Reverse-reverse peptides

Abstract

The present disclosure relates to the field of unconventional neurotrophic factors and the treatment of degenerative, chronic or progressive diseases and disorders as well as monogenic genetic diseases with ER stress as a pathogenic compound. More particularly, the present disclosure relates to modified peptides, particularly reverse-reverse peptides. The present disclosure also relates to pharmaceutical compositions comprising said peptides. Furthermore, the disclosure relates to said peptides and pharmaceutical compositions for use as a medicament and for the treatment of degenerative, chronic or progressive diseases and disorders as well as monogenic genetic diseases with ER stress as a pathogenic compound, as well as methods for the treatment of said diseases and disorders.

Description

[0001] This application is a divisional application of the patent application with application number 202080088948.0, application date December 17, 2020, and invention name “Retro-Inverse Peptide”. The patent application 202080088948.0 is an application entering the Chinese national phase of the PCT international application with application number PCT / EP2020 / 086868. Technical Field

[0002] The present disclosure relates to the field of unconventional neurotrophic factors and endoplasmic reticulum (ER) localized proteins, and to the field of treatment of degenerative, chronic or progressive diseases and disorders. More particularly, the present disclosure relates to retro-inverse peptides. The present disclosure also relates to pharmaceutical compositions comprising the peptides. In addition, the present disclosure also relates to the peptides and pharmaceutical compositions for use as medicaments and for the treatment of degenerative, chronic or progressive diseases and disorders and monogenic inherited diseases with ER stress as a causative compound, and methods for treating the diseases and disorders. Background Art

[0003] Neurotrophic factors (NTFs) are a subgroup of growth factors that promote the survival and differentiation of neurons and have neuroprotective and neurorestorative properties (Hefti, 1994). NTFs are small proteins that support the growth, survival and differentiation of developing and mature neurons and protect them from damage and toxins. Brain dopamine neurotrophic factor (CDNF) and its closest relative midbrain astrocyte-derived neurotrophic factor (MANF) form a new family of unconventional NTFs that are structurally and mechanistically different from other growth factors (Lindholm and Saarma, 2010; Huttunen and Saarma, 2019). CDNF and MANF are small monomeric proteins with a molecular weight of approximately 18 kDa, mature proteins of 161 and 158 amino acids, respectively, expressed in the central nervous system as well as in non-neuronal tissues. CDNF and MANF are mainly localized in the lumen of the endoplasmic reticulum (ER). They contain an N-terminal signal peptide that directs them to the ER. Both CDNF and MANF also contain C-terminal KDEL (SEQ ID NO: 31)-like ER retention signals, which are typically absent in growth factors intended for secretion. They interact with ER proteins (such as BiP / GRP78), regulate unfolded protein response (UPR) signaling, and protect cells from ER stress-induced cell death. Both CDNF and MANF accumulate in the ER cavity of healthy cells, and destroy the C-terminal ER retention signal to cause their secretion. Detectable levels of CDNF and MANF have been found in normal human serum, and MANF has also been found in cerebrospinal fluid (CSF). Based on these characteristics, CDNF and MANF are considered to be general stress protection proteins, rather than highly specific neurotrophic factors (Huttunen and Saarma, 2019). MANF has also been described as a myocardial factor (Glembotski, 2011).

[0004] CDNF and MANF are currently the most effective proteins for treating degenerating dopamine neurons in the 6-OHDA model of Parkinson's disease in rats (Lindholm and Saarma, 2010). When applied before the toxin, these two factors effectively prevented 6-OHDA-induced dopamine neuron loss and Parkinson's-like motor symptoms (Lindholm et al., 2007; Voutilainen et al., 2009). More importantly, when applied at a stage when 6-OHDA-induced Parkinson's disease symptoms were already very severe, the application of either factor after injury effectively restored normal motor behavior and dopaminergic innervation of the striatum (Lindholm et al., 2007; Voutilainen et al., 2011). CDNF also protects and repairs dopamine neurons in the mouse MPTP model of Parkinson's disease (Airavaara et al., 2012), and in the severe 6-OHDA model, it is more effective than glial cell line-derived neurotrophic factor (GDNF) (Airavaara et al., 2012; Voutilainen et al. 2011). The mechanism behind the neuronal protection of these factors is not fully understood, but it has been shown that they activate pathways designed to reduce oxidative stress and ER stress and inhibit apoptotic cell death. Many pathophysiological conditions (including diabetes and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS)) are associated with ER stress. Therefore, the effects of CDNF and MANF have been shown in various central nervous system diseases (WO 2009133247; WO 2007068803; and Airavaara et al., 2009). Non-cell autonomous mechanisms, including the regulation of immune and glial cell responses, have been shown to contribute to the cytoprotective effects of CDNF and MANF (Sousa-Victor et al., 2018). Specifically, CDNF and MANF have been shown to inhibit 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 share approximately 60% amino acid sequence homology, but they have highly similar three-dimensional structures. Both CDNF and MANF are composed of two independently folded domains connected by a flexible loop region (Lindholm and Saarma, 2010). The secondary structure is mainly α-helices, with five α-helices in the N-terminal domain and three α-helices in the C-terminal domain. Three disulfide bridges stabilize the N-terminal domain, while the C-terminal CRAC (SEQ ID NO: 32) sequence in CDNF and CKGC (SEQ ID NO: 33) in MANF form internal disulfide bridges. This CXXC (SEQ ID NO: 34) disulfide bridge is found in both CDNF and MANF and plays an important role in the neuroprotective activity of these proteins.

[0006] CDNF is expressed in the brain, but also in many other tissues, including, for example, skeletal muscle, liver, heart, lung, pancreas, testis, salivary glands, and the enteric nervous system (Lindholm et al., 2007). MANF is expressed in the brain, but also in peripheral tissues such as the pancreas and heart.

[0007] Natural peptides, such as those disclosed in publications WO 2013 / 3034805 and WO 2018 / 202957, are rarely useful as pharmaceutical products.

[0008] Document WO 2013 / 3034805 A1 discloses MANF and CDNF fragments of 4-40 amino acids in length, comprising the sequence CKGC (SEQ IN NO: 33) or CRAC (SEQ ID NO: 32). Document WO 2018 / 202957A1 discloses CDNF fragments of at least 50 amino acids in length. Hellmann et al., 2011, disclose active C-terminal fragment constructs of MANF comprising residues 96-158. Fletcher and Hughes 2006 disclose a brain-derived neurotrophic factor (BDNF)-derived peptide containing CRAC (SEQ ID NO: 32) having engineered cysteine ​​for ring generation. Therefore, there is still a need in the art for therapeutic agents with improved metabolic stability and distribution characteristics. Summary of the invention

[0009] One object of the present disclosure is to provide novel modified retro-inverso peptides. Another object of the present disclosure is to provide uses of the novel peptides.

[0010] The present disclosure provides tools with these aforementioned properties by utilizing peptides, particularly retro-inverso peptides, in novel and inventive ways.

[0011] The inventors have found that linear native CDNF and MANF peptides are poor drug molecules because they are metabolized quickly and poorly distributed when administered to humans or animals, especially parenterally. Therefore, natural unmodified peptides (such as those disclosed in the prior art) are rarely used as drug products. The inventors have developed new stable peptides derived from CDNF and MANF that summarize the cytoprotective effects of CDNF and MANF, but are very suitable for non-invasive peripheral administration. As shown in the data disclosed in the present invention, the inventors found that the reverse-inverse isomerization of CDNF and MANF peptides significantly improved their metabolic stability and distribution characteristics without losing their cytoprotective activity. In addition, the modified peptides of the present invention are shorter than those disclosed in the prior art.

[0012] The biological activity of CDNF / MANF is confined to the C-terminal domain of the protein. The present disclosure describes peptides of 8-32 amino acids derived from the C-terminal domains of CDNF and MANF, particularly in the form of retro-inverse isomerization. The short unmodified octapeptide around the CXXC (SEQ ID NO: 34) motif showed comparable cytoprotective activity to the full-length CDNF / MANF protein and the ability to penetrate cell membranes in vitro as disclosed herein.

[0013] The present inventors have shown for the first time that retro-inverse isomerization of CDNF / MANF peptides having a CXXC (SEQ ID NO: 34) motif or a specific type of CXXXC (SEQ ID NO: 21) motif has significantly improved drug properties, such as metabolic stability, blood-brain barrier (BBB) ​​permeability and in vivo pharmacokinetics. These retro-inverse isomerized peptides can be used to develop drugs for degenerative, chronic and / or progressive diseases and disorders, or monogenic genetic diseases with ER stress as a pathogenic component.

[0014] The present disclosure provides a peptide having a length of 8 to 32 amino acids or a pharmaceutically acceptable salt thereof, which comprises a retro-inverse form of an amino acid sequence of C-X1-X2-X3-C (SEQ ID NO: 21),

[0015] in

[0016] X1 is selected from the group consisting of: R, K, I, G, A and S;

[0017] X2 is absent or selected from the group consisting of: G, A, R, K, I, and S; and

[0018] X3 is selected from the group consisting of: A, G and S.

[0019] In some aspects, the peptide is a pseudopeptide. In some cases, the peptide has at least one of the following properties (e.g., 1, 2, 3, 4, 5, 6, or 7): (i) the peptide can protect TH-positive neurons from MPP+ toxicity in a dose-dependent manner; (ii) the peptide reduces the number of α-synuclein inclusions in TH-positive neurons; (iii) the peptide has improved stability in plasma compared to its parent counterpart; (iv) the peptide has improved stability in hepatocytes compared to its parent counterpart; or (v) the peptide has improved ability to cross the blood-brain barrier compared to its parent counterpart.

[0020] The present disclosure further provides said peptide for use as a medicament.

[0021] The present disclosure further provides the peptides for use in treating degenerative diseases or disorders, chronic diseases or disorders, or progressive diseases or disorders (such as neurodegenerative diseases or disorders), or monogenic genetic diseases with ER stress as a causative component.

[0022] The present disclosure further provides a pharmaceutical composition comprising the peptide and at least one of the following: a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a preservative, a stabilizer and / or a diluent.

[0023] The present disclosure further provides pharmaceutical compositions for use as medicaments.

[0024] The present disclosure further provides a pharmaceutical composition 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 causative component.

[0025] The present disclosure 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 with ER stress as a causative component in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the aforementioned peptide.

[0026] The present invention will be described in more detail below by way of preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A list of the compounds studied is shown. The compound number, SEQ ID NO, amino acid sequence indicating the Cys-Cys disulfide bond, sequence length, description of modifications, and monoisotopic mass (Da) are shown. Column 5 shows the detailed charged mass peaks seen in the MS spectrum, and column 6 shows the monoisotopic mass of the compound.

[0028] Figure 2A-2BThe neuroprotective effect of CDNF on dopaminergic TH (tyrosine hydroxylase) positive neurons injured by MPP+ (1-methyl-4-phenylpyridinium), and the effect on α-synuclein aggregation in TH positive neurons are shown. Data are expressed as a percentage of control non-injury conditions, i.e., mean + / - SEM (n = 4-6; MPP+ negative control n = 122-127 collected in multiple studies). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 Brown-Forsythe and Welch ANOVA tests, and post hoc unpaired t tests and Welch related paired comparisons, compared to MPP+ negative controls.

[0029] Figure 2A The number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of increasing concentrations of rhCDNF are shown. The upper dashed line represents the control level (100%) of the parameter obtained from non-injured cells; the lower dashed line represents the negative control level of the parameter obtained from MPP+ injured cells without additional treatment with the compound.

[0030] Figure 2B Shown is the aggregation of alpha-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells after MPP+ injury in the presence of increasing concentrations of rhCDNF. The upper dashed line represents the negative control level of the parameter obtained from MPP+ injured cells without additional treatment with CDNF; the lower dashed line represents the negative control level of the parameter obtained from non-injured cells (100%).

[0031] Figures 3A-3HThe neuroprotective effects of parent and retro-reversal compounds (compounds 1-8 with SEQ ID NOs: 1-8, respectively) on dopaminergic TH (tyrosine hydroxylase)-positive neurons injured by MPP+ (1-methyl-4-phenylpyridinium), as well as their effects on α-synuclein aggregation in TH-positive neurons, are shown. Data are expressed as a percentage of control non-injured conditions, i.e., mean + / - SEM (n = 4-6; MPP+ negative controls collected in multiple studies n = 122-127). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Brown-Forsythe and Welch ANOVA tests, and post hoc unpaired t tests and Welch correlated pairwise comparisons, compared to MPP+ negative controls. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 Brown-Forsythe and Welch ANOVA tests between parent and corresponding retro-reversal compounds at the same concentration, and post hoc unpaired t tests and Welch correlated pairwise comparisons.

[0032] Figure 3A The number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 1 (SEQ ID NO: 1) and Compound 2 (SEQ ID NO: 2) are shown. The upper dashed line represents the control level (100%) of the parameter obtained from non-injured cells; the lower dashed line represents the negative control level of the parameter obtained from MPP+ injured cells that were not additionally treated with the compound.

[0033] Figure 3B Shown is the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells after MPP+ injury in the presence of Compound 1 (SEQ ID NO: 1) and Compound 2 (SEQ ID NO: 2). The upper dashed line represents the negative control level of the parameter obtained from MPP+ injured cells without additional treatment with the compound; the lower dashed line represents the negative control level of the parameter obtained from non-injured cells (100%).

[0034] Figure 3C Shown are the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 3 (SEQ ID NO: 3) and Compound 4 (SEQ ID NO: 4).

[0035] Figure 3DShown is the aggregation of alpha-synuclein (aSyn) in TH neurons of 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).

[0036] Figure 3E Shown are the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 5 (SEQ ID NO: 5) and Compound 6 (SEQ ID NO: 6).

[0037] Figure 3F Shown is the aggregation of alpha-synuclein (aSyn) 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).

[0038] Figure 3G Shown are the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 7 (SEQ ID NO: 7) and Compound 8 (SEQ ID NO: 8).

[0039] Figure 3H Shown is the aggregation of alpha-synuclein (aSyn) 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).

[0040] Figures 4A-4LThe neuroprotective effects of parent and retro-reversal compounds (compounds 9-20 with SEQ ID NOs: 9-20, respectively) on dopaminergic TH (tyrosine hydroxylase)-positive neurons injured by MPP+ (1-methyl-4-phenylpyridinium), as well as their effects on α-synuclein aggregation in TH-positive neurons, are shown. Data are expressed as a percentage of control non-injured conditions, i.e., mean + / - SEM (n = 4-6; MPP+ negative controls collected in multiple studies n = 122-127). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Brown-Forsythe and Welch ANOVA tests, and post hoc unpaired t tests and Welch correlated pairwise comparisons, compared to MPP+ negative controls. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 Brown-Forsythe and Welch ANOVA tests between parent and corresponding retro-reversal compounds at the same concentration, and post hoc unpaired t tests and Welch correlated pairwise comparisons.

[0041] Figure 4A The number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 9 (SEQ ID NO: 9) and Compound 10 (SEQ ID NO: 10) are shown. The upper dashed line represents the control level (100%) of the parameter obtained from non-injured cells; the lower dashed line represents the negative control level of the parameter obtained from MPP+ injured cells that were not additionally treated with the compound.

[0042] Figure 4B Shown is the aggregation of α-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells after MPP+ injury in the presence of Compound 9 (SEQ ID NO: 9) and Compound 10 (SEQ ID NO: 10). The upper dashed line represents the negative control level of the parameter obtained from MPP+ injured cells without additional treatment with the compound; the lower dashed line represents the negative control level of the parameter obtained from non-injured cells (100%).

[0043] Figure 4C Shown are the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 11 (SEQ ID NO: 11) and Compound 12 (SEQ ID NO: 12).

[0044] Figure 4DShown is the aggregation of alpha-synuclein (aSyn) 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).

[0045] Figure 4E Shown are the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 13 (SEQ ID NO: 13) and Compound 14 (SEQ ID NO: 14).

[0046] Figure 4F Shown is the aggregation of alpha-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells after MPP+ injury in the presence of Compound 13 (SEQ ID NO: 13) and Compound 14 (SEQ ID NO: 14).

[0047] Figure 4G Shown are the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 15 (SEQ ID NO: 15) and Compound 16 (SEQ ID NO: 16).

[0048] Figure 4H Shown is the aggregation of alpha-synuclein (aSyn) in TH neurons of primary cultures of midbrain cells after MPP+ injury in the presence of Compound 15 (SEQ ID NO: 15) and Compound 16 (SEQ ID NO: 16).

[0049] Fig. 4I Shown are the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 17 (SEQ ID NO: 17) and Compound 18 (SEQ ID NO: 18).

[0050] Figure 4J Shown is the aggregation of alpha-synuclein (aSyn) in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 17 (SEQ ID NO: 17) and Compound 18 (SEQ ID NO: 18).

[0051] Figure 4KShown are the number of TH neurons, the total neurite network of TH neurons, and the number of synapses on TH neurites in primary cultures of midbrain cells after MPP+ injury in the presence of Compound 19 (SEQ ID NO: 19) and Compound 20 (SEQ ID NO: 20).

[0052] Figure 4L Shown is the aggregation of alpha-synuclein (aSyn) in TH neurons of primary cultures of mesencephalic cells after MPP+ injury in the presence of Compound 19 (SEQ ID NO: 19) and Compound 20 (SEQ ID NO: 20).

[0053] Figure 5A A computational molecular model of the nucleotide binding domain of GRP78 (GRP78-NBD) in complex with compound 14 is shown. GRP78-NBD is shown in a semi-transparent surface model along with its cartoon trace. The Cys-Cys bond in compound 14 (SEQ ID NO: 14) is shown in stick form.

[0054] Figure 5B The binding affinities (Kd, in μM) of a group of representative peptides to GRP78-NBD are shown in a tabular form. The binding affinities were obtained by a cell-free assay based on microthermophoresis.

[0055] Figure 5C The dependence of the neuroprotective activity of compounds 14 (SED ID NO: 14) and 20 (SEQ ID NO: 20) on the signaling activity of the unfolded protein response (UPR) pathway is shown. GSK2606414 was used to inhibit PERK signaling, and KIRA6 was used to inhibit IRE1α signaling. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 One-way ANOVA test and post hoc Fisher's LSD test were used for pairwise comparisons compared to MPP+negative control. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001 One-way ANOVA test and post hoc Fisher's LSD test were used for pairwise comparisons between the effects of individual compounds and their combination with UPR signaling inhibitors.

[0056] Figure 6A-6B The in vitro metabolic stabilities of the parent and retro-inverso compounds in rat plasma (Compounds 1-8, 13-14 and 19-20 having SEQ ID NOs: 1-8, 13-14 and 19-20) and in human plasma (Compounds 1-8 having SEQ ID NOs: 1-8) are shown.

[0057] Fig. 6A Half-life calculated based on the disappearance of the compound from rat plasma.

[0058] Figure 6B Calculated half-lives based on compound disappearance from human plasma. The labels and numbers on the bars reflect the change in half-life of the retro-inverse compounds compared to the corresponding parent compound. ND, not detected in the assay due to technical issues. The maximum calculated half-life of 789 minutes reflects experimental cut-off time limitations.

[0059] Figure 7A-7B The in vitro metabolic stabilities of the parent and retro-inverso compounds in rat hepatocytes (Compounds 1-8, 13-14, and 19-20 having SEQ ID NOs: 1-8, 13-14, and 19-20, respectively) and in human hepatocytes (Compounds 1-8 having SEQ ID NOs: 1-8) are shown.

[0060] Fig. 7A Half-lives were calculated based on the disappearance of compounds from rat liver hepatocytes.

[0061] Figure 7B Half-lives calculated based on compound disappearance in human liver hepatocytes. The labels and numbers on the bars reflect the change in half-life of the retro-inverse compounds as a percentage of the corresponding parent compound. The maximum calculated half-life at 395 minutes reflects the experimental cut-off time limit.

[0062] Figure 8 Permeation of parent and retro-inverse compounds (Compounds 1-14 and 17-20 having SEQ ID NOs: 1-14 and 17-20, respectively) through a 3D in vitro model of the blood-brain barrier is shown. The amount of compound that crosses the artificial blood-brain barrier is expressed as a percentage of the original applied concentration of the compound. Data are expressed as mean + / - SEM (n=3-4). ****p<0.0001, ns not significant, Brown-Forsythe and Welch ANOVA tests between parent and corresponding retro-inverse compounds, and post hoc unpaired t-tests and Welch correlated pairwise comparisons.

[0063] Fig. 9A Shown are plasma concentrations of retro-inverse compounds 6, 12, 14 and 20 (SEQ ID NOs: 6, 12, 14 and 20), native compound 13 (SEQ ID NO: 13) and a native control compound measured at 2 minutes, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours and 4 hours after intravenous administration to rats at a dose of 5 mg / kg. Data are expressed as mean + / - SEM (n=3).

[0064] Fig. 9BThe brain interstitial fluid (ISF; striatum) distribution kinetics of compound 20 (SEQ ID NO: 18) after 10 mg / kg intravenous bolus injection are shown. ISF concentrations have been normalized by microdialysis filter recovery -% (as determined by in vitro experiments). The compound was detected from ISF and plasma using LC-MS / MS.

[0065] Fig.10 A pairwise alignment of the C-terminal domains of CDNF and MANF is shown. The alignment was performed using the following Genbank retrieved sequences: Human CDNF Accession No. NP_001025125.2, and Human MANF Accession No. NP_006001.5. The CXXC motif is shown as a grey background, and the positions of the three alpha helices are shown.

[0066] Fig.11 The ClustalW multiple sequence alignment of the C-terminal domain of CDNF and MANF (61-63aa) from 10 different species (SEQ ID NO: 47-66, respectively) is shown. The Genbank accession number is shown in the sequence alignment. The CXXC motif is shown as a gray background, and the position of three alpha helices is shown. The conservative residues between these representative sequences (in both CDNF and MANF) are shown in bold. Below the sequence alignment, the natural variants found in the representative 10 species at each position are shown. The displayed sequence and species list can be used to identify conservative and variable positions, and show that for most non-essential amino acid residues, only limited variation is possible.

[0067] Fig.12 The structural formulas of compounds 17-20 (SEQ ID NOs: 17-20, respectively) are shown. The amino acid names as standard abbreviations are shown below the formulas along with the sequence order (NH2 to COOH in native peptides and COOH to NH2 in retro-inverse peptides). Gray arrows point to individual peptide bonds in each compound to illustrate the different order of amine and carbonyl groups in the amide peptide bonds of linear peptides composed of L-amino acids and retro-inverse peptides composed of D-amino acids.

[0068] Sequence Listing

[0069] SEQ ID NO:1MRVAELKQILHSWGEECRACAEK

[0070] SEQ ID NO:2KEACARCEEGWSHLIQKLEAVRM

[0071] SEQ ID NO:3LRVKELKKILDDWGETCKGCAEK

[0072] SEQ ID NO:4KEACGKCTEGWDDLICKLECVRL

[0073] SEQ ID NO:5GEECRACAEKT

[0074] SEQ ID NO:6TKEACARCEEG

[0075] SEQ ID NO:7GETCKGCAEKS

[0076] SEQ ID NO:8SKEACGKCTEG

[0077] SEQ ID NO:9GEECRGCAECT

[0078] SEQ ID NO:10TKEACAGRCEEG

[0079] SEQ ID NO:11GETCKGGCAEKS

[0080] SEQ ID NO:12SKEACGGKCTEG

[0081] SEQ ID NO:13EECRACAEK

[0082] SEQ ID NO:14KEACARCEE

[0083] SEQ ID NO:15ETCKGCAEK

[0084] SEQ ID NO:16KEACGKCTE

[0085] SEQ ID NO:17EECRACAE

[0086] SEQ ID NO:18EACARCEE

[0087] SEQ ID NO:19ETCKGCAE

[0088] SEQ ID NO:20EACGKCTE

[0089] SEQ ID NO:21C-X1-X2-X3-C

[0090] SEQ ID NO:22E-X4-C-X1-X2-X3-CAE

[0091] SEQ ID NO:23X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X11

[0092] SEQ ID NO:24

[0093] X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -V-X 24 -E-L-K-X 25 -I-L-X5X6-X7-X8-E-X4-C-X1-X2-X3-C-A-E-X9-X 10 -X 11

[0094] SEQ ID NO:25 RVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYA, natural human CDNF peptide

[0095] SEQ ID NO:26 RVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYA, natural human MANF peptide

[0096] SEQ ID NO:27

[0097] X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -V-X 24 -E-L-K-X 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-C-A-E-X9-X 10 -X 11

[0098] SEQ ID NO:28

[0099] 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

[0100] SEQ ID NO:29

[0101] 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

[0102] SEQ ID NO:30

[0103] 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

[0104] SEQ ID NO:31KDEL

[0105] SEQ ID NO:32CRAC

[0106] SEQ ID NO:33CKGC

[0107] SEQ ID NO:34CXXC

[0108] SEQ ID NO:35ETCKGCAE

[0109] SEQ ID NO:36 TCKGCA

[0110] SEQ ID NO:37 MWCASPVAVV AFCAGLLVSH PVLTQGQEAG GRPGADCEVC KEFLNRFYKSLIDRGVNFSL DTIEKELISF CLDTKGKENR LCYYLGATKD AATKILSEVT RPMSVHMPAM KICEKLKKLDSQICELKYEK TLDLASVDLR KMRVAELKQI LHSWGEECRA CAEKTDYVNL IQELAPKYAA THPKTEL Full-length CDNF (NCBI Reference Sequence: NP_001025125.2)

[0111] SEQ ID NO:38 MRRMWATQGL AVALALSVLP GSRALRPGDC EVCISYLGRF YQDLKDRDVTFSPATIENEL IKFCREARGK ENRLCYYIGA TDDAATKIIN EVSKPLAHHIPVEKICEKLK KKDSQICELKYDKQIDLSTV DLKKLRVKEL KKILDDWGET CKGCAEKSDY IRKINELMPK YAPKAASART DL Full-length MANF (NCBI Reference Sequence: NP_006001.5)

[0112] SEQ ID NO:39

[0113] TLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYA(49aa CDNF)

[0114] SEQ ID NO:40 RVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYA(37aa CDNF)

[0115] SEQ ID NO:41 TLDLASVDLRKMRVAELKQILHSWGEECRACAEKT(35

[0116] aa CDNF)

[0117] SEQ ID NO:42 LASVDLRKMRVAELKQILHSWGEECRACAEKT(32

[0118] CDNF)

[0119] SEQ ID NO:43

[0120] QIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYA(49aa MANF)

[0121] SEQ ID NO:44RVKELKKILDDWGETCKGCAEKSDYIRKINELMPKYA(37aa MANF)

[0122] SEQ ID NO:45QIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKS(35

[0123] aa MANF)

[0124] SEQ ID NO:46LSTVDLKKLRVKELKKILDDWGETCKGCAEKS(32aa MANF)

[0125] SEQ ID NO:47

[0126] KYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQELAPKY A ATHPKTEL, human CDNF(NP_001025125.2)

[0127] SEQ ID NO:48

[0128] KYEKKLDLASVDLLKMRVAELKQILNSWGEECRACAEKSDYVNLIKELAPKY A AMHPKTEL, equine CDNF(XP_001498617.2)

[0129] SEQ ID NO:49

[0130] KYEKKLDLASVDLSKMRVAELKQILHGWGEECRACAEKTDYVNLIKELAPK YA ATHPQTEL, bison CDNF(XP_010858254.1)

[0131] SEQ ID NO:50

[0132] KYEKKLDLASVDLSKMRVAELQILYSWGEECRACAEKTDYVNLIKELAPKYT E TPPQTEL, porcine CDNF(XP_003130787.1)

[0133] SEQ ID NO:51

[0134] KYEKKLDLASVDLSKMRVAELKQILHSWGEECIACAEKTDYVNLITELAPKY AA AHPKTEL, dog CDNF (XP_848954.2)

[0135] SEQ ID NO:52

[0136] KYGKKLDLASVDLWKMRVAELKQILQRWGEECRACAEKSDYVNLIRELAPK Y VEIYPQTEL, mouse CDNF (NP_808315.1)

[0137] SEQ ID NO:53

[0138] NYEKKLDLASVDLWKMRDAELKQILHSWGEECRACAEKNDYVNLIKELAP KY VEIHPQIEL, hamster CDNF (XP_027261009.1)

[0139] SEQ ID NO:54

[0140] KYERKLDLTSVDLSKMRVAELRKILDSWGEVCKACIEKTEFVNLIKELAPKYA PPNSRADL, alligator CDNF (XP_019343086.1)

[0141] SEQ ID NO:55

[0142] KYEKKLDLASVDLSKMRVAELKQILYSWGEECRACVEKTDYVNLIKELAPKY T ATYPKTEL, dolphin CDNF (XP_026977721.1)

[0143] SEQ ID NO:56

[0144] RYERLVLDWSTDALSKMRALELKRVLASWGEECRACLEKSEFIALIQEVAPK H SASEHRAHTEEF, zebrafish CDNF (NP_001116753.1)

[0145] SEQ ID NO:57

[0146] KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKY AP KAASARTDL, human MANF (NP_006001.5)

[0147] SEQ ID NO:58

[0148] KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKY AP KAASSRTDL, horse MANF (NP_001184244.1)

[0149] SEQ ID NO:59

[0150] KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKY AP KAASSRTDL, bison MANF (XP_010850093.1)

[0151] SEQ ID NO:60

[0152] KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKY AP KAASSRTDL, pig MANF (NP_001231584.1)

[0153] SEQ ID NO:61

[0154] KYDKQIDLRTVDLKKLRVRELKKILDDWGETCKGCAEKSDYIRKINELMPKY AP KAASSRTDL, dog MANF (XP_003639808.2)

[0155] SEQ ID NO:62

[0156] KYDKQIDLSTVDLKKLRVKELKKILDDWGEMCKGCAEKSDYIRKINELMPKY AP KAASARTDL, mouse MANF (NP_083379.2)

[0157] SEQ ID NO:63

[0158] KYDKQIDLSTVDLKKLRVKELKKILDDWGEMCKGCAEKSDYIRKINELMPKY AP KAASARTDL, hamster MANF (RLQ67668)

[0159] SEQ ID NO:64

[0160] KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKY AP KAASSRTDL, ALLIGATOR MANF(XP_014455597.1)

[0161] SEQ ID NO:65

[0162] KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSDYIRKINELMPKY AP KAASSRTDL,dolphin MANF(XP_026976745.1)

[0163] SEQ ID NO:66

[0164] KYDKQVDLSSVDLKKLKVKDLKKILEEWGESCKGCVEKSDFIRKINELMPKY A PSAAKARTDL, Zebrafish MANF(NP_001070097.1) DETAILED DESCRIPTION

[0165] The term "modified peptide" refers to a peptide or polypeptide that has been modified or synthesized. Peptide modification or synthesis options include, for example, reverse-inverse isomerization peptides, cyclic peptides, peptide mimetics, click chemistry, stapled peptides, N-terminal modifications, C-terminal modifications, isotope-labeled peptides, biotinylated and labeled peptides, fluorescent dye-labeled peptides, peptide dimers, post-translational modifications, internal quenching / FRET peptides, linkers / spacers / pegylation, peptide collection, protein conjugation, immunogenic peptides, and the incorporation of non-natural amino acids. "Non-naturally encoded amino acids" refers to amino acids that are 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 acids" are "non-natural amino acids (non-natural amino acid, unnatural amino acid)", "non-naturally occurring amino acids" and various hyphenated and non-hyphenated versions thereof. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that occur through modification (e.g., post-translational modification) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), but that themselves cannot be 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-acetylglucosyl-L-serine, N-acetylglucosyl-L-threonine, and O-phosphotyrosine.

[0166] The term "retro-inversion" refers to a peptide sequence in which one or more amino acids are D amino acids (inversion), and the peptide sequence is in reverse order (inversion). In some embodiments, there may be 1, 2, 3, 4, 5, 6, 7, 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 D amino acids. In some embodiments, the retro-inversion peptide includes amino acids with alternating chirality. In certain embodiments, the retro-inversion peptide includes all D amino acids. As used herein, chirality refers to the D and L isomers of amino acids. For illustration, a retro-inversion peptide with the sequence CX1XXXC will be reversed to have the sequence CX3XXXC, wherein at least one or more amino acids are D amino acids. Peptides made of D-amino acids are metabolically stable because they are poor substrates for proteases that have evolved to degrade proteins and peptides made of L-amino acids. However, D-peptides have reverse handedness in, for example, a helical structure, i.e., the amino acid side chains are positioned as mirror images. Reversing the sequence order in a D-peptide provides a structure that simulates an L-peptide analog in the direction of the side chain, in other words, a reverse-inverse isomerization. Short non-helical reverse-inverse peptides can functionally simulate their natural L-protein counterparts in target binding.

[0167] The principle of retro-inverse isomerization is that, for example, when a linear peptide is

[0168] NH2-Gly-L-Glu-L-Glu-L-Cys-L-Arg-L-Ala-L-Cys-L-Ala-L-Glu-L-Lys-L-Thr-COOH (SEQ ID NO: 5)

[0169] The retro-inverse isomerization form is

[0170] NH2-D-Thr-D-Lys-D-Glu-D-Ala-D-Cys-D-Ala-D-Arg-D-Cys-D-Glu-D-Glu-Gly-COOH (SEQ ID NO: 6)

[0171] Examples of the principle of reverse-inverse isomerization of compounds 17-20 (SEQ ID NOs: 17-20, respectively) are also given in Fig.12 Displayed in.

[0172] The term "pseudo-peptide" refers to the amide of an amino acid not present in a natural peptide or protein, especially the amide of an amino acid introduced into a polypeptide chain. Pseudo-peptides or amino bond substitutes are one of the various terms that can be used to describe backbone modified peptides. These synthetic analogs of peptides have a variety of potential uses, but the most expanded interest in these fields focuses on their potential to develop metabolically stable and possibly orally active peptide hormone analogs or enzyme inhibitors with enhanced bioefficacy. The term specifically includes peptide backbone modifications known to those skilled in the art (i.e., amide bond mimetics). Such modifications include modifications of amide nitrogen, α-carbon, amide carbonyl, complete replacement, extension, deletion or backbone cross-linking of amide bonds. Several peptide backbone modifications are known, including ψ[CH2S], ψ[CH2NH], ψ[CSNH2], ψ[NHCO], ψ[COCH2] and ψ[(E) or (Z)CH═CH]. In the terms used above, ψ indicates the absence of an amide bond. The structure of the substituted amide group is described in brackets.

[0173] As used herein, when two entities are "conjugated" to each other, they are connected by direct or indirect covalent or non-covalent interactions. In some aspects, the association is covalent. In other aspects, the association is non-covalent. Non-covalent interactions include hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. Indirect covalent interactions are when two entities are covalently linked, optionally through a linker group. "Conjugated" herein means a peptide conjugated or coupled to a detectable chemical or biochemical part or PEG or other part for extending plasma half-life. In some cases, one or more peptides disclosed herein can be conjugated to a carrier protein, for example. Such conjugated compositions can be monovalent or multivalent. For example, a conjugated composition can include a peptide disclosed herein conjugated to a carrier protein. Alternatively, a conjugated composition can include two or more peptides disclosed herein conjugated to a carrier.

[0174] "Blood-brain barrier" or "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 endothelial cells of the capillary wall, astrocyte end feet that wrap around capillaries, and pericytes embedded in the capillary basement membrane. The system allows some molecules to pass through passive diffusion, as well as the selective transport of molecules (such as glucose, water and amino acids) that are essential for neurological function. Macromolecules (such as proteins) typically cannot pass through the BBB. However, some peptides can pass through the BBB by various mechanisms, and some proteins containing the transporter specific recognition motifs located at the surface of cerebrovascular endothelial cells can also be transported through the BBB.

[0175] As used herein, "pharmaceutically acceptable carrier" may include one or more solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are acceptable for use in formulating a drug, such as a drug suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition.

[0176] CDNF and MANF share approximately 60% amino acid sequence homology ( Fig.10 and 11 ), but they have highly similar three-dimensional structures. Both CDNF and MANF are composed of two independently folded domains connected by a flexible loop region. The secondary structure is mainly α helices, with five α helices in the N-terminal domain and three α helices in the C-terminal domain. Three disulfide bridges stabilize the N-terminal domain, while the C-terminal CRAC (SEQ ID NO: 32) sequence in CDNF and the CKGC (SEQ ID NO: 33) in MANF form internal disulfide bridges. This CXXC (SEQ ID NO: 34) disulfide bridge is found in both CDNF and MANF. The CXXC motif is beneficial to the neuroprotective activity of MANF and CDNF. However, the data presented here show that the CXXC (SEQ ID NO: 34) motif can adapt to some modifications, such as adding small amino acids (e.g., glycine and serine), that is, a specific type of CXXXC motif can also be used. In some aspects, CDNF has a sequence derived from NP_001025125.2 (SEQ ID NO: 37). In some aspects, the MANF has a sequence derived from NP_006001.5 (SEQ ID NO: 38).

[0177] In addition to naturally occurring allelic variants derived from MANF and CDNF peptides, changes can be introduced into the MANF / CDNF sequence by mutation, which results in changes in the amino acid sequence of the encoded MANF / CDNF peptide. Nucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of the MANF / CDNF peptide. A MANF / CDNF peptide or a functional fragment thereof containing one or more "non-essential" substitutions can be considered an equivalent of the wild-type MANF / CDNF peptide disclosed herein.

[0178] Each amino acid can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a congener of a natural amino acid because it has a structure similar to a natural amino acid, so that it mimics the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and / or amino acid analogs that are not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine ​​or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids.

[0179] 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, hexahydropicolinic acid, proline, pyroglutamic acid, sarcosine, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, derivatives or combinations thereof.

[0180] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0181] "Pharmaceutically acceptable salts" are intended to mean salts of the free acids or bases of the compounds represented herein that are nontoxic, 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 may have sufficiently acidic groups, sufficiently basic groups, both types of functional groups, or more than one of each type, and therefore react with many inorganic or organic bases, and inorganic and organic acids, to form pharmaceutically acceptable salts.

[0182] 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, with an inorganic acid (such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid, etc.) or with an organic acid (such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, oxaloacetic 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, pyranosidic acid (such as glucosidic acid) The free base may be treated with any compatible mixture of acids such as those given as examples herein, and any other acids and mixtures thereof deemed to be equivalent or acceptable substitutes according to the level of ordinary skill in the art.

[0183] For compounds described herein containing acidic groups (such as carboxylic acid groups), base addition salts can be prepared by any suitable method available in the art, for example, by treating such compounds 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 tetraalkyl ammonium salts.

[0184] Other examples of pharmaceutically acceptable salts include, but are not limited to, camphorsulfonate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, The pharmaceutically acceptable salts of the present invention are exemplified in the following examples: 1,4-dioic acid salt, 1,6-dioic acid salt, 1,2-dioic acid salt, 1,3-dioic acid salt, 2,4-dioic acid salt, 2,6 ...

[0185] The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties (such as solubility in polar solvents), but in other respects, the salt is equivalent to the parent form of the compound for the purposes of this application.

[0186] In embodiments of the present disclosure, the length of the peptide or fragment is in the range of 8-32 amino acids, wherein the peptide or fragment thereof comprises CX1X2X3C (SEQ ID NO: 21) as described herein. In certain embodiments, preferred peptides or fragments may be composed 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 certain embodiments, the length of the peptide or fragment is in the range of 8-23, 11-23, 12-23, 8-11 or 8-12 amino acids. In certain embodiments, the peptide or fragment is 8-31, 8-29, 8-27, 8-25, 8-23, 8-21, 8-19, 8-17, 8-15, 11-27, 11-25, 11-23, 11-21, 11-19, 11-17, 11-15, 12-25, 12-23, 12-21, 12-19, 12-17, 12-15, 23-27, 24-27, or 25-27 amino acids in length. The fragments may comprise any naturally occurring amino acid, such as alanine [Ala (A)], arginine [Arg (R)], asparagine [Asn (N)], aspartic acid [Asp (D)], cysteine ​​[Cys (C)], glutamine [Gln (Q)], glutamic acid [Glu (E)], glycine [Gly (G)], histidine [His (H)], isoleucine [Ile (I)], leucine [Leu (L)], lysine [Lys (K)], methionine [Met (M)], phenylalanine [Phe (F)], proline [Pro (P)], serine [Ser (S)], threonine [Thr (T)], tryptophan [Trp (W)], tyrosine [Tyr (Y)] and valine [Val (V)], as well as non-natural or modified amino acids.

[0187] Cyclic peptides are small disulfide-rich peptides isolated from plants. Cyclic peptides typically contain 28-37 amino acids, and they have a head-to-tail cyclized peptide backbone and an interlocking arrangement of three disulfide bonds. Although the family of plant cyclic peptides may contain cyclic peptides with potential CXXC and CXXXC motifs, they are not known to have similar cytoprotective properties as CDNF and MANF in mammalian cells, i.e., protecting against ER stress-induced cell dysfunction or cell death, such as apoptosis.

[0188] In one embodiment, the peptides claimed in the present disclosure are not related to plant cyclic peptides or the family of plant cyclic peptides.

[0189] Preferably, the peptides disclosed in the present disclosure are not from the thioredoxin and / or protein disulfide isomerase family of proteins.

[0190] The present disclosure provides a peptide consisting of 8-32 amino acids in length or a pharmaceutically acceptable salt thereof, which comprises a retro-inverse form of an amino acid sequence of C-X1-X2-X3-C (SEQ ID NO: 21),

[0191] in

[0192] X1 is selected from the group consisting of: R, K, I, G, A and S;

[0193] X2 is absent or selected from the group consisting of: G, A, R, K, I, and S; and

[0194] X3 is selected from the group consisting of: A, G and S.

[0195] In a preferred embodiment, the peptide comprises a retro-inverted form of the amino acid sequence of E-X4-C-X1-X2-X3-CAE (SEQ ID NO: 22),

[0196] in

[0197] X1 is selected from the group consisting of: R, K, I, G, A and S;

[0198] X2 is absent or selected from the group consisting of: G, A, R, K, I and S;

[0199] X3 is selected from the group consisting of: A, G and S; and

[0200] X4 is selected from the group consisting of: E, T, V, D, M and G

[0201] Based on different species (human, horse, bison, pig, dog, mouse, hamster, alligator, dolphin and zebrafish), CDNF and MANF were used as Fig.11 Based on natural variations in the CDNF and MANF sequences in (exemplary sequences in ), limited changes in the peptide sequence with respect to the X-group can be accommodated without loss of biological activity compared to the human sequence.

[0202] In another preferred embodiment, the peptide comprises X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO: 23),

[0203] in

[0204] X1 is selected from the group consisting of: R, K, I, G, A and S;

[0205] X2 is absent or selected from the group consisting of: G, A, R, K, I and S;

[0206] X3 is selected from the group consisting of: A, G and S;

[0207] X4 is selected from the group consisting of: E, T, V, D, M and G;

[0208] X5 is absent or selected from the group consisting of: H, D, Q, R, Y, N and S;

[0209] X6 is absent or selected from the group consisting of: S, D, G, N and R;

[0210] X7 does not exist or is W;

[0211] X8 does not exist or is G;

[0212] X9 does not exist or is K;

[0213] X 10 is absent or is selected from the group consisting of: T, S, A, I, and N; and

[0214] X 11 Not present or selected from D and E.

[0215] In another preferred embodiment, the peptide comprises a retro-inverse form of the amino acid sequence

[0216] 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: 24).

[0217] in

[0218] X1 is selected from the group consisting of: R, K, I, G, A and S;

[0219] X2 is absent or selected from the group consisting of: G, A, R, K, I and S;

[0220] X3 is selected from the group consisting of: A, G and S;

[0221] X2 is absent or selected from the group consisting of: G, A, R, K, I and S;

[0222] X3 is selected from the group consisting of: A, G and S;

[0223] X4 is selected from the group consisting of: E, T, V, D, M and G;

[0224] X5 is absent or selected from the group consisting of: H, D, Q, R, Y, N and S;

[0225] X6 is absent or selected from the group consisting of: S, D, G, N and R;

[0226] X7 does not exist or is W;

[0227] X8 does not exist or is G;

[0228] X9 does not exist or is K;

[0229] X 10 is absent or is selected from the group consisting of: T, S, A, I, and N; and

[0230] X 11 Not present or selected from D and E,

[0231] X 12 Not present or selected from the group consisting of: L, I and V;

[0232] X 13 does not exist or is D;

[0233] X 14 Not present or selected from L and W;

[0234] X 15 Not present or selected from the group consisting of: A, S, T, E and N;

[0235] X 16 Not present or selected from S and T;

[0236] X 17 Not present or selected from V and D;

[0237] X 18 Not present or selected from D and A;

[0238] X 19 does not exist or is L;

[0239] X 20Not present or selected from the group consisting of: R, K, S and W;

[0240] X 21 does not exist or is K;

[0241] X 22 Not present or selected from the group consisting of: M, L, I and V;

[0242] X 23 does not exist or is R;

[0243] X 24 selected from the group consisting of: A, K, T, L and V;

[0244] X 25 is selected from the group consisting of: Q, K, and R; and

[0245] X 26 Selected from I and V.

[0246] In some embodiments, the peptide comprises a peptide of 8-23 amino acids in length within SEQ ID NO: 24, wherein the peptide comprises a CX1X2X3C (SEQ ID NO: 21) motif.

[0247] In another preferred embodiment, the peptide comprises a retro-inverse form of an amino acid sequence within an amino acid sequence selected from the group consisting of:

[0248] 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:35),

[0249] 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:36),

[0250] 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: 37) and

[0251] X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X 22 -X 23 -VX 24 -ELKX 25 -X 26 -L-X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE (SEQ ID NO: 38). In some embodiments, the peptide comprises a peptide of 8-23 amino acids in length within one of SEQ ID NOs: 35-38, wherein the peptide comprises a CX1X2X3C (SEQ ID NO: 21) motif.

[0252] In some cases, the peptide comprises an amino acid sequence that may be within the amino acid sequence of SEQ ID NO:39 or the amino acid sequence of SEQ ID NO:43.

[0253] Preferably, the peptide, in particular the D-peptide, consists of a sequence selected from the group consisting of KEACARCEEGWSHLIQKLEAVRM (SEQ ID NO:2), KEACGKCTEGWDDLIKKLEKVRL (SEQ ID NO:4), TKEACARCEEG (SEQ ID NO:6), SKEACGKCTEG (SEQ ID NO:8), TKEACAGRCEEG (SEQ ID NO:10), SKEACGGKCTEG (SEQ ID NO:12), KEACARCEE (SEQ ID NO:14), KEACGKCTE (SEQ ID NO:16), EACARCEE (SEQ ID NO:18) and EACGKCTE (SEQ ID NO:20), wherein all amino acids of the peptide are D-amino acids.

[0254] In one embodiment, the peptide protects against endoplasmic reticulum (ER) stress-induced cellular dysfunction or cell death, such as apoptosis.

[0255] In some embodiments, the N-terminus of the peptide is acetylated. In some embodiments, the C-terminus of the peptide is amidated. In some embodiments, the N-terminus of the peptide is acetylated and the C-terminus of the peptide is amidated.

[0256] In some embodiments, the peptide is a pseudopeptide.

[0257] In one embodiment, the peptide is cyclic.

[0258] In some cases, the length of the peptide is 11-32 amino acids. In some cases, the length of the peptide is 12-32 amino acids. In some cases, the length of the peptide is 12-23 amino acids. In some cases, the length of the peptide is 8-23 amino acids. In some cases, the length of the peptide is 8-13 amino acids. In some cases, the length of the peptide is 8-12 amino acids.

[0259] In a preferred embodiment, in the modified peptide, cysteine ​​(C) is in reduced form or in disulfide bridged form.

[0260] In certain embodiments, the peptides described herein bind to GRP78.

[0261] In some aspects, the peptides described herein are at least 1.5 times more stable than their parent counterparts. In one embodiment, the peptides described herein are at least 2, 3, or 4 times more stable than their parent counterparts. In some aspects, the half-life of the peptides described herein is at least 1.5 times higher than their parent counterparts. In one embodiment, the half-life of the peptides described herein is at least 2, 3, or 4 times higher than their parent counterparts.

[0262] In some aspects, the peptide can comprise a bond connecting the N-terminus of the peptide to the C-terminus.

[0263] In some aspects, the N-terminus of the peptide can be acetylated. In some aspects, the C-terminus of the peptide is amidated. In some aspects, the N-terminus of the peptide can be acetylated, and the C-terminus of the peptide can be amidated.

[0264] In another preferred embodiment, the peptide is conjugated to a detectable moiety, a chemical moiety or a biochemical moiety or polyethylene glycol (PEG).

[0265] The peptide may be conjugated to a detectable chemical or biochemical moiety such as a fluorophore (e.g., fluorescein or rhodamine). Radioactive labeling of the peptide may be used, for example, for SPECT or PET imaging. As used herein, "detectable chemical or biochemical moiety" means a chemical tag that exhibits an amino acid sequence or a detectable chemical or biochemical moiety for the purpose of facilitating detection of the peptide; such as a detectable molecule selected from the following: a visible, fluorescent, chemiluminescent or other detectable chemical tag; an enzyme detectable in the presence of a substrate, such as alkaline phosphatase with NBT plus BCIP or a peroxidase with a suitable substrate; a detectable protein, such as green fluorescent protein. Preferably, the tag does not prevent or hinder the penetration of the fragment into the target cell, or otherwise alter the biological activity of the compound.

[0266] It is also preferred to modify the C-terminal CDNF fragment or C-terminal MANF fragment by N and / or C-terminal modification to further increase the stability and / or cell permeability of the peptide or fragment. Acetylation-amidation (i.e., N-terminal acetylation and C-terminal amidation) of the CDNF fragment or MANF fragment terminal is one of the options known in the art (see, e.g., Marino et al. 2015, ACS Chem. Biol. [ACS Chemical Biology] 10: 1754-1764).

[0267] In some cases, a peptide as described herein has at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following properties: (i) the peptide can dose-dependently protect TH-positive neurons from MPP+ toxicity; (ii) the peptide reduces the number of α-synuclein inclusions in TH-positive neurons; (iii) the peptide has improved stability in plasma compared to its parent counterpart; (iv) the peptide has improved stability in hepatocytes compared to its parent counterpart; or (v) the peptide has improved ability to cross the blood-brain barrier compared to its parent counterpart.

[0268] One embodiment provides a peptide as described herein for use as a medicament.

[0269] Since CDNF / MANF peptides effectively protect dopamine neurons from death, prior art such as WO2009133247 and EP 1969003 show that the peptides can be used to treat central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease (PD), multiple system atrophy, amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease (HD), traumatic brain injury, drug addiction and stroke.

[0270] CDNF and MANF regulate the signaling of the unfolded protein response (UPR) pathway and protect cells from ER stress-related cell death. ER stress is known to play an important pathophysiological role in a variety of chronic diseases (such as neurodegenerative and metabolic diseases and acute injuries) (Wang and Kaufman, 2016). GRP78 (also known as BiP and HSPA5) is the main ER luminal chaperone and the main regulator of UPR (Bertolotti et al., 2000; Wang and Kaufman, 2016). The dynamic association and dissociation of GRP78 with UPR receptors IRE1α, PERK and ATF6 is a key step in regulating the signaling activity of UPR receptors under ER stress. The interaction of MANF with GRP78 regulates its cellular activity (Yan et al., 2019).

[0271] Thus, the present disclosure relates to a method for treating a degenerative, chronic or progressive disease or disorder, such as a CNS disease or disorder, or a monogenic inherited disease with ER stress as a pathogenic component, wherein a pharmaceutically effective amount of a peptide having a length of 8-32 amino acids comprising the sequence C-X1-X2-X3-C (SEQ ID NO: 21), E-X4-C-X1-X2-X3-CAE (SEQ ID NO: 22), X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X1-X2-X3-CAE ... 10 -X11 (SEQ ID NO: 23) 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 -LX 5- X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO:24), wherein the peptide is a retro-inverted form of the amino acid sequence.

[0272] Another embodiment provides the peptide for use in treating a degenerative, chronic or progressive disease or disorder, such as a neurodegenerative disease or disorder.

[0273] 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 neuropathy, and a range of diseases and disorders thereof.

[0274] Neurodegenerative diseases can be partially overlapping, dynamic, nonlinear progressive "dimensions" present in a broad spectrum of brain proteinopathies. Variability in the expression of several combinations of multiple proteinopathies within the central nervous system may occur. Therefore, the coexistence of mixed neuropathologies may be observed in patients. The genetic spectrum of neurodegenerative diseases may differ, for example different diseases may be manifested in identical twins with the same genotype.

[0275] Another embodiment provides the peptide for use in treating a monogenic genetic disease selected from the group consisting of: Wolcott-Rallison syndrome, Wolfram syndrome, Marinesco- Syndrome, Machado-Joseph disease and degenerative retinal diseases such as retinitis pigmentosa, and hereditary nephrotic syndromes such as primary nephrotic syndrome and autosomal dominant polycystic kidney disease. The monogenic hereditary diseases are diseases with ER stress as a pathogenic component.

[0276] One embodiment provides a peptide for use according to the present disclosure, wherein the peptide is administered by peripheral administration such as intravenous, intraarterial, subcutaneous, intranasal, intraocular, intratympanic or topical administration, enteral, parenteral or topical routes, including oral, rectal, sublingual or buccal administration, intraperitoneal, intramuscular, intraarticular, transdermal, intracochlear, topical intraocular or inhalation administration, or intracranial, intrathecal, epidural or intralesional administration.

[0277] In one embodiment, the peptide is administered by subcutaneous administration.

[0278] Pharmaceutical composition

[0279] One or more peptides disclosed herein can be formulated for use as or in a pharmaceutical composition.Such compositions can be formulated or adapted for administration to a subject via any route, such as any route approved by the appropriate authorities.

[0280] One embodiment provides a pharmaceutical composition comprising a peptide as described herein and at least one of: a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a preservative, a stabilizer and / or a diluent.

[0281] In one embodiment, the present disclosure further relates to a pharmaceutical composition comprising a peptide having a length of 8-32 amino acids, wherein the peptide comprises the sequence C-X1-X2-X3-C (SEQ ID NO: 21), E-X4-C-X1-X2-X3-CAE (SEQ ID NO: 22), X5-X6-X7-X8-E-X4-C-X1-X2-X3-CAE-X9-X 10 -X 11 (SEQ ID NO: 23) 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:24), wherein the peptide is a retro-inverse form of the amino acid sequence of any of the foregoing sequences.

[0282] In some cases, the pharmaceutical composition may include an effective amount of one or more peptides. As used herein, the terms "effective amount" and "effective treatment" refer to the amount or concentration of one or more compounds or pharmaceutical compositions described herein used over a period of time (including acute or chronic administration and periodic or continuous administration) that is effective for producing a desired effect or physiological result in the case of administration.

[0283] In one embodiment of the invention, the peptides may be incorporated into pharmaceutical compositions. Such compositions of the present disclosure are prepared for storage (Remington's Pharmaceutical Sciences, 22nd ed., Allen, Loyd V., Jr., (2012)) by mixing the peptides of the desired purity with optional physiologically acceptable carriers (such as nanocarriers), excipients, buffers, or stabilizers in the form of lyophilized cakes or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; 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, Pluronic, polyethylene glycol (PEG), or excipients for enhancing nose-to-brain delivery such as chitosan, methylated pectin, alkyl sugar-based mucosal absorption enhancers, and hydroxy fatty acyl esters of PEG.

[0284] The actual dosage (e.g., effective amount) of the peptide administered to the patient can be determined by physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, the patient's own disease and route of administration. The physician responsible for administration can determine the concentration of one or more active ingredients in the composition and one or more appropriate dosages for individual subjects.

[0285] The peptides may also be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 22nd edition, Allen, Loyd V., Jr., ed. (2012). Controlled release gel formulations may also be used.

[0286] In one embodiment, the pharmaceutical composition may include, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may, for example, account for about 2% to about 75% of the unit weight, or about 25% to about 60%, and any range derivable therein.

[0287] In other non-limiting examples, the dosage of the pharmaceutical composition or formulation can include about 1 ng / kg / body weight of the peptide, about 5 ng / kg / body weight, about 10 ng / kg / body weight, about 50 ng / kg / body weight, about 100 ng / kg / body weight, about 200 ng / kg / body weight, about 350 ng / kg / body weight, about 500 ng / kg / body weight, 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 10 0 μ g / kg / body weight, about 200 μ g / kg / body weight, about 350 μ g / kg / body weight, about 500 μ g / 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 of peptide or more, and any range derivable therein. In a non-limiting example of a derivable range of the numerical values ​​listed herein, a series of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μ g / kg / body weight to about 500 mg / kg / body weight of peptide, etc. can be administered based on the above numerical values.

[0288] The method herein contemplates the administration of an effective amount of a compound or compound composition to achieve the desired or described effect. Typically, the pharmaceutical composition of the present disclosure will be administered about 1 to about 6 times a day, such as 1-2, 1-3, 1-4, 1-5, 2-3, 2-4 or 2-5 times a day, or alternatively, as a continuous infusion. The pharmaceutical composition can be administered, for example, 1, 2, 3, 4, 5 or 6 times a day. This administration can be used as a chronic or acute therapy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the treated host and a specific mode of administration. A typical preparation will contain about 5% to about 95% of the active compound (w / w). Alternatively, such preparations contain about 20% to about 80% of the active compound.

[0289] Various techniques can be used to determine the dosage. The selected dosage level can depend on a variety of factors, including, for example, the activity of the specific compound used, the route of administration, the time of administration, the excretion or metabolic rate of the specific compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific compound used, the age, sex, weight, illness, general health and / or previous medical history of the treated patient, and similar factors well known in the medical field. The dosage value can also change with the severity of the condition to be alleviated. For any specific subject, the specific dosage regimen can be adjusted over time according to individual needs and the professional judgment of the person who administers or supervises the administration of the composition.

[0290] In some aspects, the suitable daily dose of the compounds of the present disclosure can be the amount of the lowest dose at which the compound is effective to produce a therapeutic effect. Such an effective dose will generally depend on the above factors. The precise time and amount of administration of any particular compound that will produce the most effective treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of the particular compound, the patient's physiological condition (including age, sex, disease type and stage, general physical condition, response to a given dose and drug type), route of administration, etc.

[0291] A physician or veterinarian may prescribe an effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian may start the dosage of the disclosed compound used in the pharmaceutical composition at a lower level than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0292] The pharmaceutical compositions described herein can be in unit dosage form suitable for single administration of precise doses. In unit dosage form, the formulation is divided into unit doses containing an appropriate amount of one or more compounds. The unit dose can be in the form of a package containing discrete amounts of the formulation. Non-limiting examples are liquids in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose, non-reclosable containers. For example, multi-dose reclosable containers can be used in combination with preservatives. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers containing preservatives.

[0293] The term "pharmaceutically acceptable carrier or adjuvant" refers to a non-toxic carrier or adjuvant that can be administered to a patient together with the compound of the present invention and does not destroy the pharmacological activity of the compound when administered in a dose sufficient to deliver a therapeutic amount of the compound.

[0294] Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of the present disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as D-α-tocopheryl polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tweens or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphates, 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 silicon, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

[0295] Pharmaceutical compositions of the present disclosure may contain any conventional nontoxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acid, alkali or buffer to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes parenteral, epidural, subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.

[0296] The effective amount of the disclosed compound can be administered in a single dose or multiple doses (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any suitable amount) by any acceptable mode 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 disclosed compound and / or the disclosed pharmaceutical composition are formulated into a pharmaceutically acceptable dosage form. According to the disclosed compound, other drugs can be formulated for administration in any convenient manner for use in humans or veterinary medicine by analogy.

[0297] In one aspect, the present disclosure provides a pharmaceutical formulation comprising one or more of the above compounds of a therapeutically effective dose prepared with one or more pharmaceutically acceptable carriers (additives) and / or diluents. In one aspect, one or more compounds described herein are formulated for parenteral administration. For parenteral administration, one or more compounds disclosed herein can be formulated into aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders, which can be re-formulated into sterile injectable solutions or dispersions before use. Such formulations can contain sugar, alcohol, antioxidants, buffers, antibacterial agents, solutes or suspensions or thickeners that make the formulation isotonic with the blood of the intended recipient. These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. It can be ensured that the effect of microorganisms on the subject compound is prevented by including various antibacterial agents and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sugar, sodium chloride, etc. in the composition. In addition, extended absorption of injectable drug forms can be achieved by comprising agents that delay absorption, such as aluminum monostearate and gelatin. If desired, the formulation can be diluted with, for example, isotonic saline or dextrose solution before use. In certain embodiments, the compound is formulated into an aqueous solution and administered intravenously.

[0298] The pharmaceutical composition can be in the form of a solution or powder for injection. Such compositions can be formulated using suitable dispersants or wetting agents (e.g., such as Tween 80) and suspending agents according to techniques known in the art. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils are conventionally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives can be used to prepare injectables as natural pharmaceutically acceptable oils such as olive oil or castor oil (especially their polyoxyethylated forms). These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, or carboxymethyl cellulose or similar dispersants commonly used to prepare pharmaceutically acceptable dosage forms (such as emulsions and / or suspensions). Other commonly used surfactants such as Tweens or Spans and / or other similar emulsifying agents or bioavailability enhancers (which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms) may also be used for the purposes of formulation.

[0299] The pharmaceutical composition can be orally administered in any oral acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are typically also added. For oral administration in capsule form, useful diluents include lactose and dry corn starch. When oral administration of aqueous suspensions and / or emulsions, the active ingredient can be suspended or dissolved in an oil phase combined with an emulsifier and / or suspending agent. If desired, some sweeteners and / or flavorings and / or coloring agents can be added.

[0300] Pharmaceutical compositions of the present disclosure can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compositions of the present disclosure with suitable non-irritating excipients, which are solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0301] Alternatively or additionally, the pharmaceutical composition may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared as saline solutions, employing benzyl alcohol or other suitable preservatives, absorption enhancers (to enhance bioavailability), fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0302] In some cases, one or more of the peptides disclosed herein can be conjugated, for example, to a carrier protein. Such conjugated compositions can be monovalent or multivalent. For example, a conjugated composition can include one peptide disclosed herein conjugated to a carrier protein. Alternatively, a conjugated composition can include two or more peptides disclosed herein conjugated to a carrier.

[0303] Methods of using the peptides described herein are provided. For example, the methods provided herein can include administering a peptide as described herein to a patient. Patients can include both mammals and non-mammals.

[0304] A pharmaceutically acceptable carrier can be selected based on the chosen route of administration and standard pharmaceutical practice. For example, the composition can be formulated into a suitable pharmaceutical preparation, such as a solution, suspension, tablet, dispersible tablet, pill, capsule, powder, sustained release formulation or elixir for oral administration, or a sterile solution or suspension for parenteral administration and intraperitoneal injection, as well as transdermal patch formulations, dry powder inhalants and ointments (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, 4th Edition 1985, 126). The peptides and / or immunoglobulins can be formulated into dosage forms according to standard practice in the pharmaceutical formulation art. See Alphonso Gennaro, Remington's Pharmaceutical Sciences, 18th Edition (1990), Mack Publishing Co., Easton, Pa.

[0305] For parenteral administration, the pharmaceutical composition can contain a suitable carrier or diluent, such as water, oil (especially vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol or diols, such as propylene glycol or polyethylene glycol. Solutions for parenteral administration preferably contain water-soluble salts of the peptide and / or active agent. Stabilizers, antioxidants and preservatives can also be added. Suitable antioxidants include sulfites, ascorbic acid, citric acid and their salts and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl or propyl paraben and chlorobutanol. Compositions for parenteral administration can take the form of aqueous or non-aqueous solutions, dispersions, suspensions or emulsions.

[0306] For oral administration, the pharmaceutical composition can contain one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms. For example, the pharmaceutical composition can contain at least one excipient, such as a filler, binder, wetting agent, disintegrant, solution retarder, absorption promoter, wetting agent, absorbent or lubricant.

[0307] The present disclosure also features a pharmaceutical composition that can further include a neural cell. The neural cell can be, for example, a neuron, a neural stem cell, or a neuronal precursor cell.

[0308] The present disclosure relates to a pharmaceutical composition for use as a medicament, comprising a peptide as described herein and at least one of: a pharmaceutically acceptable carrier, excipient, preservative, stabilizer and / or diluent.

[0309] In the method of treatment, a pharmaceutically effective amount of a peptide as defined herein is administered to a patient. In other words, the peptides according to the present disclosure are used to treat degenerative, chronic or progressive diseases or disorders, such as CNS diseases or disorders, monogenic genetic diseases (with ER stress as a pathogenic component).

[0310] The pharmaceutical composition is used to treat a degenerative, chronic or progressive disease or disorder, such as a neurodegenerative disease or disorder.

[0311] 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 neuropathy.

[0312] In one embodiment, the pharmaceutical composition is administered by subcutaneous administration.

[0313] The route of administration of the peptides is in accordance with known methods and general routes of injection or infusion by intravenous, intraarterial, subcutaneous, intranasal, intraocular, intratympanic or topical administration, enteral, parenteral or topical routes, including oral, rectal, sublingual or buccal administration, intracranial, intrathecal or epidural, intraperitoneal, intramuscular, intraarticular, transdermal, intracochlear, topical intraocular, intralesional or inhalation administration, or by sustained release systems as described below.

[0314] Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the peptide, in the form of molded articles (e.g., films) or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels as described by Langer et al., J. Biomed. Mater. Res. [Biomedical Materials Research], 15: 167-277 (1981) and Langer, Chem. Tech. [Chemical Technology], 12: 98-105 (1982), or polyvinyl alcohol, polylactic acid (U.S. Patent No. 3,773,919, EP 58,481) or nondegradable ethylene-vinyl acetate (Langer et al., supra).

[0315] The present disclosure also relates to a method for treating a degenerative, chronic or progressive disease or disorder (such as a CNS disease or disorder), a monogenic inherited disease (having ER stress as a pathogenic component), wherein a pharmaceutically effective amount of a peptide as defined herein is administered to a patient. Preferably, the fragment is administered peripherally. Oral administration is also a preferred form of administration.

[0316] The present disclosure also relates to the use of a 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 with ER stress as a causative component.

[0317] The present disclosure relates to a method for treating a degenerative, chronic, or progressive disease or disorder, such as a neurodegenerative disease or disorder, in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a peptide as described herein.

[0318] In one embodiment, 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, and peripheral neuropathy comprises administering to a subject a pharmaceutical composition comprising a peptide as described herein.

[0319] The present disclosure relates to a method for treating a monogenic genetic disease selected from the group consisting of: Wolcott-Rallison syndrome, Wolfram syndrome, Marinesco- Syndrome, Machado-Joseph disease and degenerative retinal diseases such as pigmentary retinitis), and hereditary nephrotic syndromes such as primary nephrotic syndrome and autosomal dominant polycystic kidney disease), the method comprising administering to the subject a pharmaceutical composition comprising a peptide as described herein. The monogenic hereditary disease has ER stress as a pathogenic component.

[0320] The subject in need may be a human.

[0321] The peptides of the present disclosure or pharmaceutical compositions comprising the peptides can be administered continuously by infusion or bolus. In general, the fragments should be formulated and administered for site-specific delivery, where the barrier permits. Administration can be continuous or periodic. Administration can be accomplished by implantable pumps with constant or programmable flow or by periodic injections. Peripheral or systemic administration is preferred because the present disclosure shows that retro-inverse peptides can effectively penetrate neuronal cell membranes and cross the blood-brain barrier (BBB) ​​in vitro and in vivo (respectively Figure 8 and 9B Other preferred routes of administration are subcutaneous, intrathecal, intracerebroventricular, intranasal or transdermal administration.

[0322] In another embodiment, the present disclosure provides a method for promoting the survival of dopaminergic neurons, the method comprising contacting dopaminergic neurons with a peptide of 8-32 amino acids comprising the sequence SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, wherein the peptide comprises a retro-inverted form of the amino acid sequence. Preferably, the method is performed in vitro, as shown in the experimental section below. The dopaminergic neurons are preferably cultured non-human neurons, such as mouse or rat sympathetic neurons, or human neurons derived from induced pluripotent cells (iPSC).

[0323] Based on the results provided by the present disclosure, the present disclosure also relates to a peptide with a length of 8-32 amino acids, comprising the sequence SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, wherein the peptide comprises a retro-inverted form of the amino acid sequence, for treating degenerative, chronic or progressive diseases or disorders (such as CNS diseases or disorders), monogenic genetic diseases (with ER stress as a pathogenic component).

[0324] Method for preparing peptides

[0325] Methods for synthesizing the disclosed compounds are known in the art. The following exemplary methods may be used. It should be understood that the various steps may be performed in an alternating sequence or order to obtain the desired compound. Synthetic chemistry transformations and protecting group methods (protection and deprotection) used to synthesize the compounds described herein are known in the art and include, for example, methods such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3rd ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0326] The peptides disclosed herein can be prepared by chemical synthesis methods, which are well known to those of ordinary skill. See, for example, Fields et al., Synthetic Peptides: A User's Guide, Chapter 3, Grant, ed., WH Freeman & Co., New York, USA, 1992, p. 77.

[0327] One way to prepare the peptides described herein is to use solid phase peptide synthesis (SPPS). The C-terminal amino acid is connected to a cross-linked polystyrene resin via an acid-labile bond with a linker molecule. This resin is insoluble in the solvent used for synthesis, making it relatively simple and rapid to wash away excess reagents and by-products. The N-terminus is protected by an Fmoc group, which is stable in acid but can be removed by alkali. Any side chain functional groups are protected by alkali-stable, acid-labile groups.

[0328] The publications and other materials used herein illustrate the background of the invention and, in particular, provide additional details regarding its practice, and are incorporated herein by reference.

[0329] It is obvious to those skilled in the art that, with the advancement of technology, the basic idea of ​​the present disclosure can be implemented in various ways. Therefore, the present disclosure and its embodiments are not limited to the following examples, but they can be varied within the scope of the claims.

[0330] Example

[0331] Example 1

[0332] Neuroprotective effects of parental and retro-reversal compounds on MPP+ injured dopaminergic TH-positive neurons

[0333] The neuroprotective effects of compounds 1-20 (SEQ ID NO: 1-20, maternal and retro-inverse) were tested in an in vitro model in which primary cultures of rat embryonic midbrain neurons were stressed with MPP+, the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP+ kills dopaminergic (TH-positive) neurons via a variety of toxic mechanisms, including mitochondrial dysfunction, production of peroxynitrite, oxidative stress, ER stress, and induction of apoptosis. Since peptides derived from the C-terminal domains of CDNF and MANF were modified into retro-inverse peptides, their neuroprotective activity was tested in such a model: full-length CDNF protein has been shown to protect TH+ dopamine neurons from neuroprotective effects, their synaptic and neurite networks from MPP+-induced damage ( Figure 2A ) and reduce the accumulation of α-synuclein aggregates in TH+ neurons ( Figure 2B ).

[0334] Materials and Methods

[0335] Materials and Methods for Synthesis and Characterization of Peptides.

[0336] Standard solid phase peptide synthesis methods were used to generate linear (L-aa) and retro-inverse (D-aa) peptides.

[0337] General Protocol for Synthetic Peptides - SPPS

[0338] Solid phase peptide synthesis was performed on an automatic peptide synthesizer (Biotage Initiator+Alstra or Activotec Activo-P11). Standard Fmoc-protected amino acids were used for peptide extension: Ala, Arg (Pbf), Asp (tBu), Gln (Trt), Glu (OtBu), Gly, His (Trt), Ile, Lys (Boc), Leu, Met, Ser (tBu), Thr (tBu), Trp (Boc), Val and Cys (StBu). Removal of the Fmoc group was performed using 20% ​​piperidine in DMF, and coupling was performed using 4 equivalents of the corresponding amino acid, 3.9 equivalents of HBTU, 4 equivalents of HOBt and 8 equivalents of DIPEA under microwave irradiation. The crude peptide was deprotected and cleaved from the resin by treatment with TFA / H2O / iPr3SiH for 2 hours followed by precipitation in cold Et2O.

[0339] In the case of retro-inverse peptides, standard Fmoc protected D-amino acids are used instead of Fmoc protected L-amino acids. Before starting the Biofage starting peptide synthesizer, note the reverse sequence in order to obtain the desired retro-inverse peptide.

[0340] Quality control of the peptides was obtained using standard liquid chromatography-mass spectrometry methods. Figure 1 (Column 5) shows the peaks identified in the MS analysis of the peptides.

[0341] Culture of midbrain neurons.

[0342] Rat dopaminergic neurons were cultured as described by Visanji et al., 2008. Briefly, the midbrain obtained from 15-day-old rat embryos (Janvier, France) was dissected, and the ventral part of the midbrain flexure (an area rich in dopaminergic neurons in the developing brain) was used for cell preparation. At 37°C (the final concentration of the solution was 0.05% trypsin and 0.02% EDTA), the midbrain cells were dissociated by trypsin digestion for 20 minutes. The reaction was terminated by adding Dulbecco's modified Eagle's medium (DMEM) containing DNase I grade II (0.5 mg / mL) and 10% fetal calf serum (FCS). The cells were then mechanically dissociated by flowing through a 10 ml pipette 3 times. The cells were then centrifuged at 180 x g for 10 minutes at +4°C on a BSA (3.5%) layer in L15 culture medium. The cell pellet was resuspended in a defined culture serum-free medium consisting of Neurobasal (Invitrogen) supplemented with B27 (2%), L-glutamine (2mM) and 2% PS solution and 10ng / ml brain-derived neurotrophic factor (BDNF) and 1ng / mL glial-derived neurotrophic factor (GDNF). Viable cells were counted in a Neubauer hemocytometer using a trypan blue exclusion test. Cells were seeded in 96-well plates (previously coated with poly-L-lysine) at a density of 40,000 cells / well and maintained in a humid incubator at 37°C in a 5% CO2 / 95% air atmosphere. Half of the culture medium was replaced with fresh culture medium every 2 days. On a 96-well plate, only 60 wells were used. In order to avoid any edge effects, the first and last rows and columns were not used for culture and were filled with sterile water.

[0343] Test compounds and MPP+ exposure.

[0344] On day 6 of culture, CDNF or compound 1-20 (SEQ ID NO: 1-20, parent and retro-reversed) were dissolved in culture medium and then pre-incubated with midbrain neurons for 4 hours prior to MPP+ application. 4 hours after pre-incubation of compound, MPP+ was added to a final concentration of 4 μM and diluted in control culture medium in which compound was still present for 48 hours.

[0345] Immunostaining: TH neuron survival neurite network and α-syn aggregation in TH neurons.

[0346] At 48 hours after intoxication, cells were fixed by 4% paraformaldehyde in PBS (pH 7.3) for 20 minutes at room temperature. Cells were washed twice in PBS and then blocked for permeabilization and nonspecific sites with PBS containing 0.1% saponin and 1% FCS for 15 minutes at room temperature. Next, cells were incubated with (a) monoclonal antibody anti-tyrosine hydroxylase (TH) produced in mice at a dilution of 1:10000 and (b) polyclonal antibody anti-α-synuclein (aSyn) antibody produced in rabbit at a dilution of 1:400 in PBS containing 1% FCS, 0.1% saponin for 2 hours at room temperature. These antibodies were exposed for 1 hour at room temperature in PBS containing 1% FCS, 0.1% saponin with secondary antibodies Alexa Fluor 488 goat anti-mouse IgG at a dilution of 1:800 and Alexa 568 goat anti-rabbit IgG at a dilution of 1:400.

[0347] Synaptic immunostaining: TH neurons and PSD-95 (overlap between TH / PSD-95 neurons).

[0348] 48 hours after intoxication, the cell culture supernatant was removed and the cells were fixed by 4% paraformaldehyde in PBS (pH 7.3) for 20 minutes at room temperature. The cells were washed twice in PBS and then blocked for 15 minutes at room temperature with a PBS solution containing 0.1% saponin and 1% FCS to permeabilize and nonspecific sites. The cells were then incubated a) at 1:10000 dilution in PBS containing 1% FCS, 0.1% saponin with a monoclonal anti-tyrosine hydroxylase (TH) antibody produced in mice for 2 hours at room temperature, and b) at 1:200 dilution in PBS containing 1% FCS, 0.1% saponin with a polyclonal anti-postsynaptic density protein-95 (PSD-95) antibody produced in rabbit for 2 hours at room temperature. This antibody specifically stains synapses. The antibodies were exposed with Alexa Fluor 488 goat anti-mouse IgG at a dilution of 1:800 and Alexa Fluor 568 goat anti-rabbit IgG at a dilution of 1:400 in PBS containing 1% FCS, 0.1% saponin for 1 hour at room temperature.

[0349] For each case, images representing the entire well area were automatically acquired using ImageXpress (Molecular Devices) at 10x (20 images for TH and a-syn) or 40x magnification (60 images for TH and PSD-95). The following readouts were automatically determined using the Custom Module Editor (Molecular Devices):

[0350] - Analysis of the total number of TH neurons (TH-positive neurons),

[0351] - Total neurite network of TH-positive neurons (in μm)

[0352] - aSyn aggregation (overlap between TH and aSyn staining)

[0353] - Number of synaptic TH-positive neurons (overlap of TH and PSD95, in μm 2 as units).

[0354] The total number of TH+ neurons, the total neurite network of TH+ neurons, and the number of synapses of TH+ neurons were Figure 2A (CDNF), Figure 3A (Compounds 1 and 2), Figure 3C (Compounds 3 and 4), Figure 3E (Compounds 5 and 6), Figure 3G (Compounds 7 and 8), Figure 4A (Compounds 9 and 10) and Figure 4C (Compounds 11 and 12), Figure 4E (Compounds 13 and 14), Figure 4G (Compounds 15 and 16), Fig. 4I (Compounds 17 and 18), Figure 4K (Compounds 19 and 20).

[0355] α-Synuclein aggregates in TH-positive neurons of primary cultures of midbrain cells after MPP+ injury Figure 2B (CDNF), Figure 3B (Compounds 1 and 2), Figure 3D (Compounds 3 and 4), Figure 3F (Compounds 5 and 6), Figure 3H (Compounds 7 and 8), Figure 4B (Compounds 9 and 10) and Figure 4D (Compounds 11 and 12), Figure 4F (Compounds 13 and 14), Figure 4H (Compounds 15 and 16), Figure 4J (compounds 17 and 18), Figure L (compounds 19 and 20).

[0356] The data show that many retro-inverse peptides protect TH-positive neurons and their neurites and synapses from MPP+ toxicity. In addition, these retro-inverse compounds effectively reduce the number of aSyn inclusions in TH-positive neurons, the accumulation of which is strongly induced by MPP+. In most cases, the efficacy of retro-inverse peptides (compounds 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20) is comparable to its parent compound (compounds 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, respectively) and full-length CDNF protein (Figure 2).

[0357] Example 2

[0358] Interaction with the ER stress response pathway

[0359] CDNF and MANF protect cells from ER stress-induced cell dysfunction or cell death, such as apoptosis, by regulating the cell's response to ER stress. Yan et al. (2019) showed that the C-terminus of MANF binds to the nucleotide binding domain (NBD) of GRP78 and regulates its cellular activity. This data suggests that MANF (and CDNF) have a regulatory interaction with GRP78, the most abundant chaperone protein in the ER lumen, rather than a substrate-like interaction. The binding of compounds to GRP78-NBD was evaluated in a cell-free binding assay using purified recombinant GRP78-NBD and synthetic peptides. Since GRP78 also acts as a key ligand for three receptors of the unfolded protein response (UPR) pathway, IRE1α, PERK, and ATF6, the dependence of the neuroprotective effect of the compound on UPR signaling was also tested. Figure 5A Molecular modeling of compound 6 in the MANF binding pocket of GRP78-NBD is shown. Figure 5B Shown are the binding affinities of selected compounds to GRP78-NBD in a cell-free binding assay. Figure 5C It was shown that the neuroprotective effects of compounds 14 and 20 were abolished in the presence of pharmacological inhibitors of PERK (GSK2606414) and IRE1α (KIRA6).

[0360] Materials and Methods

[0361] Molecular modeling.

[0362] GRP78-NBD in complex with the different compounds mentioned here was analyzed based on the previously solved structure of the GRP78-NBD:MANF complex using the PRIME module of the Schrodinger suite version 2018-4 (Schrodinger Ltd., USA) via the MAESTRO interface. Figure 5B) was modeled. The generated model was manually checked using the template structure (PDB:6HAB, Yan et al., 2019). The model was also verified by viewing its Ramachandran plot.

[0363] Cell-free binding assay.

[0364] His-tagged GRP78-NBD was recombinantly overexpressed and purified from E. coli cells and labeled with NHS-red dye (Nanotemper Technologies, Inc.). The His tag was cleaved from the protein using TEV protease. The binding of different peptides (serial dilutions) to the labeled untagged GRP78-NBD was measured in a Monolith NT standard capillary in a PBS environment using a Monolith NA device (Nanotemper Technologies, Inc.) at high power. Figure 5B Shown are tabulated data obtained from MST binding experiments.

[0365] Culture of midbrain neurons.

[0366] Neuronal cell culture, MPP+ intoxication, immunostaining and analysis of the neuroprotective effects of compounds were performed as in Example 1. PERK inhibitor GSK2606414 (2 μM, Sigma) or IRE1α inhibitor KIRA6 (2 μM, Sigma) were added to the culture 1 hour before the addition of the test compound.

[0367] These data suggest that retro-inverso compounds bind to GRP78, a key ER stress regulatory target molecule, and that the neuroprotective effects of retro-inverso compounds are dependent on UPR signaling activity.

[0368] Example 3

[0369] In vitro metabolic stability of the parent and retro-inverso compounds in rat plasma

[0370] The metabolic stability of the retro-inverso peptide and native control peptide was studied using rat plasma over a period of 120 minutes with an initial test concentration of 1 μM. Samples were analyzed using LC / QE-orbitrap-MS. The calculated half-life was based on the disappearance of the compound from rat plasma.

[0371] Materials and Methods

[0372] Parent or retro-inverse compounds 1-8, 13-14 and 19-20 (SEQ ID NO: 1-8, 13-14 and 19-20) were incubated with rat plasma (Spraque-Dawley, male; 400 μl) at a concentration of 1 μM at 37°C for different time points (0, 20, 40, 60 or 120 minutes). The incubation was terminated by acetonitrile. The collected samples were centrifuged at 2272×g for 20 minutes and analyzed. A stock solution was prepared using 50% DMSO, and the compound was spiked into the incubation at 1 / 100 to have a final DMSO content of 0.5%. The samples were analyzed by UHPLC / PDA using high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) to monitor the disappearance of the compound. To analyze compounds 13-14 and 19-20, stock solutions were prepared with PBS, and the samples were analyzed by UHPLC-ToF mass spectrometry. 1 μM of enalapril was used as a disappearance rate control. The analytical method was optimized by using the parent compound to obtain the best chromatographic characteristics (peak shape and retention time) and mass spectrometry ionization. The ion chromatogram was extracted from the total ion chromatogram using the calculated monoisotope accurate mass with a 5mDa window. Disappearance was based on the LC / MS peak area, with 0 minute marked as 100%. Excel software was used to obtain the first-order rate constant k (minute-1) of metabolism from the slope of the time compared to the logarithm (% of the remaining compound) graph. The in vitro half-life (t 1 / 2 ) is defined as: 1 / 2 =ln2 / k.

[0373] exist Fig. 6A In the figure, each pair of bars shows the data for an unmodified peptide (parent) and the corresponding modified peptide (retro-inverse). Some of the tested retro-inverse peptides (compounds 8, 14 and 20) showed significantly improved stability in rat plasma compared to their parent compounds (compounds 7, 13 and 19, respectively).

[0374] Example 4

[0375] In vitro metabolic stability of the parent and retro-inverse compounds in human plasma

[0376] Metabolic stability was studied using human plasma over a 120 minute period with an initial test concentration of 1 μM. Samples were analyzed using LC / QE-orbitrap-MS. Calculated half-lives were based on compound disappearance from human plasma.

[0377] Materials and Methods

[0378] Parent or retro-inverted compounds 1-8 (SEQ ID NO: 1-8) were incubated with human plasma (mixed sex, 400 μl) at a concentration of 1 μM at 37°C for different time points (0, 20, 40, 60 or 120 minutes). The incubation was terminated by acetonitrile. The collected samples were centrifuged at 2272×g for 20 minutes and analyzed. The samples were analyzed by UHPLC / PDA using high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) to monitor the disappearance of the compounds. 1 uM of propantheline bromide was used as a disappearance rate control. The analytical method was optimized using the parent compound to obtain the best chromatographic characteristics (peak shape and retention time) and mass spectrometry ionization. Ion chromatograms were extracted from the total ion chromatogram using a calculated monoisotopic accurate mass with a 5 mDa window. Disappearance was based on LC / MS peak area, with 0 minutes marked as 100%. The first order rate constant k (min-1) for metabolism was obtained from the slope of a plot of time versus log (% of compound remaining) using Excel software. The in vitro half-life (t 1 / 2 ) is defined as: 1 / 2 =ln2 / k.

[0379] exist Figure 6B In the Figures, each pair of bars shows data for an unmodified peptide (parent) and the corresponding modified peptide (retro-inverse).

[0380] The stability of the linear and retro-inverso peptides in human plasma was better compared to that in rat plasma, as indicated by the maximum reported half-life in human plasma (maximum half-life for the particular test was 795 minutes). One tested retro-inverso compound (Compound 8) showed improved stability in both rat and human plasma compared to its parent compound (Compound 7).

[0381] Example 5

[0382] In vitro metabolic stability of the parent and retro-inverso compounds in rat hepatocytes

[0383] The metabolic stability was studied using rat (Spraque-Dawley, male) liver hepatocytes for 0-60 minutes (n=2) with an initial test concentration of 1 μM. The samples were analyzed using LC / QE-orbitrap-MS. The calculated half-life was based on compound disappearance.

[0384] Materials and Methods

[0385] Parent or retro-inverted compounds 1-8, 13-14 and 19-20 (SEQ ID NO: 1-8, 13-14 and 19-20) were incubated with pooled cryopreserved rat hepatocytes (Spraque-Dawley, male; 400 μl, 1 million viable cells / ml for compounds 1-8 or 100 μl, 100,000 viable cells / ml for compounds 13-14 and 19-20) at 37°C for different time points (0, 10, 20, 40 or 60 minutes) at a concentration of 1 μM. Cell density and viability were determined by trypan blue exclusion. Incubation was terminated by acetonitrile. The collected samples were centrifuged at 2272×g for 20 minutes and analyzed. The samples were analyzed by UHPLC / PDA using high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) (compounds 1-8) or HHPLC-ToF mass spectrometry (for compounds 13-14 and 19-20) to monitor the disappearance of the compounds. 1uM of verapamil was used as a disappearance rate control. The analytical method was optimized using the parent compound to obtain the best chromatographic characteristics (peak shape and retention time) and mass spectrometry ionization. Ion chromatograms were extracted from the total ion chromatogram using a calculated monoisotopic accurate mass with a 5mDa window. Disappearance was based on LC / MS peak area, with 0 minutes marked as 100%. Using Excel software, the first-order rate constant k (minutes-1) of metabolism was obtained from the slope of the time versus logarithm (% of the remaining compound) graph. The in vitro half-life (t 1 / 2 ) is defined as: 1 / 2 =ln2 / k.

[0386] exist Fig. 7A In the Figures, each pair of bars shows data for an unmodified peptide (parent) and the corresponding modified peptide (retro-inverse).

[0387] All tested retro-inverso peptides (compounds 2, 4, 6, 8, 14 and 20) showed significantly improved stability in rat hepatocytes compared to their parent compounds (compounds 1, 3, 5, 7, 13 and 19, respectively).

[0388] Example 6

[0389] In vitro metabolic stability of the parent and retro-inverso compounds in human hepatocytes

[0390] Metabolic stability was studied using rat (mixed sex, male) liver hepatocytes for 0-60 min (n=2) with an initial test concentration of 1 μM. Samples were analyzed using LC / QE-orbitrap-MS. Calculated half-lives were based on compound disappearance.

[0391] Materials and Methods

[0392] Parent or retro-inverted compounds 1-8 (SEQ ID NO: 1-8) were incubated with pooled cryopreserved human hepatocytes (mixed sex; 400 μl, 1 million viable cells / ml) at a concentration of 1 μM at 37°C for different time points (0, 10, 20, 40 or 60 minutes). Cell density and viability were determined by trypan blue exclusion. The incubation was terminated by acetonitrile. The collected samples were centrifuged at 2272×g for 20 minutes and analyzed. The samples were analyzed by UHPLC / PDA using high-resolution mass spectrometry (QE-Orbitrap-MS in DDI mode) to monitor the disappearance of the compounds. 1 uM of verapamil was used as a disappearance rate control. The analytical method was optimized using the parent compound to obtain the best chromatographic characteristics (peak shape and retention time) and mass spectrometry ionization. Ion chromatograms were extracted from the total ion chromatogram using a calculated monoisotopic accurate mass with a 5 mDa window. Disappearance was based on LC / MS peak area, with 0 minutes marked as 100%. The first order rate constant k (min-1) for metabolism was obtained from the slope of a plot of time versus log (% of compound remaining) using Excel software. The in vitro half-life (t 1 / 2 ) is defined as: 1 / 2 =ln2 / k.

[0393] exist Figure 7B In the Figures, each pair of bars shows data for an unmodified peptide (parent) and the corresponding modified peptide (retro-inverse).

[0394] All tested retro-inverso peptides (compounds 2, 4, 6, and 8) showed significantly improved stability in human hepatocytes compared to their parent compounds (compounds 1, 3, 5, and 7, respectively).

[0395] Example 7

[0396] Permeation properties of parent and retrograde-inverse compounds in a 3D in vitro model of the blood-brain barrier

[0397] Due to the interest in developing CDNF and MANF-derived peptides for the treatment of CNS diseases via peripheral administration routes, the ability of compounds to cross the blood-brain barrier was tested in an established in vitro model of the blood-brain barrier. For this purpose, retro-reverso and parent compounds 1-14 and 17-20 (SEQ ID NOs: 1-14 and 17-20) were incubated at 500 nM for 2 hours in a two-chamber in vitro blood-brain barrier model (n=4), followed by sample collection and LC-MS / MS analysis.

[0398] exist Figure 8In the figure, each pair of bars shows the data of the unmodified peptide (parent) and the corresponding modified peptide (retro-inverse). Compared with the parent compound, retro-inverse compounds 2, 4, 6 and 12 (SEQ ID NO: 2, 4, 6 and 12) were observed to have better BBB passage.

[0399] The four retro-inverse compounds tested (compounds 2, 4, 6 and 12) showed improved ability to cross the in vitro blood-brain barrier compared to their parent compounds (compounds 1, 3, 5 and 11, respectively).

[0400] Materials and Methods

[0401] Primary culture of astrocytes.

[0402] Rat astrocytes were prepared from E15 embryos. In brief, pregnant female rats (Wistar, Janvier Laboratory) at 15 days of gestation were deeply anesthetized in a (CO2 chamber) and then killed by cervical dislocation. The fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium containing 2% penicillin (10,000U / mL) and streptomycin (10mg / mL) solution (PS) and 1% bovine serum albumin (BSA). The whole brain was treated with trypsin-EDTA solution at 37°C for 20 minutes at a final concentration of 0.05% trypsin and 0.02% EDTA. The dissociated cells were cultured in DMEM 10% fetal bovine serum. Purified astrocytes were used at the 4th generation (P4).

[0403] Culture of human endothelial cells. One vial of HBMEC (primary human brain microvascular endothelial cells, ACBRI 376) at a specific passage 8 (P8) was used.

[0404] Primary culture of cortical neurons. Rat cortical neurons were cultured as described by Callizot et al., 2013, but with modifications. Briefly, pregnant female rats (Wistar, Janvier Laboratory) at 15 days of gestation were deeply anesthetized in a CO2 chamber and then killed by cervical dislocation. The fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium containing 2% penicillin (10,000U / mL) and streptomycin (10mg / mL) solution (PS) and 1% bovine serum albumin (BSA). The cortex was treated with trypsin-EDTA solution at 37°C for 20 minutes at a final concentration of 0.05% trypsin and 0.02% EDTA. Dissociation was terminated by adding Dulbecco's modified Eagle's medium (DMEM) with 4.5g / L glucose containing DNAse I grade II (final concentration 0.5mg / mL) and 10% fetal calf serum (FCS). Mechanically dissociate cells by forcing flow through the tip of a 10-ml pipette three times. At 4 ° C, cells were centrifuged at 515 x g for 10 minutes. The precipitation was resuspended in a defined culture medium consisting of a neurobasal culture medium containing 2% B27 supplement solution, 2mmol / L L-glutamine, 2% PS solution, 10ng / mL brain-derived neurotrophic factor (BDNF). Using the trypan blue exclusion test, live cells were counted in a Neubauer hemocytometer. In a 24-well plate with an insert, cortical neurons were seeded at a density of 255,000 / well at the bottom of a well pre-coated with poly-L-lysine, and cultured at 37 ° C in an air (95%)-CO2 (5%) incubator. Culture medium was replaced every other day.

[0405] Co-culture of endothelial cells, astrocytes and primary cortical neurons. The procedure was performed as previously published (Xue et al., 2013 but modified, Callizot et al., 2017). Briefly, on day 0, purified astrocytes (P4) were rapidly thawed in a 37°C water bath. The cells were immediately placed in DMEM containing 10% FCS. The cell suspension was centrifuged at 515 x g for 5 minutes at 4°C, and the pellet was suspended in DMEM F12 containing 10% FCS. The cells were seeded on the outside of the insert membrane (PET, 1 μm) at a density of 45,000 cells / insert and cultured at 37°C in an air (95%)-CO2 (5%) incubator. Thirty-six (36) hours after astrocyte seeding, HBMEC (P8) were rapidly thawed in a water bath at 37°C and immediately placed in DMEM containing 10% FCS. The cell suspension was centrifuged at 515 x g for 5 minutes at 4°C, and the pellet was suspended in an EGM-2 bullet kit containing 5% FCS, 1% PS solution, 1.4 μM hydrocortisone, 5 μg / mL acidic ascorbic acid, 1% lipid mixture, 10 mM HEPES, 1 ng / mL bFGF. The cells were seeded on the inner side of the insert membrane (PET, 1 μm) at a density of 50,000 cells / insert and cultured at 37°C in an air (95%)-CO2 (5%) incubator. Thirty-six (36) hours after HBMEC seeding (72 hours after astrocyte seeding), cortical neurons were seeded at a density of 170,000 / well on the bottom of the well pre-coated with poly-L-lysine and cultured at 37°C in an air (95%)-CO2 (5%) incubator.

[0406] Parent or retro-inverse compounds are used.

[0407] Five (5) days after HBMEC seeding, after the first test of the integrity of the endothelial cell layer, test compounds 1-14 and 17-20 (SEQ ID NOs: 1-14 and 17-20) were added to the chamber and incubated at a concentration of 500 nM for 2 hours.

[0408] Quantification of test compounds.

[0409] Detection and quantification of each compound in the abluminal supernatant was further performed by mass spectrometry (MS) analysis.After thawing of the samples, a 100 μL aliquot of each cell culture sample was analyzed by quantification of peptides by mass spectrometry.

[0410] The calculated pass percentage represents the percentage of compound applied to the abluminal compartment, as measured in the abluminal compartment at the end of the application.

[0411] Example 8

[0412] In vivo pharmacokinetic profiles of retrograde-inverse compounds following peripheral administration to rats

[0413] The clearance and elimination of peptides can be mediated by multiple processes in vivo, including metabolism and renal elimination (Li et al., 2015; Lin et al., 2009). Since in vitro studies have shown that retro-inverso peptides have improved metabolic stability, their in vivo pharmacokinetic properties were tested by peripherally (intravenously) administering a single dose level of the compound and then measuring the presence of the compound in plasma at different time points after peripheral administration.

[0414] Fig. 9A Plasma concentrations of retro-inverse compounds, a native compound and a native 27 amino acid control compound at various time points after intravenous administration are shown. All tested retro-inverse compounds showed increased plasma retention times compared to the native compound.

[0415] Table 1 discloses the in vivo pharmacokinetic properties of the retro-inverso compounds and the native 27-amino acid control compound following intravenous administration to rats.

[0416] Table 1. In vivo pharmacokinetics of retrograde-inverso compounds and control native peptides following a 5 mg / kg intravenous bolus injection in male Sprague-Dawley rats.

[0417]

[0418]

[0419] Fig. 9B The brain distribution kinetics of Compound 20 in male Sprague-Dawley rats are shown. microdialysis The probe was inserted into the rat ventral striatum and perfused with aCSF. Compound 18 was administered as a single 10 mg / kg intravenous push, and microdialysis samples were collected at 20-minute intervals over 4 hours. The brain interstitial fluid (ISF) concentration of the compound was determined by LC-MS / MS and normalized to the recovery rate-% of the microdialysis membrane (determined by in vitro experiments).

[0420] All tested retro-inverso compounds showed improved plasma half-life, volume of distribution and mean residence time compared to a control native peptide consisting of 27 L-amino acids. In addition, brain microdialysis studies showed that compound 20 penetrated into the brain parenchyma after a single intravenous bolus.

[0421] Materials and Methods

[0422] Test compounds were administered intravenously (iv) to male Spraque-Dawley rats (approximately 6 weeks old, n=3 for each compound) at 5 mg / kg. Blood samples were collected from the jugular vein by implanting an indwelling catheter (250 μl of blood) into a labeled polypropylene tube containing an anticoagulant (heparin) at 2 minutes, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours after compound administration, and kept on wet ice for up to 30 minutes. Blood samples were centrifuged for plasma separation (4°C, 21100G, 5 minutes). 500 ng / ml tolbutamide containing 10% TFA in acetonitrile or MeCN was used as an internal standard solution. Standard samples were prepared as rat plasma by spiking the matrix into the analyte at a concentration of 2-10 000 ng / ml, respectively, otherwise treated as samples. 200 μl of internal standard was added to a 50 μl aliquot of sample plasma. The samples were mixed (150 rpm, 15 minutes) and centrifuged (3000 rpm, 15 minutes). The analytical method was optimized for reaction monitoring chromatographic (peak shape and retention time) shifts and mass spectrometric properties (ionization efficiency, MS / MS detection). The supernatant was analyzed by UHPLC-TOF mass spectrometry using electrospray ionization.

[0423] The test compound was administered intravenously (iv) to male Spraque-Dawley rats (n=3) at 0.5 mg / kg. The compounds administered were compound 6 (SEQ ID NO: 6), compound 12 (SEQ ID NO: 12), compound 13 (SEQ ID NO: 13), compound 14 (SEQ ID NO: 14), compound 20 (SEQ ID NO: 18) and a parent control compound consisting of 27 L-amino acids. Plasma samples were collected and subsequently analyzed by LC-MS / MS. Pharmacokinetic parameters were calculated from the plasma concentrations at different time points.

[0424] A separate group of awake animals treated intravenously with compound 20 (SEQ ID NO: 20) was subjected to a brain microdialysis study. One week before the microdialysis experiment, Sprague-Dawley rats were implanted with a guide cannula in the striatum at the following coordinates: AP + 0.6 mm; L-3.0 mm; V-2.8 mm, providing a final V-6.8 mm for 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, cutoff 50 kDa) 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 minutes, compound 18 was administered as a single 10 mg / kg intravenous push, and samples were collected at 20-minute intervals over 4 hours. The intracerebral fluid concentration of the compound was analyzed by UHPLC-MS / MS. Additional in vitro testing was performed to determine the recovery of the test compound from tubing, connectors, and microdialysis probes when perfused with aCSF under conditions similar to the in vivo testing. The determined recovery percentage (27.4%) was used to correct the data obtained in the microdialysis studies.

[0425] Reference List

[0426] Patent Literature

[0427] EP 58,481;

[0428] US 3,773,919;

[0429] WO 2007068803;

[0430] WO 2009133247;

[0431] WO 2013 / 3034805;

[0432] WO 2018 / 202957

[0433] Non-patent literature

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Claims

1. A peptide consisting of 8 to 23 amino acids in length or a pharmaceutically acceptable salt thereof, wherein the peptide consists of an amino acid sequence selected from any one of the following: KEACGKCTEGWDDLIKKLEKVRL(SEQ ID NO:4), SKEACGKCTEG (SEQ ID NO: 8), SKEACGGKCTEG (SEQ ID NO: 12), KEACGKCTE (SEQ ID NO: 16) or EACGKCTE (SEQ ID NO: 20), wherein all amino acids of the peptide are D-amino acids. The peptide according to claim 1 , wherein the N-terminus of the peptide is acetylated. The peptide according to claim 1 , wherein the C-terminus of the peptide is amidated. The peptide according to claim 1 , wherein the N-terminus of the peptide is acetylated, and the C-terminus of the peptide is amidated.

5. The peptide of claim 1, which is conjugated to a fluorophore, a detectable chemical tag, an enzyme detectable in the presence of a substrate, a detectable protein, or polyethylene glycol (PEG).

6. Use of the peptide according to claim 1 in the preparation of a medicament for treating the following diseases: Parkinson's disease, Alzheimer's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease, traumatic brain injury and retinitis pigmentosa.

7. The use according to claim 6, wherein the peptide is administered by peripheral administration, local administration, enteral administration or parenteral administration.

8. Use according to claim 7, wherein the peripheral administration is selected from subcutaneous and intranasal administration.

9. The use according to claim 7, wherein the parenteral administration is selected from intravenous, intraarterial, intraocular, intratympanic, intraperitoneal, intramuscular, intraarticular, intracranial, intrathecal, epidural and intralesional administration.

10. The use according to claim 7, wherein the topical administration is selected from transdermal administration, topical intraocular administration and inhalation administration.

11. The use according to claim 7, wherein the enteral administration is selected from oral, rectal, sublingual and buccal administration.

12. The use according to claim 8, wherein the peptide is administered by subcutaneous administration.

13. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 5 and at least one of: a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutically acceptable carrier and the pharmaceutically acceptable excipient are selected from preservatives, stabilizers and / or diluents.

15. Use of the pharmaceutical composition according to claim 13 in the preparation of a medicament for treating the following diseases: 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 and retinitis pigmentosa.

16. The use according to claim 15, wherein the composition is administered by peripheral administration, topical administration, enteral administration or parenteral administration.

17. The use according to claim 16, wherein the peripheral administration is selected from subcutaneous and intranasal administration.

18. The use according to claim 16, wherein the parenteral administration is selected from intravenous, intraarterial, intraocular, intratympanic, intraperitoneal, intramuscular, intraarticular, intracranial, intrathecal, epidural and intralesional administration.

19. The use according to claim 16, wherein the topical administration is selected from transdermal administration, topical intraocular administration and inhalation administration.

20. The use according to claim 16, wherein the enteral administration is selected from oral, rectal, sublingual and buccal administration.

21. The use according to claim 17, wherein the composition is administered by subcutaneous administration.

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