure 3F] 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells following MPP+ injury in the presence of Compound 3 (SEQ ID NO: 3) and Compound 4 (SEQ ID NO: 4).

[0050] [Figure 3G] 1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 5 (SEQ ID NO: 5) and Compound 6 (SEQ ID NO: 6).

[0051] [Figure 3H] 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 5 (SEQ ID NO: 5) and Compound 6 (SEQ ID NO: 6).

[0052] [Figure 3I] 1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 7 (SEQ ID NO: 7) and Compound 8 (SEQ ID NO: 8).

[0053] [Figure 3J] 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 7 (SEQ ID NO: 7) and Compound 8 (SEQ ID NO: 8).

[0054] [Figure 3K]1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 9 (SEQ ID NO: 9) and Compound 10 (SEQ ID NO: 10).

[0055] [Figure 3L] 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells following MPP+ injury in the presence of Compound 9 (SEQ ID NO: 9) and Compound 10 (SEQ ID NO: 10).

[0056] Figures 4A-4L show the neuroprotective effects of linear and macrocyclic compounds (compounds 11-26, with SEQ ID NOs: 11-26, respectively) on MPP+-injured dopaminergic TH-positive neurons and their effects on alpha-synuclein aggregation in TH-positive neurons. Data are expressed as mean ± SEM as a percentage of the control non-injured condition (n = 4-6; MPP+-negative control n = 122-127, pooled across multiple experiments). * p<0.05, ** p<0.01, *** p<0.001, **** *p<0.0001 vs. MPP + negative control by Brown-Forsythe and Welch ANOVA test with post hoc independent t-test using Welch's correlation pairwise comparisons. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 between linear and corresponding macrocycle compounds at the same concentrations by Brown-Forsythe and Welch ANOVA test with post hoc independent t-test using Welch's correlation pairwise comparisons.

[0057] [Figure 4A] 1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 11 (SEQ ID NO: 11) and Compound 12 (SEQ ID NO: 12). The upper dotted line indicates the control level (100%) of the parameter obtained from non-injured cells; the lower dotted line indicates the negative control level of the parameter obtained from MPP+ injured cells not further treated with compounds.

[0058] [Figure 4B] Figure 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 11 (SEQ ID NO: 11) and Compound 12 (SEQ ID NO: 12). The upper dotted line indicates the negative control level of alpha-synuclein aggregation in MPP+ injured cells that were not further treated with test compound; the lower dotted line represents the negative control level of the parameter (100%) obtained from non-injured cells.

[0059] [Figure 4C] 1 shows the number of TH neurons in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 13 (SEQ ID NO: 13) and Compound 14 (SEQ ID NO: 14).

[0060] [Figure 4D] 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 13 (SEQ ID NO: 13) and Compound 14 (SEQ ID NO: 14).

[0061] [Figure 4E] 1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 15 (SEQ ID NO: 15), Compound 16 (SEQ ID NO: 16), Compound 17 (SEQ ID NO: 17), and Compound 18 (SEQ ID NO: 18).

[0062] [Figure 4F] Figure 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 15 (SEQ ID NO: 15), Compound 16 (SEQ ID NO: 16), Compound 17 (SEQ ID NO: 17) and Compound 18 (SEQ ID NO: 18).

[0063] [Figure 4G]1 shows the number of TH neurons in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 19 (SEQ ID NO: 19), Compound 20 (SEQ ID NO: 20), Compound 21 (SEQ ID NO: 21), and Compound 22 (SEQ ID NO: 22).

[0064] [Figure 4H] Figure 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 19 (SEQ ID NO: 19), Compound 20 (SEQ ID NO: 20), Compound 21 (SEQ ID NO: 21) and Compound 22 (SEQ ID NO: 22).

[0065] [Figure 4I] 1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 23 (SEQ ID NO: 23) and Compound 24 (SEQ ID NO: 24).

[0066] [Figure 4J] 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 23 (SEQ ID NO: 23) and Compound 24 (SEQ ID NO: 24).

[0067] [Figure 4K] 1 shows the number of TH neurons, the total neurite network of TH neurons, and the number of synapses of TH neurites in primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 25 (SEQ ID NO: 25) and Compound 26 (SEQ ID NO: 26).

[0068] [Figure 4L] 1 shows alpha-synuclein aggregation in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 25 (SEQ ID NO: 25) and Compound 26 (SEQ ID NO: 26).

[0069] [Figure 5A]1 shows a computerized molecular model of the nucleotide-binding domain of GRP78 (GRP78-NBD) complexed with compound 12. GRP78-NBD is shown as a semi-transparent surface model with its schematic trace. The Cys-Cys bond in compound 12 (SEQ ID NO: 12) is shown as a gray bar.

[0070] [Figure 5B] The binding affinities (Kd, in μM) of a representative set of compounds to GRP78-NBD are tabulated. Binding affinities are obtained by a microscale thermophoresis-based cell-free assay.

[0071] [Figure 5C] The neuroprotective activity of compounds 10 (SEQ ID NO: 10) and 14 (SEQ ID NO: 14) depends on the activity of unfolded protein response (UPR) pathway signaling. GSK2606414 was used to inhibit PERK signaling, and KIRA6 was used to inhibit IRE1 alpha signaling. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, pairwise comparisons to MPP + negative control by one-way ANOVA with post-hoc Fisher's LSD test. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, pairwise comparisons between compounds alone and in combination with UPR signaling inhibitors by one-way ANOVA with post-hoc Fisher's LSD test.

[0072] Figures 6A-6B show the in vitro metabolic stability of linear and macrocyclic compounds in rat (compounds 1-14 and compounds 23-26 having SEQ ID NOs: 1-14 and 23-26, respectively) and human plasma (compounds 1-8, 11-14 and compounds 23-26 having SEQ ID NOs: 1-8, 11-14 and 23-26, respectively).

[0073] [Figure 6A] Calculated half-lives based on compound elimination in rat plasma are shown.

[0074] [Figure 6B]Calculated half-lives based on compound disappearance in human plasma are shown. The brackets and numbers above the columns indicate the change in plasma half-life of the macrocyclic compounds as a percentage of the corresponding linear compounds. The highest calculated half-life of 789 min represents the experimental cutoff time limit.

[0075] Figures 7A-7B show the in vitro metabolic stability of linear and macrocyclic compounds in rats (compounds 1-14 and compounds 23-26 having SEQ ID NOs: 1-14 and 23-26, respectively) and human hepatocytes (compounds 1-8, 11-14 and compounds 23-26 having SEQ ID NOs: 1-8, 11-14 and 23-26, respectively).

[0076] [Figure 7A] Calculated half-lives based on compound elimination in rat hepatocytes are shown.

[0077] [Figure 7B] Calculated half-lives based on compound elimination in human hepatocytes are shown. The brackets and numbers above the columns indicate the change in half-life of the macrocyclic compound in hepatocytes as a percentage of the corresponding linear compound. The highest calculated half-life of 395 min represents the experimental cutoff time limit.

[0078] [Figure 8] Figure 1 shows the penetration of linear and macrocyclic compounds (compounds 1-8, 11-14, and compounds 23-26, with sequence numbers 1-8, 11-14, and 23-26, respectively) through a 3D in vitro model of the blood-brain barrier. The amount of compound passing through the artificial blood-brain barrier is expressed as a percentage of the initially applied concentration. Data are shown as mean ± SEM (n = 3-4). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns non-significant, Brown-Forsythe and Welch ANOVA test and post-hoc unpaired t-test using Welch correlation pairwise comparisons between linear and corresponding macrocyclic compounds.

[0079] [Figure 9A]Plasma concentrations of macrocyclic compounds 2, 10, and 14 (SEQ ID NOs: 2, 10, and 14) and linear compounds 3 and 9 (SEQ ID NOs: 3 and 9) measured at 2, 5, 15, 30 minutes, 1 hour, 2 hours, and 4 hours after intravenous administration to rats at a dose of 5 mg / kg are shown. Data are presented as mean ± SEM (n=3).

[0080] [Figure 9B] Figure 1 shows the distribution kinetics of Compound 14 (SEQ ID NO: 14) in the brain interstitial fluid (ISF; striatum) after 10 mg / kg intravenous bolus injection. ISF concentrations are normalized by microdialysis filter recovery-% (determined by in vitro experiments). Compounds were detected in ISF and plasma using LC-MS / MS.

[0081] [Figure 10] Pairwise alignment of human CDNF and MANF C-terminal domains (61-63 aa) is shown. Alignment was performed using the following Genbank reads: accession number NP_001025125.2 for human CDNF and accession number NP_006001.5 for human MANF. The CXXC motif is shown in gray background, and the positions of the three α-helices are indicated.

[0082] [Figure 11] Figure 1 shows a ClustalW multiple sequence alignment of the C-terminal domains of CDNF and MANF (61-63 aa) from 10 different species (SEQ ID NOs: 53-72, respectively). Genbank accession numbers are shown in the sequence alignment. The CXXC motif is shown in gray background, and the positions of the three α-helices are indicated. Those bases that are conserved between these representative sequences (within both CDNF and MANF) are shown in bold. Below the sequence alignment, natural variants at each position found in the 10 representative species are shown. Using the list of sequences and species shown, conserved and variable positions can be identified, indicating that only limited variation is possible for most non-essential amino acid residues.

[0083] Sequence Listing SEQ ID NO: 1 VDLRKMRVAELKQILHSWGEECRACAE SEQ ID NO: 2 VDLRKMRVAELKQILHSWGEECRACAE, head-to-tail cyclic SEQ ID NO: 3 VDLKKLRVKELKKILDDWGETCKGCAE SEQ ID NO: 4 VDLKKLRVKELKKILDDWGETCKGCAE, head-to-tail circular SEQ ID NO: 5 MRVAELKQILHSWGEECRACAEK SEQ ID NO: 6 MRVAELKQILHSWGEECRACAEK, head-to-tail circular SEQ ID NO: 7 LRVKELKKILDDWGETCKGCAEK SEQ ID NO: 8 LRVKELKKILDDWGETCKGCAEK, head-to-tail circular SEQ ID NO: 9 KSILDDWGETCKGCAE SEQ ID NO: 10 KSILDDWGETCKGCAE, head-to-tail cyclic SEQ ID NO: 11 WGEECRACAEKT SEQ ID NO: 12 WGEECRACAEKT, head-to-tail circular SEQ ID NO: 13 WGETCKGCAEKS SEQ ID NO: 14 WGETCKGCAEKS, head-to-tail circular SEQ ID NO: 15 GEECRACAEKT SEQ ID NO: 16 GEECRGACAEKT SEQ ID NO: 17 GEECRAACAEKT SEQ ID NO: 18 GEECRSACAEKT SEQ ID NO: 19 GETCKGCAEKS SEQ ID NO: 20 GETCKGGCAEKS SEQ ID NO: 21 GETCKAGCAEKS SEQ ID NO: 22 GETCKSGCAEKS SEQ ID NO: 23 WGEECRGACAEKT SEQ ID NO: 24 WGEECRGACAEKT, head-to-tail circular SEQ ID NO: 25 WGETCKGGCAEKS SEQ ID NO: 26 WGETCKGGCAEKS, head-to-tail circular SEQ ID NO: 27 C-X1-X2-X3-C SEQ ID NO: 28 E-X4-C-X1-X2-X3-CAE Sequence number 29 X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 SEQ ID NO: 30X 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-X5X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 SEQ ID NO: 31 RVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYA, native human CDNF peptide SEQ ID NO: 32 RVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYA, native human MANF peptide SEQ ID NO: 33X 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: 34X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X22 -X 23 -VX 24 -ELKX 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 Allocation number 35 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 Allocation number 36 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 Allocation number 37 KDEL Allocation number 38 CRAC Allocation number 39 CKGC Allocation number 40 CXXC Allocation number 41 ETCKGCAE Allocation number 42 TCKGCA SEQ ID NO: 43 MWCASPVAVV AFCAGLLVSH PVLTQGQEAG GRPGADCEVC KEFLNRFYKS LIDRGVNFSL DTIEKELISF CLDTKGKENR LCYYLGATKD AATKILSEVT RPMSVHMPAM KICEKLKKLD SQICELKYEK TLDLASVDLR KMRVAELKQI LHSWGEECRA CAEKTDYVNL IQELAPKYAA THPKTEL, full length CDNF (NCBI Reference Sequence: NP_001025125.2) SEQ ID NO: 44 MRRMWATQGL AVALALSVLP GSRALRPGDC EVCISYLGRF YQDLKDRDVT FSPATIENEL IKFCREARGK ENRLCYYIGA TDDAATKIIN EVSKPLAHHI PVEKICEKLK KKDSQICELK YDKQIDLSTV DLKKLRVKEL KKILDDWGET CKGCAEKSDY IRKINELMPK YAPKAASART DL, full length MANF (NCBI Reference Sequence: NP_006001.5) SEQ ID NO: 45 TLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYA (49 aa CDNF) SEQ ID NO: 46 RVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYA (37aa CDNF) SEQ ID NO: 47 TLDLASVDLRKMRVAELKQILHSWGEECRACAEKT (35 aa CDNF) SEQ ID NO: 48 LASVDLRKMRVAELKQILHSWGEECRACAEKT (32 CDNF) SEQ ID NO: 49 QIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYA (49aa MANF) SEQ ID NO: 50 RVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYA (37aa MANF) SEQ ID NO: 51 QIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKS (35aa MANF) SEQ ID NO: 52 LSTVDLKKLRVKELKKILDDWGETCKGCAEKS (32aa MANF) SEQ ID NO: 53 KYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYA ATHPKTEL, human CDNF (NP_001025125.2) SEQ ID NO: 54 KYEKKLDLASVDLLKMRVAELKQILNSWGEECRACAEKSDYVNLIKELAPKYA AMHPKTEL, Equine CDNF (XP_001498617.2) SEQ ID NO: 55 KYEKKLDLASVDLSKMRVAELKQILHGWGEECRACAEKTDYVNLIKELAPKYA ATHPQTEL, Bison CDNF (XP_010858254.1) SEQ ID NO: 56 KYEKKLDLASVDLSKMRVAELQILYSWGEECRACAEKTDYVNLIKELAPKYTE TPPQTEL, porcine CDNF (XP_003130787.1) SEQ ID NO: 57 KYEKKLDLASVDLSKMRVAELKQILHSWGEECIACAEKTDYVNLITELAPKYAA AHPKTEL, Canine CDNF (XP_848954.2) SEQ ID NO: 58 KYGKKLDLASVDLWKMRVAELKQILQRWGEECRACAEKSDYVNLIRELAPKY VEIYPQTEL, mouse CDNF (NP_808315.1) SEQ ID NO: 59 NYEKKLDLASVDLWKMRDAELKQILHSWGEECRACAEKNDYVNLIKELAPKY VEIHPQIEL, Hamster CDNF (XP_027261009.1) SEQ ID NO: 60 KYERKLDLTSVDLSKMRVAELRKILDSWGEVCKACIEKTEFVNLIKELAPKYA PPNSRADL, Alligator CDNF (XP_019343086.1) SEQ ID NO: 61 KYEKKLDLASVDLSKMRVAELKQILYSWGEECRACVEKTDYVNLIKELAPKYT ATYPKTEL, Dolphin CDNF (XP_026977721.1) SEQ ID NO: 62 RYERLVLDWSTDALSKMRALELKRVLASWGEECRACLEKSEFIALIQEVAPKH SASEHRAHTEEF, zebrafish CDNF (NP_001116753.1) SEQ ID NO: 63 KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAP KAASARTDL, human MANF (NP_006001.5) SEQ ID NO: 64 KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAP KAASSRTDL, Horse MANF (NP_001184244.1) SEQ ID NO: 65 KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAP KAASSRTDL, Bison MANF (XP_010850093.1) SEQ ID NO: 66 KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAP KAASSRTDL, Pig MANF (NP_001231584.1) SEQ ID NO: 67 KYDKQIDLRTVDLKKLRVRELKKILDDWGETCKGCAEKSDYIRKINELMPKYAP KAASSRTDL, dog MANF (XP_003639808.2) SEQ ID NO: 68 KYDKQIDLSTVDLKKLRVKELKKILDDWGEMCKGCAEKSDYIRKINELMPKYAP KAASARTDL, mouse MANF (NP_083379.2) SEQ ID NO: 69 KYDKQIDLSTVDLKKLRVKELKKILDDWGEMCKGCAEKSDYIRKINELMPKYAP KAASARTDL, hamster MANF (RLQ67668) Sequence number 70 KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAP KAASSRTDL, Alligator MANF (XP_014455597.1) Sequence number 71 KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYAP KAASSRTDL, dolphin MANF (XP_026976745.1) SEQ ID NO: 72 KYDKQVDLSSVDLKKLKVKDLKKILEEWGESCKGCVEKSDFIRKINELMPKYA PSAAKARTDL, zebrafish MANF (NP_001070097.1) DETAILED DESCRIPTION OF THE INVENTION

[0084] Detailed Description of the Invention The term "modified peptide" refers to a peptide or polypeptide that has been modified. Peptide modifications or synthesis options include, for example, macrocyclic peptides, peptidomimetics, N-terminal modifications, C-terminal modifications, isotope-labeled peptides, biotinylated and tagged peptides, fluorescent dye-labeled peptides, peptide dimers, post-translational modifications, internal quenching / FRET peptides, linkers / spacers / PEGylation, peptide pooling, protein conjugates, immunogenic peptides, and the incorporation of non-naturally encoded amino acids. A "non-naturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that can be used synonymously with the term "non-naturally encoded amino acid" are "unnatural amino acid," "unnatural amino acid," "non-naturally occurring amino acid," and various hyphenated and non-hyphenated versions thereof. The term "non-naturally encoded amino acid" includes, but is not limited to, amino acids that result from modification (e.g., post-translational modification) of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but that are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0085] The term "macrocyclic peptide" refers to a polypeptide chain having a cyclic ring structure. As used herein, if the two cysteine ​​residues in the CX1X2X3C motif are present in a disulfide-bridged form, the macrocyclic peptide includes a ring structure in addition to the ring structure formed by the disulfide bridge. In some embodiments, the macrocyclic peptide includes a ring structure formed by more than five amino acid residues. The ring structure can include bonds including N-terminus-C-terminus (head-to-tail), head-to-side chain, side chain-to-tail, and side chain-to-side chain bonds. In some embodiments, the present application provides peptides having a head-to-tail bond. The bond can be formed by linking one amino acid of the peptide to another amino acid of the peptide (e.g., both ends, side chain-to-end) through an amide bond or other chemically stable bond such as a lactone, ether, thioether, or disulfide. In some embodiments, bicyclic and monocyclic peptides can be cyclized through a disulfide bond between two cysteines. Cyclization options include, for example: Cys-Cys (up to four disulfide bonds per peptide, site-specific or thermodynamic cyclization), cyclized peptides via amides (head-to-tail or side chain-to-side chain), thioethers (Cys-bromoacetate).

[0086] The term "pseudopeptide" refers to an amide of an amino acid that does not occur in natural peptides or proteins, particularly one introduced into a polypeptide chain. Pseudopeptide or amino bond surrogate are among a variety of terms that can be used to refer to backbone-modified peptides. Synthetic analogs of these peptides have diverse potential applications, but much of the interest in these areas has focused on the possibility of developing metabolically stable, potentially orally active peptide hormone analogs or enzyme inhibitors with enhanced biological potency. The term specifically includes peptide backbone modifications (i.e., amide bond mimetics) known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the α-carbon, the amide carbonyl, complete replacement of the amide bond, extensions, deletions, or backbone crosslinks. Several peptide backbone modifications are known, including ψ[CHS], ψ[CHNH], ψ[CSNH], ψ[NHCO], ψ[COCH], and ψ[(E) or (Z)CH=CH]. In the above nomenclature, ψ indicates the absence of an amide bond. The structure that replaces the amide group is specifically shown in parentheses.

[0087] As used herein, when two entities are "conjugated" to one another, they are linked by direct or indirect covalent or non-covalent interactions. In some embodiments, the bond is covalent. In other embodiments, the bond is non-covalent. Non-covalent interactions include hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. An indirect covalent interaction is when two entities are covalently linked, optionally via a linker group. "Conjugate" herein refers to when a peptide is conjugated or attached to a detectable chemical or biochemical moiety, or to PEG or other moieties used to extend plasma half-life. In some instances, one or more peptides disclosed herein can be conjugated to, for example, a carrier protein. Such conjugate compositions can be monovalent or multivalent. For example, a conjugate composition can include one peptide disclosed herein conjugated to a carrier protein. Alternatively, a conjugate composition can include two or more peptides disclosed herein conjugated to a carrier.

[0088] 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 capillary wall endothelial cells, astrocyte endfeet that ensheath capillaries, and pericytes embedded in the capillary basement membrane. This system allows the passage of certain molecules by passive diffusion and the selective transport of molecules important for neuronal function, such as glucose, water, and amino acids. Large molecules, such as proteins, generally cannot cross the BBB. However, some peptides can cross the BBB via various mechanisms, and some proteins containing specific recognition motifs for transporter proteins present on the surface of cerebrovascular endothelial cells can also be transported across the BBB.

[0089] As used herein, " pharmaceutically acceptable carrier " can include one or more solvents, buffer solutions, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., which are acceptable for use in the preparation of pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Auxiliary active ingredients can also be included in the composition.

[0090] CDNF and MANF not only share approximately 60% amino acid sequence homology (Figures 10 and 11), but also have very similar three-dimensional structures. Both CDNF and MANF consist of two independently folding domains connected by a flexible loop region. The secondary structure is primarily α-helical, 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, and the C-terminal CRAC (SEQ ID NO: 38) sequence in CDNF and CKGC (SEQ ID NO: 39) sequence in MANF form an internal disulfide bridge. This CXXC (SEQ ID NO: 40) disulfide bridge is found in both CDNF and MANF. The CXXC (SEQ ID NO: 40) motif is beneficial for the neuroprotective activity of MANF and CDNF. However, the data presented here indicate that the CXXC (SEQ ID NO: 40) motif can accommodate certain 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 embodiments, CDNF has a sequence derived from NP_001025125.2 (SEQ ID NO: 43). In some embodiments, MANF has a sequence derived from NP_006001.5 (SEQ ID NO: 44).

[0091] In addition to naturally occurring allelic variants derived from MANF and CDNF peptides, changes that alter the amino acid sequence of the encoded MANF / CDNF peptide can be introduced by mutation into the MANF / CDNF sequence. Nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made into the sequence of the MANF / CDNF peptide. A MANF / CDNF peptide or functional fragment thereof containing one or more "non-essential" substitutions can be viewed as equivalent to the wild-type MANF / CDNF peptide disclosed herein.

[0092] Each amino acid can be a natural amino acid or an unnatural amino acid. The term "unnatural amino acid" refers to an organic compound that is a relative of the natural amino acids in that it has a structure similar to the natural amino acids so as to mimic the structure and reactivity of the natural amino acids. The unnatural amino acid can be a modified amino acid and / or an amino acid analog that is not one of the 20 commonly occurring naturally occurring amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine.

[0093] Examples of suitable amino acids include, but are not limited to, alanine, alloisoleucine, arginine, asparagine, aspartic acid, cysteine, cyclohexylalanine, 2,3-diaminopropionic acid, 4-fluorophenylalanine, glutamine, glutamic acid, glycine, histidine, homoproline, isoleucine, leucine, lysine, methionine, naphthylalanine, norleucine, phenylalanine, phenylglycine, pipecolic acid, proline, pyroglutamic acid, sarcosine, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, derivatives, or combinations thereof.

[0094] The term "pharmaceutically acceptable" is used herein to refer to compounds, substances, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0095] "Pharmaceutically acceptable salt" is intended to mean a free acid or base salt of a compound described herein that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to a subject. See generally, SM Berge, 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 undue toxicity, irritation, or allergic response. The compounds described herein possess sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one of each type, and thus can react with numerous inorganic or organic bases and inorganic and organic acids to form pharmaceutically acceptable salts.

[0096] For compounds described herein that contain a basic group, such as an amine, pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example, by converting the free base to a carboxylic acid such as a carboxylic acid or a carboxylic acid with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, or a pyranosidyl acid, such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid, glucuronic acid, or galacturonic acid. The hydroxy acids may be prepared by treating with any suitable mixture of acids, such as alpha-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, naphthoic acid, or cinnamic acid, organic acids, such as lauryl sulfonic acid, p-toluene sulfonic acid, methane sulfonic acid, or sulfonic acids, such as ethane sulfonic acid, or any other acids and mixtures thereof, which are recognized as equivalent or acceptable substitutes in the light of the level of ordinary skill in the art.

[0097] For compounds described herein that contain an acidic group, such as a carboxylic acid group, base addition salts can be prepared by any method available in the art, e.g., by treating such a compound with a sufficient amount of the desired base, either neat 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 tetra-alkylammonium salts.

[0098] Other examples of pharmaceutically acceptable salts include camsylate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyrate-1,4-diol, hexyne-1,6 ... The salts include, but are not limited to, diacid salts, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-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.

[0099] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent forms of the compounds differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to the parent forms of the compounds for purposes of this application.

[0100] In embodiments of the invention, the length of the macrocyclic peptide or fragment ranges from 8 to 32 amino acids, wherein the macrocyclic peptide or fragment thereof comprises CX1X2X3C (SEQ ID NO: 27), as described herein. In certain 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 within the range of 8 to 31, 8 to 29, 8 to 27, 8 to 25, 8 to 23, 8 to 21, 8 to 19, 8 to 17, 8 to 15, 11 to 27, 11 to 25, 11 to 23, 11 to 21, 11 to 19, 11 to 17, 11 to 15, 13 to 25, 13 to 23, 13 to 21, 13 to 19, 13 to 17, 13 to 15, 23 to 27, 24 to 27, or 25 to 27 amino acids. In some embodiments, the length of the peptide or fragment is within the range of 12 to 27, 13 to 27, 12 to 23, 13 to 23, or 23 to 27 amino acids. The peptide or fragment thereof may comprise any of the 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 unnatural or modified amino acids.

[0101] Cyclotides are small disulfide-rich peptides isolated from plants. Cyclotides generally contain 28–37 amino acids and have a head-to-tail cyclized peptide backbone and an interlocking arrangement of three disulfide bonds. Although the plant cyclotide family may contain macrocyclic peptides with potential CXXC and CXXXC motifs, none are known to possess cytoprotective properties similar to those of CDNF and MANF in mammalian cells, i.e., to protect against ER stress-induced cellular dysfunction or cell death, e.g., apoptosis.

[0102] In some embodiments, the macrocyclic peptides of the present invention are not related to plant cyclotides or families of plant cyclotides.

[0103] Preferably, the peptides disclosed herein are not derived from proteins of the thioredoxin and / or protein disulfide isomerase family.

[0104] The present invention relates to C-X1-X2-X3-C (SEQ ID NO: 27). [During the ceremony, X1 is selected from the group consisting of R, K, I, G, A and S; X2 is absent or selected from the group consisting of G, A, R, K, I, and S; and X3 is selected from the group consisting of A, G and S. The present invention provides a macrocyclic peptide having a length of 8 to 32 amino acids, which comprises the amino acid sequence shown below, or a pharmaceutically acceptable salt thereof.

[0105] In certain embodiments, the macrocyclic peptide has a bond between the N-terminus and C-terminus of the peptide.

[0106] In a preferred embodiment, the macrocyclic peptide is E-X4-C-X1-X2-X3-CAE (SEQ ID NO: 28) [During the ceremony, X1 is selected from the group consisting of R, K, I, G, A and S; X2 is absent or selected from the group consisting of G, A, R, K, I and S; X3 is selected from the group consisting of A, G, and S; and X4 is selected from the group consisting of E, T, V, D, M and G. It contains the amino acid sequence of

[0107] In certain embodiments, the macrocyclic peptide has a bond between the N-terminus and C-terminus of the peptide.

[0108] Based on the natural variations in CDNF and MANF sequences in different species (human, horse, bison, pig, dog, mouse, hamster, alligator, dolphin and zebrafish CDNF and MANF are used as example sequences in Figure 10), limited changes in the peptide sequence with respect to the X group compared to the human sequence can be accommodated without loss of biological activity.

[0109] In another preferred embodiment, the macrocyclic peptide is X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO: 29) [During the ceremony, X1 is selected from the group consisting of R, K, I, G, A and S; X2 is absent or selected from the group consisting of G, A, R, K, I and S; X3 is selected from the group consisting of A, G and S; X4 is selected from the group consisting of E, T, V, D, M and G; X5 is absent or selected from the group consisting of H, D, Q, R, Y, N and S; X6 is absent or selected from the group consisting of S, D, G, N, and R; X7 is absent or W; X8 is absent or G; X9 is absent or K; X 10is absent or selected from the group consisting of T, S, A, I, and N; and X 11 is absent or selected from D and E. It contains the amino acid sequence of

[0110] In certain embodiments, the macrocyclic peptide has a bond between the N-terminus and C-terminus of the peptide.

[0111] In another preferred embodiment, the macrocyclic peptide is 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) [During the ceremony, X1 is selected from the group consisting of R, K, I, G, A and S; X2 is absent or selected from the group consisting of G, A, R, K, I and S; X3 is selected from the group consisting of A, G and S; X4 is selected from the group consisting of E, T, V, D, M and G; X5 is absent or selected from the group consisting of H, D, Q, R, Y, N and S; X6 is absent or selected from the group consisting of S, D, G, N, and R; X7 is absent or W; X8 is absent or G; X9 is absent or K; X 10is absent or selected from the group consisting of T, S, A, I and N; X 11 is absent or selected from D and E; X 12 is absent or selected from the group consisting of L, I and V; X 13 is absent or D; X 14 is absent or selected from L and W; X 15 is absent or selected from the group consisting of A, S, T, E and N; X 16 is absent or selected from S and T; X 17 is absent or selected from V and D; X 18 is absent or selected from D and A; X 19 is absent or is L; X 20 is absent or selected from the group consisting of R, K, S and W; X 21 is absent or is K; X 22 is absent or selected from the group consisting of M, L, I and V; X 23 is absent or is R; X 24 is selected from the group consisting of A, K, T, L and V; X 25 is selected from the group consisting of Q, K and R; and X 26 is absent or selected from I and V. The amino acid sequence includes an amino acid sequence within the amino acid sequence of

[0112] In certain embodiments, the macrocyclic peptide has a bond between the N-terminus and C-terminus of the peptide.

[0113] In certain embodiments, the macrocyclic peptide comprises an 8-27 amino acid long peptide within SEQ ID NO: 28, wherein the macrocyclic peptide comprises a CX1X2X3C (SEQ ID NO: 27) motif.

[0114] In another preferred embodiment, the macrocyclic peptide is 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), 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 (SEQ ID NO: 34), 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 X 13 -X 14 -X 15 -X16 -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 (Sequence number 36) The amino acid sequence is within the amino acid sequence selected from the group consisting of:

[0115] In some embodiments, the macrocyclic peptide comprises an 8-27 amino acid long peptide within any of SEQ ID NOs: 33-36, wherein the peptide comprises a CX1X2X3C (SEQ ID NO: 27) motif.

[0116] In certain instances, the macrocyclic peptide comprises an amino acid sequence that is within the amino acid sequence of SEQ ID NO:45 or within the amino acid sequence of SEQ ID NO:49.

[0117] 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).

[0118] In certain embodiments, the macrocyclic peptides protect against endoplasmic reticulum (ER) stress-induced cellular dysfunction or cell death, eg, apoptosis.

[0119] In certain embodiments, the macrocyclic peptide has a bond between the N-terminus and C-terminus of the peptide.

[0120] In some instances, the macrocyclic peptide is 11-32 amino acids in length. In some instances, the peptide is 12-32 amino acids in length. In some instances, the peptide is 12-27 amino acids in length. In some instances, the peptide is 8-27 amino acids in length. In some instances, the peptide is 8-13 amino acids in length. In some instances, the peptide is 8-12 amino acids in length.

[0121] In a preferred embodiment, the macrocyclic peptide cysteine ​​(C) is in reduced or disulfide-bridged form.

[0122] In certain embodiments, the macrocyclic peptides described herein bind to GRP78.

[0123] In some embodiments, the macrocyclic peptides described herein are at least 1.5 times more stable than their linear counterparts. In some embodiments, the peptides described herein are at least 2, 3, or 4 times more stable than their linear counterparts.

[0124] In some embodiments, the macrocyclic peptides described herein have a half-life that is at least 1.5 times longer than their linear counterparts. In some embodiments, the peptides described herein have a half-life that is at least 2 times, 3 times, or 4 times longer than their linear counterparts.

[0125] In some embodiments, the macrocyclic peptide may include a bond connecting the N-terminus to the C-terminus of the peptide.

[0126] In certain embodiments, the N-terminus of the peptide may be acetylated.

[0127] In certain embodiments, the C-terminus of the peptide may be amidated.

[0128] In some embodiments, the N-terminus of the peptide may be acetylated and the C-terminus of the peptide may be amidated.

[0129] In other preferred embodiments, the macrocyclic peptide is conjugated to a detectable moiety, a chemical moiety, a biochemical moiety, or polyethylene glycol (PEG).

[0130] The macrocyclic peptide can be conjugated to a detectable chemical or biochemical moiety, such as a fluorophore (e.g., fluorescein or rhodamine). For example, radiolabeling of the peptide can be used for SPECT or PET imaging. As used herein, "detectable chemical or biochemical moiety" refers to a chemical tag that displays a desired amino acid sequence or a detectable chemical or biochemical moiety that facilitates the detection of the peptide, such as a visible, fluorescent, chemiluminescent, or other detectable chemical tag; an enzyme that is detectable in the presence of a substrate, such as alkaline phosphatase in combination with NBT and BCIP, or peroxidase in combination with an appropriate substrate; a detectable protein, such as green fluorescent protein. Preferably, the tag does not prevent or interfere with the penetration of the fragment into target cells or otherwise alter the biological activity of the compound.

[0131] N- and / or C-terminal modifications of the C-terminal CDNF fragment or C-terminal MANF fragment are also preferred to further increase the stability and / or cell permeability of the peptide or fragment. Acetylation-amidation of the CDNF fragment or MANF fragment (i.e., N-terminal acetylation and C-terminal amidation) is one option known in the art (see, for example, Marino et al. 2015, ACS Chem. Biol. 10: 1754-1764).

[0132] For side chain-to-side chain cyclized peptides, acetylation-amidation of the peptide termini (ie, N-terminal acetylation and C-terminal amidation) increases the stability and cell permeability of the peptide.

[0133] In certain instances, the macrocyclic peptide has at least one (e.g., one, two, three, four, five, six, or seven) of the following properties: (i) the peptide induces TH-positive neurons to become MPPs; + (ii) the peptide reduces the number of alpha-synuclein inclusions in TH-positive neurons; (iii) the peptide has improved plasma stability compared to its linear counterpart; (iv) the peptide has improved hepatocyte stability compared to its linear counterpart; or (v) the peptide has improved ability to cross the blood-brain barrier compared to its linear counterpart.

[0134] Some embodiments provide a macrocyclic peptide as described herein for use as a pharmaceutical.

[0135] CDNF / MANF peptides Dopa Because the peptides potently protect mitochondrial neurons from death, prior art such as WO2009133247 and EP1969003 indicates 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 lobar degeneration, dementia with Lewy bodies, mild cognitive impairment, Huntington's disease (HD), traumatic brain injury, drug addiction, and stroke.

[0136] CDNF and MANF regulate signaling in the unfolded protein response (UPR) pathway and protect cells from ER stress-associated cell death. ER stress is known to play an important pathophysiological role in various chronic diseases, such as neurodegenerative and metabolic disorders, as well as acute injuries (Wang and Kaufman, 2016). GRP78 (also known as BiP and HSPA5) is a major ER luminal chaperone and a key regulator of the UPR (Bertolotti et al., 2000; Wang and Kaufman, 2016). The dynamic association and dissociation of GRP78 with UPR receptors IRE1α, PERK, and ATF6 is a key step in controlling the signaling activity of UPR receptors under ER stress. The interaction between MANF and GRP78 regulates their cellular activity (Yan et al., 2019).

[0137] Accordingly, the present invention provides a method for treating a degenerative, chronic or progressive disease or disorder, such as a CNS disease or disorder, or a monogenic inherited disease having ER stress as a pathogenic factor, comprising administering to a subject a gene encoding a gene encoding a gene having 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-X5X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 The present invention relates to a method for treating a patient comprising administering to the patient a pharmaceutically effective amount of a macrocyclic peptide having a length of 8 to 32 amino acids and comprising (SEQ ID NO: 30) or a pharmaceutically acceptable salt thereof.

[0138] Another embodiment provides a macrocyclic peptide for use in the treatment of a degenerative, chronic or progressive disease or disorder, such as a neurodegenerative disease or disorder.

[0139] The neurodegenerative disease or disorder is preferably a central nervous system disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, 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 neuropathies and the spectrum of diseases and disorders thereof.

[0140] Neurodegenerative diseases may have overlapping, dynamic, nonlinear, progressive "dimensions" resulting in widespread brain proteinopathy. Variability may occur in the expression of several combinations of proteins within the central nervous system. Thus, mixed neuropathology may be observed in patients. The genetic spectrum of neurodegenerative diseases may vary, for example, identical twins with the same genotype may manifest different diseases.

[0141] Another embodiment provides a macrocyclic peptide for use in treating a monogenic genetic disease selected from the group consisting of degenerative retinal diseases such as Wolcott-Rallison syndrome, Wolfram syndrome, Marinesco-Sjögren syndrome, Machado-Joseph disease, and retinitis pigmentosa, and inherited nephrotic syndromes such as primary nephrotic syndrome and autosomal dominant polycystic kidney disease, wherein the monogenic genetic disease has ER stress as a pathogenic factor.

[0142] One embodiment provides a macrocyclic peptide for use in accordance with the present invention, wherein the peptide is administered by peripheral administration, such as intravenous, intra-arterial, subcutaneous, intranasal, intraocular, intratympanic or topical administration, enteral, parenteral or topical routes, including oral, rectal, sublingual or buccal administration, intraperitoneal, intramuscular, intra-articular, transdermal, intracochlear, topical ocular or inhalation administration, or by inhalation administration, or intracranial, intrathecal, epidural or intralesional administration.

[0143] In certain embodiments, the macrocyclic peptide is administered by subcutaneous administration.

[0144] Pharmaceutical Composition One or more of the macrocyclic peptides disclosed herein can be formulated for use as and into pharmaceutical compositions. Such compositions can be formulated or adapted for administration to a subject by any route, e.g., any route approved by the appropriate authorities.

[0145] Some embodiments provide pharmaceutical compositions comprising a macrocyclic peptide described herein and at least one of the following pharmaceutically acceptable carriers, pharmaceutically acceptable additives, preservatives, stabilizers and / or diluents:

[0146] In some embodiments, the present invention further provides a method for the preparation of a nucleotide sequence comprising the sequences 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) or a pharmaceutically acceptable salt thereof.

[0147] In certain instances, a pharmaceutical composition can comprise an effective amount of one or more macrocyclic peptides. As used herein, the terms "effective amount" and "effective for treatment" refer to an amount or concentration of one or more compounds or pharmaceutical compositions described herein, utilized over a period of time (including acute or chronic administration and regular or continuous administration), that is effective, within the context of its administration, to produce the intended effect or physiological outcome.

[0148] In some embodiments of the present invention, the macrocyclic peptides can be incorporated into pharmaceutical compositions. Such compositions of the present invention can be prepared for storage as lyophilized cakes or aqueous solutions by combining the peptide of the desired purity with a physiologically acceptable carrier (e.g., nanocarrier), additive, buffer, or stabilizer (Remington's Pharmaceutical Sciences, 22nd edition, Allen, Loyd V., Jr, Ed., (2012)). Acceptable carriers, additives, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include phosphate, citrate, and other organic acid buffers; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; 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 dextrins; 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, Pluronics, polyethylene glycol (PEG), or additives that enhance nose-to-brain delivery, such as chitosan, methylated pectin, alkyl sugar-based mucosal absorption enhancers, and hydroxy fatty-acyl esters of PEG.

[0149] The actual dosage (e.g., effective amount) of peptide administered to a patient may be determined by physical and physiological factors such as body weight, severity of the condition, the type of disease being treated, previous or current therapeutic interventions, any idiopathies the patient may have, and the route of administration. The administering practitioner can determine the concentration of active ingredient in the composition and the appropriate dose for each individual subject.

[0150] Peptides can be encapsulated in microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example, prepared by conventional techniques or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively). Such techniques are disclosed in Remington's Pharmaceutical Sciences, 22nd edition, Allen, Loyd V., Jr, Ed., (2012). Controlled release gel formulations can also be applied.

[0151] In some embodiments, the pharmaceutical composition may contain, for example, at least about 0.1% of the active compound, hi other embodiments, the active compound may comprise from about 2% to about 75% by weight, or from about 25% to about 60%, for example, and any range therebetween.

[0152] In other non-limiting examples, one dose of the pharmaceutical composition or formulation may contain about 1 ng / kg / body weight, 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 100 μg / kg / body weight, about 200 μg Per administration, the macrocyclic peptide may comprise about 1 mg / 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 of the peptide (any range therebetween). In non-limiting examples of the ranges listed herein, ranges such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight of the peptide may be administered based on the above values.

[0153] The methods herein contemplate the administration of an effective amount of a compound or combination composition to achieve the desired or described effect. Generally, pharmaceutical compositions of the present invention are administered about 1 to about 6 times daily, e.g., 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, or 2-5 times daily, or as a continuous infusion. Pharmaceutical compositions may be administered, for example, 1, 2, 3, 4, 5, or 6 times per day. Such administration can be used for chronic or acute treatment. The amount of active ingredient that may be combined with carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. Typical formulations contain from about 5% to about 95% active compound (w / w). Alternatively, such formulations contain from about 20% to about 80% active compound.

[0154] Dosage can be determined using various techniques.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 excretion or metabolic rate of the specific compound used, the duration of treatment, other drugs, compounds and / or substances used in combination with the specific compound used, the age, sex, weight, condition, general health and / or medical history of the patient being treated, and such factors well known in the medical field.Dosage value can also vary according to the severity of the condition to be alleviated.For a specific subject, specific dosage regimen can be adjusted over time according to individual need and the professional judgment of the person who administers or supervises the administration of the composition.

[0155] In some embodiments, the appropriate daily dose of the compound of the present invention may be the dose of the compound that is the lowest effective dose to produce a therapeutic effect.Such an effective dose generally depends on the above factors.The exact administration time and amount of a particular compound that will produce the most effective treatment for a patient depends on the activity, pharmacokinetics and bioavailability of the particular compound, the physiological condition of the patient (including age, sex, disease type and stage, general physical condition, response to a certain dose and type of administration), route of administration, etc.

[0156] A physician or veterinarian can prescribe an effective amount of the pharmaceutical composition required, for example, by starting the administration of a compound of the present invention in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increasing the dosage until the desired effect is achieved.

[0157] The pharmaceutical compositions described herein can be in unit dosage form suitable for single administration of precise dosage amounts.In 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 a package containing a discrete amount of the formulation.Non-limiting examples are liquids in vials or ampoules.Aqueous suspension compositions can be packaged in single-dose non-reclosable containers.Multiple-dose reclosable containers can be used, for example, in combination with preservatives.Parenteral injection preparations can be provided in unit dosage form, for example, in ampoules or multi-dose containers with preservatives.

[0158] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with a compound of the invention and that does not destroy the pharmacological activity of the compound and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the compound.

[0159] Pharmaceutically acceptable carriers, adjuvants, and vehicles that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-alpha-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins such as human serum albumin, buffers such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated 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 carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0160] The pharmaceutical compositions of the present invention can contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle.In some cases, the pH of the formulation can be adjusted with pharmaceutically acceptable acid, base or buffer to enhance the stability of the formulated compound or its delivery form.The term parenteral used herein includes parenteral, epidural, subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0161] An effective amount of the compound of the present invention can be administered in a single or multiple doses, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or any other suitable number of doses permitted by the method of administration. The number of doses can be within the range defined by any two of the above values. Regardless of the route of administration selected, the compound of the present invention and / or pharmaceutical composition of the present invention will be formulated into a pharmaceutically acceptable dosage form. The compound of the present invention, like other pharmaceuticals, can be formulated for administration in any conventional manner used in human or veterinary medicine.

[0162] In some embodiments, the present invention provides pharmaceutical formulations comprising a therapeutically effective amount of one or more of the compounds described above, formulated with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. In some embodiments, one or more of the compounds described herein are formulated for parenteral administration, where one or more of the compounds disclosed herein may be formulated as aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, or as sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use. Such formulations may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes to render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity against the target compounds can be ensured by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. If desired, the formulation can be diluted, for example, with an isotonic saline solution or dextrose solution before use. In some instances, the compound is formulated as an aqueous solution and administered intravenously.

[0163] Pharmaceutical compositions may be in the form of an injectable solution or powder. Such compositions may be formulated by techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80, etc.) and suspending agents. Sterile injectable preparations may be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol solutions. Particularly acceptable vehicles and solvents that may be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are commonly used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in injectable preparations, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants or carboxymethylcellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants, such as Tweens or Spans, and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

[0164] The pharmaceutical composition can be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.For oral use of tablets, commonly used carriers include lactose and corn starch.Lubricants such as magnesium stearate are also generally added.For oral administration in capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions and / or emulsions are orally administered, the active ingredient can be suspended or dissolved in an oily phase in combination with an emulsifying and / or suspending agent.If desired, certain sweeteners and / or flavorings and / or colorings can be added.

[0165] The pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compound of the present invention with a suitable non-irritating additive that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0166] Alternatively or additionally, pharmaceutical compositions may be administered by nasal aerosol or inhalation. Such compositions are prepared by techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and / or other solubilizing or dispersing agents known in the art.

[0167] In some instances, one or more peptides disclosed herein can be conjugated to, for example, a carrier protein. Such conjugate compositions can be monovalent or multivalent. For example, a conjugate composition can include one peptide disclosed herein conjugated to a carrier protein. Alternatively, a conjugate composition can include two or more peptides disclosed herein conjugated to a carrier.

[0168] Provided herein are methods of using the peptides described herein. For example, the methods provided herein include administering a peptide described herein to a patient. Patients can include both mammals and non-mammals.

[0169] Pharmaceutically acceptable carriers can be selected based on the selected route of administration and standard pharmaceutical practice. For example, the compositions can be formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained-release preparations or elixirs for oral administration, or sterile solutions or suspensions or suspensions for parenteral administration and intraperitoneal injection, as well as transdermal patch preparations, dry powder inhalers and ointments (see, for example, Ansel, Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126). Peptides and / or immunoglobulins can be formulated into dosage forms according to standard practice in the field of pharmaceutical formulations. See Alphonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Edition (1990), Mack Publishing Co., Easton, Pa.

[0170] For parenteral administration, pharmaceutical compositions may contain suitable carriers or diluents, such as water, oils (especially vegetable oils), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or glycols, such as propylene glycol or polyethylene glycol. Solutions for parenteral administration preferably contain a water-soluble salt of the peptide and / or active agent. Stabilizers, antioxidants, and preservatives may also be added. Suitable antioxidants include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Parenteral administration carriers may take the form of aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions.

[0171] For oral administration, the pharmaceutical composition may contain one or more solid inactive ingredients for producing tablets, capsules, pills, powders, granules, or other suitable oral dosage forms. For example, the pharmaceutical composition may contain at least one additive such as a filler, binder, wetting agent, disintegrant, solution retarder, absorption enhancer, wetting agent, absorbent, or lubricant.

[0172] The present invention also relates to pharmaceutical compositions that may further comprise neural cells, which may be, for example, neurons, neural stem cells, or neural progenitor cells.

[0173] The present invention relates to pharmaceutical compositions comprising the macrocyclic peptides described herein and at least one of the following pharmaceutically acceptable carriers, additives, preservatives, stabilizers and / or diluents for use as pharmaceuticals.

[0174] In the method of treatment, a pharmaceutically effective amount of a macrocyclic peptide as defined herein is administered to a patient, i.e., the macrocyclic peptides of the invention are for use in treating degenerative, chronic or progressive diseases or disorders, such as CNS diseases or disorders, or monogenic inherited diseases having ER stress as a pathogenic factor.

[0175] The pharmaceutical composition is for use in the treatment of a degenerative, chronic or progressive disease or disorder, such as a neurodegenerative disease or disorder.

[0176] The neurodegenerative disease or disorder is a central nervous system disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, 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.

[0177] In certain embodiments, the pharmaceutical composition is administered by subcutaneous administration.

[0178] The routes of administration of the macrocyclic peptides are by known methods as well as by injection or infusion via intravenous, intra-arterial, subcutaneous, intranasal, intraocular, intratympanic or topical administration, enteral, parenteral or local routes including oral, rectal, sublingual or buccal administration, intracranial, intrathecal or epidural, intraperitoneal, intramuscular, intra-articular, transdermal, intracochlear, topical ocular, intralesional or inhalation administration or by the common routes of sustained release systems as described below.

[0179] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the peptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, the hydrogels described in Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982), or polyvinyl alcohol, polylactide (U.S. Pat. No. 3,773,919, EP 58,481), or nondegradable ethylene-vinyl acetate (Langer et al., supra).

[0180] The present invention also relates to a method of treating degenerative, chronic or progressive diseases or disorders, such as CNS diseases or disorders, monogenic inherited diseases having ER stress as a pathogenic factor, wherein a pharmaceutically effective amount of a macrocyclic peptide as defined herein is administered to a patient. Preferably, the fragment is administered peripherally.

[0181] The present invention also relates to the use of a macrocyclic peptide as defined herein for the manufacture of a medicament for the treatment of a degenerative, chronic or progressive disease or disorder, such as a CNS disease or disorder, or a monogenic inherited disease having ER stress as a pathogenic factor.

[0182] The present invention also relates to a method of treating a degenerative, chronic, or progressive disease or disorder, such as a neurodegenerative disease or disorder, in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a macrocyclic peptide described herein.

[0183] In certain embodiments, a method for treating a neurodegenerative disease or disorder, such as a central nervous system disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, 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, comprises administering to a subject a pharmaceutical composition comprising a macrocyclic peptide described herein.

[0184] The present invention relates to a method for treating a monogenic genetic disease selected from the group consisting of degenerative retinal diseases such as Wolcott-Rallison syndrome, Wolfram syndrome, Marinesco-Sjögren syndrome, Machado-Joseph disease, and retinitis pigmentosa, and inherited nephrotic syndromes such as primary nephrotic syndrome and autosomal dominant polycystic kidney disease, wherein the monogenic genetic disease has ER stress as a pathogenic factor, comprising administering to a subject a pharmaceutical composition comprising a macrocyclic peptide as described herein.

[0185] The subject in need of treatment may be a human.

[0186] The macrocyclic peptides of the present invention or pharmaceutical compositions containing the peptides can be administered continuously by infusion or by bolus injection. Generally, if the disorder allows, the fragments should be formulated and administered for site-specific delivery. Administration can be continuous or periodic. Administration can be achieved by implantable pumps with metered or programmed flow rates or by periodic injections. Peripheral or systemic administration is preferred because the present invention has demonstrated that macrocyclic peptides can efficiently penetrate neuronal membranes and the blood-brain barrier in vitro and in vivo (Figures 8 and 9B, respectively). Other preferred administration routes are subcutaneous, intrathecal, intracerebroventricular, intranasal, or transdermal administration.

[0187] In another embodiment, the present invention provides a method for promoting survival of dopaminergic neurons, comprising contacting the dopaminergic neurons with a macrocyclic peptide comprising a sequence of 8-32 amino acids set forth in 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 described in the Experimental Section below. The dopaminergic neurons are preferably cultured non-human neurons, such as mouse or rat sympathetic neurons, or induced pluripotent cell (iPSC)-derived human neurons.

[0188] Based on the results provided herein, the present invention also relates to macrocyclic peptides having a length of 8 to 32 amino acids comprising the sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30 peptide for use in the treatment of degenerative, chronic or progressive diseases or disorders such as CNS diseases or disorders or monogenic inherited diseases having ER stress as a pathogenic factor.

[0189] Method for producing macrocyclic peptides Methods for synthesizing the compounds of the present invention are known in the art. The following exemplary methods can be used. It is recognized that the various steps can be performed in an alternate order or sequence to obtain the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Syntheses; and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.

[0190] The peptides of the present invention can be prepared by chemical synthesis methods well known to those skilled in the art. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W.H. Freeman & Co., New York, NY, 1992, p. 77. One method for preparing the peptides described herein is to use solid-phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid-labile bond using a linker molecule. This resin is solvent-independent, relatively simple, and fast, allowing for washing away excess reagents and by-products. The N-terminus is protected with the acid-stable but base-removable Fmoc group. Any side chain functional groups are protected with base-stable, acid-labile groups.

[0191] Publications and other materials used herein to illuminate the background of the invention, and in particular to provide further details regarding the practice, are incorporated herein by reference.

[0192] It is obvious to those skilled in the art that with the advancement of technology, the basic idea of ​​the present invention can be implemented in various ways. Therefore, the present disclosure and its embodiments are not limited to the following examples, and may vary within the scope of the claims. The present invention further includes the following aspects. 1. CX 1 -X 2 -X 3 -C (SEQ ID NO: 27) [During the ceremony, X 1 is selected from the group consisting of R, K, I, G, A and S; X 2 is absent or selected from the group consisting of G, A, R, K, I, and S; and X 3 is selected from the group consisting of A, G and S. or a pharmaceutically acceptable salt thereof. 2. EX 4 -CX 1 -X 2 -X 3 -CAE (SEQ ID NO: 28) [During the ceremony, X 1 is selected from the group consisting of R, K, I, G, A and S; X 2 is absent or selected from the group consisting of G, A, R, K, I and S; X 3 is selected from the group consisting of A, G and S; and X 4 is selected from the group consisting of E, T, V, D, M and G. Item 1. A macrocyclic peptide comprising the amino acid sequence: 3. X 5 -X 6 -X 7 -X 8 -EX 4 -CX 1 -X 2 -X 3 -CAEX 9 -X 10 -X 11 (SEQ ID NO: 29) [During the ceremony, X 1 is selected from the group consisting of R, K, I, G, A and S; X 2 is absent or selected from the group consisting of G, A, R, K, I and S; X 3 is selected from the group consisting of A, G and S; X 4 is selected from the group consisting of E, T, V, D, M and G; X 5 is absent or selected from the group consisting of H, D, Q, R, Y, N and S; X 6 is absent or selected from the group consisting of S, D, G, N and R; X 7 is absent or W; X 8 is absent or is G; X 9 is absent or is K; X 10 is absent or selected from the group consisting of T, S, A, I, and N; and X 11 is absent or selected from D and E. The macrocyclic peptide of item 1 or 2, comprising the amino acid sequence: 4. 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 -LX 5 X 6 -X 7 -X 8 -EX 4 -CX 1 -X 2 -X3 -CAEX 9 -X 10 -X 11 (SEQ ID NO: 30) [During the ceremony, X 1 is selected from the group consisting of R, K, I, G, A and S; X 2 is absent or selected from the group consisting of G, A, R, K, I and S; X 3 is selected from the group consisting of A, G and S; X 4 is selected from the group consisting of E, T, V, D, M and G; X 5 is absent or selected from the group consisting of H, D, Q, R, Y, N and S; X 6 is absent or selected from the group consisting of S, D, G, N and R; X 7 is absent or W; X 8 is absent or is G; X 9 is absent or is K; X 10 is absent or selected from the group consisting of T, S, A, I and N; X 11 is absent or selected from D and E; X 12 is absent or selected from the group consisting of L, I and V; X 13 is absent or D; X 14 is absent or selected from L and W; X 15 is absent or selected from the group consisting of A, S, T, E and N; X 16 is absent or selected from S and T; X 17 is absent or selected from V and D; X 18 is absent or selected from D and A; X 19 is absent or is L; X 20 is absent or selected from the group consisting of R, K, S and W; X 21 is absent or is K; X 22 is absent or selected from the group consisting of M, L, I and V; X 23 is absent or is R; X 24 is selected from the group consisting of A, K, T, L and V; X 25 is selected from the group consisting of Q, K and R; and X 26 is absent or selected from I and V. 4. The macrocyclic peptide according to any one of Items 1 to 3, comprising an amino acid sequence within the amino acid sequence of 5. 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) 5. The macrocyclic peptide according to any one of items 1 to 4, which consists of a sequence selected from the group consisting of: 6. A macrocyclic peptide according to any one of items 1 to 5, which protects against endoplasmic reticulum (ER) stress-induced cell dysfunction or cell death. 7. The macrocyclic peptide according to any one of items 1 to 6, wherein the macrocyclic peptide binds to GRP78. 8. The macrocyclic peptide according to any one of items 1 to 7, wherein the cysteine ​​is in a reduced form or a disulfide-bridged form. 9. The macrocyclic peptide according to any one of items 1 to 8, wherein the macrocyclic peptide is a pseudopeptide. 10. The macrocyclic peptide of any of paragraphs 1 to 9 conjugated to a detectable chemical moiety, a biochemical moiety, or polyethylene glycol (PEG). 11. The peptide has the following properties: (i) TH-positive neurons were identified as MPPs. + It can provide dose-dependent protection from toxicity; (ii) reduce the number of alpha-synuclein inclusions in TH-positive neurons; (iii) have improved plasma stability compared to their linear counterparts; (iv) has improved stability in hepatocytes compared to its linear counterpart; or (v) have an improved ability to cross the blood-brain barrier compared to their linear counterparts 11. The macrocyclic peptide according to any one of items 1 to 10, having at least one of the following: 12. The macrocyclic peptide according to any one of items 1 to 11, for use as a medicine. 13. The macrocyclic peptide of any of paragraphs 1 to 12 for use in treating a degenerative disease or disorder, such as a neurodegenerative disease or disorder, a chronic disease or disorder, or a progressive disease or disorder. 14. A macrocyclic peptide for use according to paragraph 13, wherein the neurodegenerative disease or disorder is a central nervous system disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, 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, and peripheral neuropathies, and the spectrum of diseases and disorders thereof. 15. The macrocyclic peptide of any of items 1 to 11 for use in treating a monogenic genetic disease having endoplasmic reticulum (ER) stress as a pathogenic factor, selected from the group consisting of degenerative retinal diseases such as Wolcott-Rallison syndrome, Wolfram syndrome, Marinesco-Sjögren syndrome, Machado-Joseph disease, and retinitis pigmentosa, and hereditary nephrotic syndromes such as primary nephrotic syndrome and autosomal dominant polycystic kidney disease. 16. A macrocyclic peptide for use according to any of paragraphs 12 to 15, wherein the peptide is administered by peripheral administration, such as intravenous, intra-arterial, subcutaneous, intranasal, intraocular, intratympanic or topical administration, by enteral, parenteral or local routes, including oral, rectal, sublingual or buccal administration, by intraperitoneal, intramuscular, intra-articular, transdermal, intracochlear, topical ocular or inhalation administration, or by intracranial, intrathecal, epidural or intralesional administration. 17. A macrocyclic peptide for use according to paragraph 16, wherein the peptide is administered by subcutaneous administration. 18. A pharmaceutical composition comprising the macrocyclic peptide of any one of items 1 to 11 and at least one of the following pharmaceutically acceptable carriers, pharmaceutically acceptable additives, preservatives, stabilizers and / or diluents: 19. A pharmaceutical composition according to item 18 for use as a medicine. 20. The pharmaceutical composition of paragraph 18 for use in treating a degenerative disease or disorder, such as a neurodegenerative disease or disorder, a chronic disease or disorder, or a progressive disease or disorder. 21. A pharmaceutical composition for use according to paragraph 20, wherein the neurodegenerative disease or disorder is a central nervous system disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, 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, and peripheral neuropathies, and the spectrum of diseases and disorders thereof. 22. The pharmaceutical composition according to item 18, for use in treating a monogenic genetic disease having endoplasmic reticulum (ER) stress as a pathogenic factor, selected from the group consisting of degenerative retinal diseases such as Wolcott-Rallison syndrome, Wolfram syndrome, Marinesco-Sjögren syndrome, Machado-Joseph disease, and retinitis pigmentosa, and hereditary nephrotic syndromes such as primary nephrotic syndrome and autosomal dominant polycystic kidney disease. 23. A pharmaceutical composition for use according to any of paragraphs 18 to 22, wherein the composition is administered by peripheral administration such as intravenous, intra-arterial, subcutaneous, intranasal, intraocular, intratympanic or topical administration, by enteral, parenteral or local routes including oral, rectal, sublingual or buccal administration, by intraperitoneal, intramuscular, intra-articular, transdermal, intracochlear, topical ocular or inhalation administration, or by intracranial, intrathecal, epidural or intralesional administration. 24. A pharmaceutical composition for use according to paragraph 23, wherein the composition is administered by subcutaneous administration. [Example]

[0193] Example 1 MPP+Injury Do Neuroprotective effects of linear and macrocyclic compounds on paminergic TH-positive neurons The neuroprotective effects of compounds 1-26 (SEQ ID NOs: 1-26) were tested in an in vitro model in which primary cultures of rat embryonic midbrain neurons were challenged with MPP+, the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP+ kills dopaminergic (TH-positive) neurons through multiple toxic mechanisms, including mitochondrial dysfunction, peroxynitrite production, oxidative stress, ER stress, and induction of apoptosis. Peptides derived from the C-terminal domains of CDNF and MANF were modified into pseudopeptides, demonstrating the neuroprotective activity of full-length CDNF protein against MPP+-induced injury (Figure 3A) and reduced accumulation of alpha-synuclein aggregates in TH+ neurons (Figure 3B). Dopa Min has been shown to be neuroprotective in protecting neurons, synapses, and neurite networks, and has been tested in models

[0194] Materials and methods for the synthesis and characterization of head-to-tail cyclic derivatives and linear peptides of CDNF and MANF General Protocol - Resin Filling Resins bearing the auxiliary moiety (crypto-thioester Gly-Cys(Hnb)-NH) were prepared for all syntheses. For this purpose, moc-Gly-OH and Fmoc-Cys(StBu)-OH were coupled to TentaGel® 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 Fmoc group removal by treatment with 20% piperidine in DMF, reductive amination was performed using 2-hydroxy-5-nitrobenzaldehyde and sodium cyanoborohydride according to the original method (Terrier et al., 2016). The coupling of the first amino acid (Ala or Gly) was then carried out 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 with acetic anhydride / DIPEA. This resin with auxiliary groups was further used for solid-phase peptide synthesis.

[0195] General Protocol - SPPS 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). The Fmoc group was removed using 20% ​​piperidine in DMF, and capping 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 peptide was deprotected and cleaved from the resin by treatment with TFA / H2O / iPr3SiH for 2 h, followed by precipitation with cold Et2O and lyophilization.

[0196] General Protocol - Native Chemical Ligation (NCL) and SS Oxidation Head-to-tail ring closure was performed using NCL between the N-terminal Cys and the C-terminal crypto-thioester Gly-Cys (Hnb). NCL buffer (6M Gn * HCl, 25mM MPAA, 50mM TCEP * HCl-containing 0.2 M phosphate buffer, pH 6.4, was prepared according to a published protocol (Terrier et al. 2016). The linear peptide was dissolved in the buffer at 1 mM concentration, and the reaction was stirred overnight at 38 °C. The cyclic peptide was isolated 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 disulfide bridges were formed. The target peptide was then purified by RP-HPLC.

[0197] The purity of the peptides was assessed by standard HPLC methods and the identity was confirmed using standard LC-MS methods. The masses of the charged ions obtained from the peptides in this method can be seen in Figure 1.

[0198] Materials and methods for the synthesis of linear derivatives of CDNF and MANF. Linear peptides were synthesized using the general SPPS protocol described previously. Purity and identity were determined by the same method. The quality of the peptides was confirmed using an LC-MS method. The charge masses obtained in this method are shown in Figure 1.

[0199] Confirmation of cyclization of head-to-tail cyclic derivatives of CDNF and MANF using NMR. All head-to-tail cyclic peptides were dissolved in 20 mM sodium phosphate pH 6.0, 10 vol% D2O, 1 mM EDTA, 100 mM NaCl, 3 μM NaN3, 0.5 mM DSS buffer at 25 °C. 1 H / 13 C 1D and 2D NMR spectra were recorded on a 500 MHz spectrometer.

[0200] Head-to-tail cyclic peptide compounds are primarily 1D 1 H, 2D 1 H TOCSY, 2D 1 H ROESY and 2D 1 H- 13 It has been analyzed by C HSQC NMR (Figures 2A-2P). 1 H and 2D 1 H TOCSY NMR data were recorded for compounds 2, 4, 6, 8, 10, 12, 14, 24, and 26 to confirm head-to-tail cyclization. All backbone amides must have one proton each, which is absent in the corresponding linear peptides, whose N-termini have one acid proton (peptide acid) or two amide protons (peptide amide). Peak contours for the N-terminal amide protons are assigned for compounds 2, 4, 6, 8, 10, 12, 14, 24, and 26 and are shown in Figures 2B, 2D, 2F, 2H, 2J, 2L, 2N, 2P, and 2R, respectively. In all cases, the observed number of amide protons matches the number expected for a head-to-tail cyclized peptide of the specific amino acid (as opposed to the corresponding linear analogs, which do not include the N-terminus). This, in itself, indicates that the compounds are cyclized. However, when the data were interpreted in light of the compounds' LC-MS data (shown in Figure 1), the compounds were doubly confirmed to be cyclized.

[0201] Due to spectral overlap between NH and Hα, some of the i1-i23 / 27 ROE correlations were not clearly confirmed (compounds 2, 6, and 8). For the remaining peptides (compounds 4, 10, 12, 14, 24, and 26), the 2D ROE correlations through the space between i1:HN and i(12 / 13 / 23):Hα, i.e., the continuous HN(i)-Hα(i-1) correlations, were sufficient to independently support the cyclized structure. 1 H ROESY data was also collected (and these bonded protons are less than 5 Å apart). 2D data for these compounds (compounds 4, 10, 12, 14, 24, and 26) 1 Zoom-in of the H ROESY amide fingerprint region is shown in Figures 2D, 2J, 2L, 2N, 2P, and 2R, respectively, and significant consecutive ROE cross-peaks are identified by the corresponding HN Marked by frequency.

[0202] Materials and Methods Related to Testing the Neuroprotective Activity of Compounds Culture of midbrain neurons. Rat dopaminergic neurons were cultured as described by Visanji et al., 2008. Then, midbrains obtained from 15-day-old rat embryos (Janvier, France) were excised, and the ventral part of the midbrain flexure, a developing region of the brain rich in dopaminergic neurons, was used for cell preparation. Midbrain cells were dissociated by trypsinization at 37°C for 20 minutes (a solution of 0.05% trypsin and 0.02% EDTA in a final concentration). DNAase The reaction was stopped by adding Dulbecco's modified Eagle's medium (DMEM) containing Grade II (0.5 mg / mL) and 10% fetal calf serum (FCS). The cells were then mechanically dissociated by passing them three times with a 10 ml pipette. The cells were then centrifuged at 180 × g for 10 min at +4 °C on a layer of BSA (3.5%) in L15. The cell pellet was then transferred to Neuron 100 (Nuclear Cell Culture Medium) supplemented with B27 (2%), L-glutamine (2 mM), and 2% PS solution and 10 ng / mL brain-derived neurotrophic factor (BDNF) and 1 ng / mL glial-derived neurotrophic factor (GDNF). The cells were resuspended in a defined serum-free culture medium consisting of 100% ribosomal (Invitrogen). Viable cells were counted using a Neubauer hemocytometer using a trypan blue exclusion test. Cells were seeded at a density of 40,000 cells / well in 96-well plates (precoated with poly-L-lysine) and maintained in a humidified incubator at 37°C with a 5% CO2 / 95% air atmosphere. Half of the medium was replaced with fresh medium every two days. Only 60 wells of a 96-well plate were used. To avoid any edge effects, the first column and row were not used for culture and were filled with sterile water.

[0203] Test Compound and MPP+ Exposure. CDNF and linear and macrocyclic compounds 1-24 (SEQ ID NOs: 1-26) were tested. On day 6 of culture, compounds were dissolved in culture medium and then preincubated with midbrain neurons for 4 hours before application of MPP+. After 4 hours of compound preincubation, MPP+ was added to a final concentration of 4 μM diluted in control medium, still in the presence of compound, for 48 hours.

[0204] Immunostaining: TH neuron survival, neurite network and α-syn aggregation in TH neurons. After 48 hours of intoxication, cells were fixed with 4% paraformaldehyde in PBS, pH 7.3, for 20 minutes at room temperature. Cells were washed twice with PBS, permeabilized, and nonspecific sites were blocked with PBS containing 0.1% saponin and 1% FCS for 15 minutes at room temperature. Next, cells were incubated with (a) a mouse-produced monoclonal antibody anti-tyrosine hydroxylase (TH) at a 1:10,000 dilution and (b) a rabbit-produced polyclonal antibody anti-alpha-synuclein (α-syn) at a 1:400 dilution in PBS containing 1% FCS and 0.1% saponin for 2 hours at room temperature. These antibodies were probed with secondary antibodies, Alexa Fluor 488-conjugated goat anti-mouse IgG at a 1:800 dilution and Alexa 568-conjugated goat anti-rabbit IgG at a 1:400 dilution in PBS containing 1% FCS and 0.1% saponin for 1 hour at room temperature.

[0205] Synaptic immunostaining: TH neurons and PSD-95). After 48 hours of intoxication, the cell culture supernatant was removed and the cells were fixed with 4% paraformaldehyde in PBS, pH 7.3, for 20 minutes at room temperature. The cells were washed twice with PBS, permeabilized, and nonspecific sites were blocked with 0.1% saponin and 1% FCS in PBS for 15 minutes at room temperature. The cells were then incubated with a) a monoclonal anti-tyrosine hydroxylase (TH) antibody at a 1:10,000 dilution in 1% FCS, 0.1% saponin in PBS for 2 hours at room temperature, and b) a polyclonal anti-postsynaptic density protein-95 (PSD-95) antibody at a 1:200 dilution in 1% FCS, 0.1% saponin in PBS for 2 hours at room temperature. This antibody specifically stains synapses. The antibodies were probed with Alexa Fluor 488-conjugated goat anti-mouse IgG at 1:800 and Alexa Fluor 568-conjugated goat anti-rabbit IgG diluted 1:400 in PBS containing 1% FCS, 0.1% saponin for 1 hour at room temperature.

[0206] For each condition, images representing the entire well area were automatically acquired in ImageXpress (Molecular Devices) using 10x (for 20 figures, TH and α-syn) or 40x (for 60 figures, TH and PSD-95) magnification. The following readouts were automatically determined using the Custom Module Editor (Molecular Devices): - Analysis of the total number of TH neurons (TH-positive neurons) - Total neurite network of TH-positive neurons (μm) - Number of α-syn inclusions in TH-positive neurons (total number of TH and α-syn stainings) - Number of synapses in TH-positive neurons (overlap of TH and PSD-95, μm 2 ).

[0207] The total number of TH+ neurons, the total neurite network of TH+ neurons, and the number of synapses of TH+ neurons are shown 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).

[0208] The total number of TH+ neurons is shown in Figure 4E (compounds 15, 16, 17, and 18) and Figure 4G (compounds 19, 20, 21, and 22).

[0209] α-Synuclein aggregation in TH-positive neurons of primary cultures of MPP+-injured midbrain cells is shown in 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).

[0210] These data demonstrate that many macrocyclic peptides protect TH-positive neurons, their neurites, and synapses from MPP+ toxicity in a dose-dependent manner. Furthermore, these macrocyclic compounds effectively reduced the number of α-Syn inclusions, the accumulation of which is strongly induced by MPP+ in TH-positive neurons. In most cases, the efficacy of the macrocyclic peptides (compounds 2, 6, 8, 10, 12, 14, 24, and 26) was comparable to that of their linear counterparts (compounds 1, 5, 7, 9, 11, 13, 23, and 25, respectively).

[0211] Example 2 Interaction with the ER stress response pathway CDNF and MANF protect cells from R-stress-induced cellular dysfunction or cell death, such as apoptosis, by modulating the cellular response to ER stress. Yan et al. (2019) demonstrated that the C-terminus of MANF binds to the nucleotide-binding domain (NBD) of GRP78 and regulates its cellular activity. These data suggest that MANF (and CDNF) have a regulatory, rather than substrate-like, interaction with GRP78, the most abundant chaperone protein in the ER lumen. The binding of compounds to GRP78-NBD was assessed in cell-free binding assays using purified recombinant GRP78-NBD and synthetic peptides. Because GRP78 acts as a key ligand for three receptors in the unfolded protein response (UPR) pathway, IRE1α, PERK, and ATF6, we also tested the dependency of the neuroprotective effects of compounds on UPR signaling. Figure 5A shows molecular modeling of the MANF-binding pocket of GRP78-NBD. Figure 5B shows the binding affinity of selected compounds to GRP78-NBD in a cell-free binding assay. Figure 5C shows that the neuroprotective effects of compounds 10 and 14 are abolished in the presence of pharmacological inhibitors of PERK (GSK2606414) and IRE1 alpha (KIRA6).

[0212] material and method Molecular modeling. GRP78-NBD in complex with various compounds described here (Figures 2A-2P) was modeled based on the previously solved structure of the GRP78-NBD:MANF complex using the PRIME module of the Schrodinger suite version 2018-4 (Schroedinger LLC, USA) via the MAESTRO interface. The generated model was manually verified using the template structure (PDB: 6HAB, Yan et al., 2019). The model was also validated by examining its Ramachandran diagram.

[0213] Cell-free binding assay. His-tagged GRP78-NBD was recombinantly overexpressed in E. coli cells, purified, and labeled using NHS-Red dye (Nanotemper Technologies GmbH). The His tag was cleaved from the protein using TEV protease. Binding of various peptides (serial dilutions) to label untagged GRP78-NBD was measured with a Monolith NT standard capillary using a Monolith NA device (Nanotemper Technologies GmbH) at high power output in a PBS environment. Figure 5B shows a table of data from the MST binding experiments.

[0214] Midbrain neuron culture. Neuronal culture, MPP+ intoxication, immunostaining, and analysis of the neuroprotective effects of compounds were performed as in Example 1. The PERK inhibitor GSK2606414 (2 μM, Sigma) or the IRE1 alpha inhibitor KIRA6 (2 μM, Sigma) was added to the cultures 1 hour before the addition of test compounds.

[0215] This data indicates that the macrocycles bind to the key ER stress-regulated target molecule GRP78 and that the neuroprotective effects of the macrocycles depend on UPR signaling activity.

[0216] Example 3 In vitro metabolic stability of linear and macrocyclic compounds in rat plasma. In this example, the metabolic stability of the macrocyclic compounds was tested. Because metabolic stability is important for compounds intended for parenteral administration in vivo, rat plasma stability studies were performed on both the macrocyclic and linear compounds.

[0217] material and method Linear and macrocyclic compounds 1–14 and 23–26 (SEQ ID NOs: 1–14 and 23–26) were incubated at 1 μM with rat plasma (Sprague-Dawley, male; 400 μl) at various time points (0, 20, 40, 60, or 120 min) at 37°C. The incubation was stopped with acetonitrile. Collected samples were centrifuged at 2272 × g for 20 min and analyzed. Stock solutions were prepared using 50% DMSO, and compounds were added at 1 / 100 for incubation to achieve a final DMSO content of 0.5%. Samples were analyzed by UHPLC / PDA with high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) to monitor compound disappearance. Enalapril at 1 μM was used as a disappearance rate control. For the analysis of compounds 9, 10, 13, and 14, stock solutions were prepared in PBS, and samples were analyzed by UHPLC-ToF mass spectrometry. The analytical method was optimized using the parent compounds for optimal chromatographic properties (peak shape and retention) and mass spectrometric ionization. Ion chromatograms were extracted from total ion chromatograms using calculated monoisotopic accurate masses with a 5 mDa width. Disappearance was based on LC / MS peak areas, with 0 min taken as 100%.

[0218] The first-order rate constant of metabolism, k (min -1 ) was obtained from the slope of the time vs. log (% compound remaining) plot using Excel software. The in vitro half-life (t 1 / 2 ) to t 1 / 2 = ln2 / k. The calculated half-life is based on the elimination of the compound from rat plasma.

[0219] In Figure 6A, each pair of bars shows data from an unmodified peptide (linear) and the 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).

[0220] Example 4 In vitro metabolic stability of linear and macrocyclic compounds in human plasma. Metabolic stability was tested using human plasma at an initial test concentration of 1 μM for 120 minutes. Samples were analyzed using LC / QE-orbitrap-MS. Calculated half-lives are based on the disappearance of the compound in human plasma.

[0221] material and method Linear or macrocyclic compounds 1-8, 11-14, and 23-24 (SEQ ID NOs: 1-8, 11-14, and 23-26) were incubated at 1 μM concentration with human plasma (gender-mixed, 400 μl) for various time points (0, 20, 40, 60, or 120 min) at 37°C. The incubation was stopped with acetonitrile. Collected samples were centrifuged at 2272 × g for 20 min and analyzed. Samples were analyzed by UHPLC / PDA with high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) to monitor compound disappearance. 1 μM propantheline bromide was used as a disappearance rate control. The analytical method was optimized using the parent compounds for optimal chromatographic properties (peak shape and retention) and mass spectrometric ionization. Ion chromatograms were extracted from total ion chromatograms using calculated monoisotopic accurate masses with a 5 mDa width. The disappearance was based on the LC / MS peak area, with 0 min being 100%. The first-order rate constant of metabolism, k (min -1 ) was obtained from the slope of the time vs. log (% compound remaining) plot using Excel software. The in vitro half-life (t 1 / 2 ) to t 1 / 2 = ln2 / k. In Figure 6B, each pair of bars shows data from an unmodified peptide (linear) and the corresponding modified peptide (cyclic).

[0222] Peptide stability in human plasma was better for both linear and macrocyclic compounds compared to stability in rat plasma, as indicated by the reported half-lives in human plasma (maximum test-specific half-life was 795 min). Two macrocyclic compounds (compounds 2 and 26) showed improved stability in human plasma compared to their rat counterparts.

[0223] Example 5 In vitro metabolic stability of linear and macrocyclic compounds in rat hepatocytes Hepatic metabolism plays a central role in compound clearance ,large Cyclic and linear compounds Metabolic stability of was tested in cultured rat hepatocytes.

[0224] material and method Linear or macrocyclic compounds 1–14 and 23–26 (SEQ ID NOs: 1–14 and 21–26) were incubated at 1 μM with pooled cryopreserved rat hepatocytes (Sprague-Dawley, male; 400 μl, 1 million viable cells / ml for compounds 1–8, 11–12, and 23–26, or 100 μl, 100,000 viable cells / ml for compounds 9–10 and 13–14) at 37°C for various time points (0, 10, 20, 40, or 60 min). Cell density and viability were determined by trypan blue exclusion. Incubations were terminated with acetonitrile. Collected samples were centrifuged at 2272 × g for 20 min and analyzed. Samples were analyzed by UHPLC / PDA with high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) (compounds 1–8, 11–12, and 23–26) or HHPLC-ToF mass spectrometry (for compounds 9–10 and 13–14) to monitor compound disappearance. Verapamil 1 μM was used as a disappearance rate control. The analytical method was optimized using the parent compound for optimal chromatographic properties (peak shape and retention) and mass spectrometric ionization. Ion chromatograms were extracted from total ion chromatograms using calculated monoisotopic accurate masses with a 5 mDa width. Disappearance was based on LC / MS peak areas, with 0 min taken as 100%. The first-order rate constant of metabolism, k (min), was calculated as -1 ) was obtained from the slope of the time vs. log (% compound remaining) plot using Excel software. The in vitro half-life (t 1 / 2 ) to t 1 / 2 = ln2 / k. Calculated half-lives are based on compound elimination in rat hepatocytes.

[0225] In Figure 7A, each pair of bars shows data from an unmodified peptide (linear) and the corresponding modified peptide (cyclic). In vitro hepatocyte metabolic stability increased from >10 min to >395 min.

[0226] All macrocyclic compounds (compounds 2, 4, 6, 8, 10, 12, 14, 24, and 26) showed significantly improved stability in rat hepatocytes compared to their linear counterparts (compounds 1, 3, 5, 7, 9, 11, 13, 23, and 25, respectively).

[0227] Example 6 In vitro metabolic stability of linear and macrocyclic compounds in human hepatocytes material and method Linear or macrocyclic compounds 1–8, 11–14, and 23–26 (SEQ ID NOs: 1–8, 11–14, and 23–26) were incubated at 1 μM concentration with pooled cryopreserved human hepatocytes (gender-mixed; 400 μl, 1 million viable cells / ml) for various time points (0, 10, 20, 40, or 60 min) at 37°C. Cell density and viability were determined by trypan blue exclusion. The incubation was terminated with acetonitrile. Collected samples were centrifuged at 2272 × g for 20 min and analyzed. Samples were analyzed by UPLC / PDA with high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) to monitor compound disappearance. 1 μM verapamil was used as a disappearance rate control. The analytical method was optimized using the parent compound for optimal chromatographic properties (peak shape and retention) and mass spectrometric ionization. Ion chromatograms were extracted from total ion chromatograms using calculated monoisotopic accurate masses with a 5 mDa width. Disappearance was based on LC / MS peak area, with 0 min taken as 100%. The first-order rate constant of metabolism, k (min -1 ) was obtained from the slope of the time vs. log (% compound remaining) plot using Excel software. The in vitro half-life (t 1 / 2 ) to t 1 / 2 = ln2 / k. Calculated half-lives are based on compound elimination in human hepatocytes.

[0228] In Figure 7B, each pair of bars shows data from an unmodified peptide (linear) and the corresponding modified peptide (cyclic).

[0229] Nearly all macrocyclic compounds (compounds 2, 4, 6, 12, 14, 24, and 26) showed significantly improved stability in human hepatocytes compared to their linear counterparts (compounds 1, 3, 5, 11, 23, and 25, respectively).

[0230] Example 7 Permeability of linear and macrocyclic compounds in a 3D in vitro model of the blood-brain barrier Due to the interest in developing CDNF- and MANF-derived peptides for treating CNS diseases via peripheral administration routes and the fact that macrocyclization of peptides can improve cell penetration and distribution properties, the ability of the compounds to cross the blood-brain barrier was tested in an established in vitro model of the blood-brain barrier. To this end, 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 acquisition and LC-MS / MS analysis.

[0231] Figure 8 shows the results; the amount of compound that crossed the artificial blood-brain barrier is expressed as a percentage of the originally applied concentration of compound. Compared to the linear compound, better BBB penetration was observed with the macrocyclic compound. BBB penetration increased from <3% (unmodified >30 aa peptide) to >15% (modified).

[0232] All macrocyclic compounds (compounds 2, 4, 6, 8, 12, 14, 24, and 26) showed improved ability to cross the in vitro blood-brain barrier compared to their linear counterparts (compounds 1, 3, 5, 7, 11, 13, 23, and 25, respectively).

[0233] material and method Primary astrocyte culture. Rat astrocytes were prepared from E15 embryos. Briefly, pregnant female rats (Wistar, Janvier Labs) on day 15 of gestation were deeply anesthetized (in a CO2 chamber) and sacrificed by cervical dislocation. Fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium supplemented with 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). Whole brains were treated with trypsin-EDTA solution at a final concentration of 0.05% trypsin and 0.02% EDTA for 20 minutes at 37°C. Dissociated cells were cultured in DMEM + 10% fetal bovine serum. Purified astrocytes were used at passage 4 (P4).

[0234] Culture of human endothelial cells. Vials of HBMEC (primary human brain microvascular endothelial cells, ACBRI 376) were used at a specific passage 8 (P8).

[0235] Primary culture of cortical neurons. Rat cortical neurons were cultured as described (Callizot et al., 2013) with modifications. Briefly, pregnant female rats (Wistar, Janvier Labs) at 15 days of gestation were deeply anesthetized in a CO2 chamber and sacrificed by cervical dislocation. Fetuses were collected and immediately placed in ice-cold L15 Leibovitz's medium supplemented with 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). Cortices were treated with a trypsin-EDTA solution at a final concentration of 0.05% trypsin and 0.02% EDTA for 20 minutes at 37°C. Dissociation was terminated by adding DNAse I Grade II (final concentration 0.5 mg / mL) and 4.5 g / L glucose-supplemented Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Cells were mechanically dissociated by passing them through a 10 ml pipette three times. They were then centrifuged at 515 × g for 10 minutes at 4 °C. The pellet was resuspended in a defined culture medium consisting of Neurobasal medium containing a 2% solution of B27 supplement, 2 mmol / L L-glutamine, 2% PS solution, and 10 ng / mL brain-derived neurotrophic factor (BDNF). Viable cells were counted using a Neubauer hemocytometer using a trypan blue exclusion test. Cortical neurons were seeded at a density of 255,000 per well of a 24-well plate onto the bottom of poly-L-lysine-precoated wells with inserts and cultured at 37 °C in an air (95%)-CO2 (5%) incubator. The culture medium was changed every other day.

[0236] Coculture of endothelial cells, astrocytes, and primary cortical neurons. The procedure was performed as previously published (Xue et al., 2013, modified by 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 × g for 5 min at 4°C, and the pellet was suspended in DMEM-F12 containing 10% FCS. Cells were seeded on the outer surface of the insert membrane (PET, 1 μm) at a density of 45,000 cells per insert and cultured at 37°C in an air (95%)-CO2 (5%) incubator. 36 h 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 515 × g for 5 minutes at 4°C, and the pellet was suspended in EGM-2 bullet kit containing 5% FCS, 1% PS solution, 1.4 μM hydrocortisone, 5 μg / mL ascorbic acid, 1% lipid mixture, 10 mM HEPES, and 1 ng / mL bFGF. Cells were seeded on the inside of insert membranes (PET, 1 μm) at a density of 50,000 cells per insert and cultured at 37°C in an air (95%)-CO2 (5%) incubator. Thirty-six hours after HBMEC seeding (72 hours after astrocyte seeding), cortical neurons were seeded on the bottom of poly-L-lysine-precoated wells at a density of 170,000 cells per well and cultured at 37°C in an air (95%)-CO2 (5%) incubator.

[0237] Application of linear or macrocyclic compounds. Five days after HBMEC seeding, after an initial test of the integrity of the endothelial cell layer, test compounds (compounds 1-8, 11-14, and 23-26, SEQ ID NOs: 1-8, 11-14, and 21-26) were added to the luminal compartment and incubated for 2 hours at a 500 nM concentration.

[0238] Quantification of test compounds. Detection and quantification of each compound in the abluminal supernatant was further performed by mass spectrometry (MS) analysis. After thawing the samples, 100 μL of each cell culture sample was analyzed by mass spectrometry peptide quantification. The calculated pass-through percentage represents the percentage of the applied compound in the abluminal compartment measured in the abluminal compartment at the end of application.

[0239] Example 8 In vivo pharmacokinetic profile of macrocyclic compounds after peripheral administration to rats Peptide clearance and elimination can be mediated by multiple in vivo processes, including metabolism and renal excretion. Because in vitro studies suggested improved stability of the macrocyclic peptides, preliminary pharmacokinetic properties were examined by administering the compounds peripherally (subcutaneously and intravenously) at single dose levels and then determining the presence of the compounds in plasma at various time points after peripheral administration.

[0240] Figure 9A shows the plasma concentrations of macrocyclic compounds 2, 10, and 14 (SEQ ID NOs: 2, 10, and 14) and linear compounds 3 and 9 (SEQ ID NOs: 3 and 9) at various time points after intravenous administration. Compound 14 showed improved blood retention behavior and was detected in plasma for at least 2 hours after administration.

[0241] Table 1 discloses the in vivo pharmacokinetic properties calculated from the plasma concentrations of the linear and cyclic compounds measured at various time points after peripheral administration to rats. [Table 1]

[0242] Figure 9B shows the brain distribution kinetics of compound 14 in male Sprague-Dawley rats. A microdialysis probe was inserted through a guide cannula implanted in the ventral striatum of the rat and perfused with CSF. Compound 14 was administered as a single 10 mg / kg intravenous bolus injection, and microdialysis samples were collected at 20-minute intervals for 4 hours. Brain interstitial fluid (ISF) concentrations of the compound were determined by LC-MS / MS and normalized to the % recovery of the microdialysis membrane (determined by in vitro experiments).

[0243] Low-molecular-weight natural linear peptides generally have a short circulation lifetime because they are removed from blood plasma within minutes by metabolic degradation and clearance mechanisms (Li et al., 2015; Lin et al., 2009). Plasma concentrations of selected linear and macrocyclic compounds were measured at various time points after intravenous administration to rats. Macrocyclic compound 14 (SEQ ID NO: 14) showed improved plasma half-life, distribution volume, and mean residence time. Furthermore, brain microdialysis studies demonstrated the penetration of compound 14 into the brain parenchyma after a single intravenous bolus injection.

[0244] material and method Test compounds were administered intravenously (iv) at 5 mg / kg to male Sprague-Dawley rats (approximately 6 weeks old, n = 3 per compound). Blood samples were collected from the jugular vein via an indwelling catheter (250 μl blood) implanted in labeled polypropylene containing anticoagulant (heparin) at 2, 5, 15, 30, 1, 2, and 4 hours after compound administration and kept on wet ice for up to 30 minutes. Blood samples were centrifuged (4°C, 21,100 xg, 5 minutes) for plasma separation. 500 ng / ml tolbutamide containing 10% TFA in acetonitrile or MeCN was used as the internal standard solution. Standard samples were prepared by adding 2 to 10,000 ng / ml of the test substance to rat plasma matrix and treating the samples in the same manner as the other samples. 200 μl of the internal standard was added to 50 μl of sample plasma. The sample was mixed (150 rpm, 15 min) and centrifuged (3000 rpm, 15 min). The analytical method was optimized for reaction monitoring of chromatographic (peak shape & retention) shifts and mass spectral properties (ionization efficiency, MS / MS detection). The supernatant was analyzed by UHPLC-TOF mass spectrometry using electrospray ionization.

[0245] Brain microdialysis studies were performed in a separate group of awake animals treated intravenously with compound 14. One week before the microdialysis experiment, Sprague-Dawley rats were implanted with a guide cannula in the striatum in the following configuration: AP +0.6 mm; L -3.0 mm; V -2.8 mm, providing a final V of -6.8 mm at the tip of the microdialysis probe. On the day of the experiment, a microdialysis probe (Eicom AI: 0.22 mm OD, 4 mm membrane length with a 50 kDa cutoff) was inserted into the guide cannula and perfused with artificial cerebrospinal fluid (aCSF) solution at a constant flow rate of 0.1 μL / min. After a stabilization period of 120–150 min, compound 18 was administered as a single 10 mg / kg intravenous bolus injection, and samples were collected at 20-min intervals for 4 h. Brain interstitial fluid concentrations of the compound were analyzed by UHPLC-MS / MS. Further in vitro studies were performed to determine the recovery of test compound from tubing, connectors, and microdialysis probes when perfused with aCSF under conditions similar to the in vivo studies. The determined recovery percentage (29.3%) was used to correct the data obtained in the microdialysis studies. [Prior art documents] [Patent documents]

[0246] [Patent Document 1] EP58,481; [Patent Document 2] US3,773,919; [Patent Document 3] WO2007068803; [Patent Document 4] WO2009133247; [Patent Document 5] WO2013 / 3034805; [Patent Document 6] WO2018 / 202957 [Non-patent literature]

[0247] [Non-Patent Document 1] Airavaara M, Shen H, Kuo CC, Peranen J, Saarma M, Hoffer B, and Wang Y. 2009. Mesencephalic astrocyte-derived neurotrophic factor reduces ischemic brain injury and promotes behavioral recovery in rats. J. Comp. Neurol. 515(1):116-24.

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Claims

1. A head-to-tail cyclized macrocyclic peptide consisting of 12 to 27 amino acids in length or a pharmaceutically acceptable salt thereof, 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) A macrocyclic peptide consisting of a sequence selected from the group consisting of:

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

3. 3. The macrocyclic peptide of claim 1 or 2, wherein the macrocyclic peptide binds to GRP78.

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

5. The macrocyclic peptide of any one of claims 1 to 4, wherein the macrocyclic peptide is a pseudopeptide.

6. 6. The macrocyclic peptide of any one of claims 1 to 5 conjugated to a detectable chemical moiety, a biochemical moiety or polyethylene glycol (PEG).

7. The peptide has the following properties: (i) TH-positive neurons were treated as MPPs. ＋ It can provide dose-dependent protection from toxicity; (ii) reducing the number of alpha-synuclein inclusions in TH-positive neurons; (iii) has improved plasma stability compared to its linear counterpart; (iv) has improved stability in hepatocytes compared to its linear counterpart; or (v) have an improved ability to cross the blood-brain barrier compared to their linear counterparts The macrocyclic peptide of any one of claims 1 to 6, having at least one of the following:

8. The macrocyclic peptide of any one of claims 1 to 7 for use as a pharmaceutical.

9. 8. The macrocyclic peptide of any of claims 1 to 7 for use in the treatment of a degenerative, chronic or progressive disease or disorder.

10. 10. The macrocyclic peptide of claim 9, wherein the disease or disorder is a neurodegenerative disease or disorder.

11. 11. The macrocyclic peptide of claim 10, wherein the neurodegenerative disease or disorder is a central nervous system disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, 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, and peripheral neuropathies and the spectrum of diseases and disorders thereof.

12. 8. The macrocyclic peptide of any one of claims 1 to 7 for use in treating a monogenic genetic disease having endoplasmic reticulum (ER) stress as a pathogenic factor, selected from the group consisting of Walcott-Rallison syndrome, Wolfram syndrome, Marinesco-Sjögren syndrome, Machado-Joseph disease, degenerative retinal diseases, and hereditary nephrotic syndromes.

13. 13. The macrocyclic peptide of claim 12, wherein the degenerative retinal disease is retinitis pigmentosa.

14. 13. The macrocyclic peptide of claim 12, wherein the hereditary nephrotic syndrome is selected from primary nephrotic syndrome and autosomal dominant polycystic kidney disease.

15. 15. The macrocyclic peptide for use according to any one of claims 8 to 14, wherein the peptide is administered peripherally, topically, enterally or parenterally.

16. 16. The macrocyclic peptide of claim 15, wherein the peripheral administration is selected from subcutaneous and intranasal administration.

17. 16. The macrocyclic peptide of claim 15, wherein the parenteral administration is selected from intravenous, intraarterial, intraocular, intratympanic, intraperitoneal, intramuscular, intraarticular, intracochlear, intracranial, intrathecal, epidural and intralesional administration.

18. 16. The macrocyclic peptide of claim 15, wherein the topical administration is selected from transdermal, topical ocular and inhalation administration.

19. 16. The macrocyclic peptide of claim 15, wherein enteral administration is selected from oral, rectal, sublingual and buccal administration.

20. 17. The macrocyclic peptide of claim 16, wherein the peptide is administered by subcutaneous administration.

21. A pharmaceutical composition comprising the macrocyclic peptide of any one of claims 1 to 7 and at least one of the following pharmaceutically acceptable carriers, pharmaceutically acceptable additives, preservatives, stabilizers and / or diluents:

22. 22. The pharmaceutical composition of claim 21 for use as a medicament.

23. 22. The pharmaceutical composition of claim 21 for use in treating a degenerative, chronic or progressive disease or disorder.

24. 24. The pharmaceutical composition of claim 23, wherein the disease or disorder is a neurodegenerative disease or disorder.

25. 25. The pharmaceutical composition of claim 24, wherein the neurodegenerative disease or disorder is a central nervous system disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, 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, and peripheral neuropathies and the spectrum of diseases and disorders thereof.

26. 22. The pharmaceutical composition of claim 21 for use in treating a monogenic inherited disease having endoplasmic reticulum (ER) stress as a pathogenic factor, selected from the group consisting of Walcott-Rallison syndrome, Wolfram syndrome, Marinesco-Sjogren syndrome, Machado-Joseph disease and degenerative retinal diseases and hereditary nephrotic syndromes.

27. 27. The pharmaceutical composition of claim 26, wherein the degenerative retinal disease is retinitis pigmentosa.

28. 27. The pharmaceutical composition of claim 26, wherein the hereditary nephrotic syndrome is selected from primary nephrotic syndrome and autosomal dominant polycystic kidney disease.

29. 29. The pharmaceutical composition of any of claims 21 to 28, wherein the composition is administered peripherally, topically, enterally or parenterally.

30. 30. The pharmaceutical composition of claim 29, wherein the peripheral administration is selected from subcutaneous and intranasal administration.

31. 30. The pharmaceutical composition of claim 29, wherein the parenteral administration is selected from intravenous, intraarterial, intraocular, intratympanic, intraperitoneal, intramuscular, intraarticular, intracochlear, intracranial, intrathecal, epidural, and intralesional administration.

32. 30. The pharmaceutical composition of claim 29, wherein the topical administration is selected from transdermal, topical ocular and inhalation administration.

33. 30. The pharmaceutical composition of claim 29, wherein enteral administration is selected from oral, rectal, sublingual, and buccal administration.

34. 30. The pharmaceutical composition for use of claim 29, wherein the composition is administered by subcutaneous administration.

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