peptides

Abstract

Provided are polyarginine polypeptide ASIC inhibitors, pharmaceutical compositions comprising the same as well as methods and uses therefor In particular, the present disclosure relates to the methods and uses of such polypeptides or pharmaceutical compositions in the treatment of ASIC-related diseases. Such polypeptides or pharmaceutical compositions are useful to treat neurological disorders, neurodegenerative diseases, psychological disorders, sexual disorders, cardiovascular diseases, respiratory diseases, inflammatory diseases, renal diseases, gastrointestinal diseases, metabolic disorders, cancer, and addiction, e.g. smoking addiction.

Description

[0001] PEPTIDES

[0002] Sequence Listing

[0003] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled P81768WO_SEQ LIST.xml, created on 05 November 2025, which is 24 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0004] Technical Field

[0005] The invention relates to the development of cyclic polyarginine peptides as inhibitors of acid-sensing ion channels (ASICs), such as ASIC1 and particularly ASIC1a, for the treatment of diseases related to ASICs.

[0006] Background

[0007] Acid-sensing ion channels (ASICs) are a group of ion channels that are activated by extracellular protons and are involved in various physiological and pathological processes. These channels are particularly significant in the context of neurological and cardiovascular health. During events such as ischemic stroke or myocardial infarction, the body experiences a drop in pH levels, leading to acidosis, which in turn activates ASICs. This activation can result in increased calcium influx into cells, exacerbating neuronal injury and contributing to cell death. Consequently, ASICs have been identified as potential therapeutic targets for conditions where acidosis plays a critical role, such as stroke and neurodegenerative diseases.

[0008] The challenge in targeting ASICs lies in developing inhibitors that can effectively modulate these channels without causing adverse effects. Current treatments for stroke and myocardial infarction are limited, with thrombolytic drugs like Alteplase being the primary option, which do not address the underlying biochemical pathways leading to tissue damage. Moreover, the development of ASIC inhibitors has been complicated by the need for molecules that can act quickly and maintain efficacy over a prolonged period. Small-molecule inhibitors, such as amiloride and diminazene aceturate, often have a narrow therapeutic window due to the rapid recovery of the receptor. In contrast, large-sized peptide inhibitors, such as Psalmotoxin-1 (PcTx1), Mamba1gine-1 (Mamba1), and Hila toxins, face challenges in production, crossing the blood-brain barrier and potential pharmacological interactions. Therefore, there is a significant need for innovative solutions that can provide rapid action and a broad therapeutic window to improve outcomes in patients suffering from ASIC-related diseases.

[0009] Early research described short linear arginine-rich peptides such as simple linear hexapeptides primarily composed of arginine residues (Ferrer-Montiel, et al., Nature Biotechnology, vol. 16, pp. 286-291, 1998); short linear arginine polymers such as R7 and R9 (Uemura, et al. (Circulation Journal, vol. 66, pp. 1155-1160, 2002); and the TAT peptide (Vaslin, etal. (Neurotoxicity Research vol. 15, pp. 123-126, 2009).

[0010] WO 2015 / 061856 describes linear cationic arginine-rich peptides (CARPS) and their application for neuroprotection.

[0011] Meloni et al. (Neuromolecular Medicine, vol. 19, pp. 271-285, 2017) assessed arginine-rich and polyarginine peptides such as cyclic R12 and linear R22 peptides fortheir potential neuroprotective effects.

[0012] Lian, etal. (Journal of the American Chemical Society, vol. 135, pp. 9830-9833, 2014) described a modular approach to fuse cyclic cell-penetrating peptides (CPPs) with cyclic target-binding domains, yielding cell-permeable bicyclic peptides that demonstrate high selectivity and nanomolar potency in inhibiting intracellular proteins such as PTP1 B and Pin 1.

[0013] Xu et al. (Journal of the American Chemical Society, vol. 139, pp. 2245-2256, 2017) discusses a class of macrocycles that employ combinatorial linker engineering to constrain short substrate-mimicking peptides for the purpose of modulating intracellular protein-protein interactions (for instance, Tankyrase).

[0014] MacDougall, et al. (Neurobiology of Disease, vol. 121, pp. 17-33, 2018) describes CARPs, such as TAT, R9, and R18, and related analogues as bioactive molecules endowed with the capacity to confer neuroprotection in stroke models. All peptides delineated therein are predominantly linear or minimally cyclised.

[0015] Marshall, et al. (Journal of Biological Chemistry, vol. 290, no. 36, pp. 22030-22048, 2015) indicated that linear polyarginine peptides, such as R9, exert their neuroprotective effects through intracellular mechanisms following cellular uptake and localised mitochondrial activity.

[0016] Summary of Invention

[0017] The present invention provides cyclic polyarginine polypeptides as further described herein, and pharmaceutically acceptable salts thereof. The cyclic polyarginine polypeptides of the present invention, and their pharmaceutically acceptable salts, are inhibitors of acidsensing ion channels (ASICs), optionally acid-sensing ion channel 1 (ASIC1) such as acidsensing ion channel 1a (ASIC1a), e.g. human acid-sensing ion channel 1a (hASIC1a). In overview, the present invention provides a cyclic polyarginine polypeptide comprising at least one motif corresponding to general formula la:

[0018] cyclo[B-(J)m-RRR-(U)n-Z] (General formula la - SEQ ID NO: 18)

[0019] wherein

[0020] R represents an arginine residue;

[0021] B, J, U and Z each represent amino acid residues other than arginine;

[0022] m and n are each independently integers from 0 to 6, wherein when m is nonzero, each instance of J may independently be the same or different, and wherein n is nonzero, each instance of U may independently be the same or different;

[0023] and wherein B and Z may independently be the same as, or different from, one another and may independently be the same as, or different from, any instance of J or U;

[0024] and wherein B is stapled to Z,

[0025] further wherein the arginine content of the cyclic polyarginine polypeptide comprising the at least one motif according to general formula la is at least 40% w / w;

[0026] or a pharmaceutically acceptable salt thereof.

[0027] More specifically, the present invention provides a cyclic polyarginine polypeptide comprising at least one SEQ ID NO: 1 motif: X(1) - X(2) - X(3) - RRR - X(4) - X(5) - X(6) (SEQ ID NO: 1), wherein: (a) X(1) and X(6) are amino acid residues other than arginine and X(1) is stapled to X(6); (b) X(2), X(3), X(4) and X(5) are, independently, either absent or amino acid residues other than arginine; and (c) the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof.

[0028] The cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof may comprise at least one SEQ ID NO: 2 motif: C(1) - X(2) - X(3) - RRR - X(4) - X(5) - C(6) (SEQ ID NO: 2), wherein C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof; optionally wherein the stapling moiety may be a disulfide bridge; and wherein X(2), X(3), X(4) and X(5) are, independently, either absent or amino acid residues other than arginine; and wherein the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w.

[0029] In one aspect, the present invention provides a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof comprising at least one SEQ ID NO: 3 motif: C(1) -RRR - C(6) (SEQ ID NO: 3), wherein C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof; and wherein the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w. The stapling moiety may be a disulfide bridge. Stated alternatively, the SEQ ID NO: 3 motif of the present invention may be represented as CRRRC (SEQ ID NO: 3), wherein both C are stapled to one another by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof; and wherein the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w. The stapling moiety may be a disulfide bridge.

[0030] SEQ ID NO: 3 may therefore be referred to as C(1) - RRR - C(6) or CRRRC interchangeably in the present specification.

[0031] The cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof may comprise a plurality of SEQ ID NO: 3 motifs, optionally wherein the cyclic polyarginine polypeptide may comprise at least 2, at least 3, at least 4, at least 5 or at least 6 SEQ ID NO: 3 motifs, further optionally wherein the cyclic polyarginine polypeptide may comprise at least 2 SEQ ID NO: 3 motifs.

[0032] The cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof may comprise between 3 and 50 arginine amino acid residues, optionally wherein the cyclic polyarginine polypeptide may comprise between 3 and 30 arginine amino acid residues.

[0033] The cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof may further comprises a spacer located at the N-terminus and / or C-terminus of the polypeptide, optionally the spacer may be a flexible spacer comprising: S, G, GS, A, Ahx, Ado, PEG, Sar, gamma-E, repetition and / or combination thereof.

[0034] The cyclic polyarginine polypeptide N-terminus or the spacer located at the N-terminus of the polypeptide and the polypeptide C-terminus or the spacer located at the C-terminus of the polypeptide may be covalently bound; optionally wherein they may be covalently bound by an amide bond.

[0035] The cyclic polyarginine polypeptide may be SEQ ID NO: 15 or a pharmaceutically acceptable salt thereof.

[0036] The arginine content of the cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof may be at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w or at least 95% w / w.

[0037] The cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof may further comprise at least one detectable label, optionally wherein the at least one detectable label may be selected from: (a) enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase, e.g., horseradish peroxidase; (b) dyes; (c) fluorescent labels, such as fluorescein and its derivatives, fluorochrome, rhodamine compounds and derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde, and fluorescamine; (d) fluorophores such as lanthanide cryptates and chelates; (e) chemoluminescent labels, such as isoluminol, luminol and the dioxetanes; (f) bio-luminescent labels, such as luciferase and luciferin; (g) sensitizers; (h) coenzymes; (i) enzyme substrates; (j) radiolabels, such as bromine77, carbon14, cobalt57, fluorine8, gallium67, gallium68, hydrogens (tritium), indium111, indium113, iodine123, iodine125, iodine126, iodine131, iodine133, mercury107, mercury203, phosphorous32, rhenium99, rhenium101, rhenium105, ruthenium95, ruthenium97, ruthenium103, ruthenium105, scandium47, selenium75, sulphur35, technetium99, technetium99, tellurium121, tellurium122, tellurium125, thulium165, thulium167, thulium168, yttrium199; (k) paramagnetic labels, such as nitroxide spin labels, e.g. methanethiosulfonate spin label (MTSSL), or lanthanide-chelating tags; (I) particles, such as latex or carbon particles; and (m) contrast agents; optionally wherein the at least one detectable label may be covalently attached via a linker, spacer and / or anchor group, e.g. a cleavable linker.

[0038] The invention further provides a pharmaceutical composition comprising a cyclic polyarginine polypeptide of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

[0039] The invention further provides the cyclic polyarginine polypeptide (or pharmaceutically acceptable salt thereof) of the invention or the pharmaceutical composition of the invention for use in therapy. The therapy may be for the treatment of an ASIC-related disease in a subject. The ASIC-related disease may be selected from neurological disorders, neurodegenerative diseases, psychological disorders, sexual disorders, cardiovascular diseases, respiratory diseases, inflammatory diseases, renal diseases, gastrointestinal diseases, metabolic disorders, cancer, and addiction, e.g. smoking addiction.

[0040] The neurological disorder may be selected from the group consisting of stroke, ischemia, traumatic brain injury, spinal cord injury, neuropathic pain, and epilepsy.

[0041] The neurodegenerative disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis.

[0042] The psychological disorder may be selected from the group consisting of anxiety, depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), and addiction.

[0043] The cardiovascular disease may be selected from the group consisting of myocardial infarction, heart failure, arrhythmia, and hypertension.

[0044] The respiratory disease may be selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS).

[0045] The inflammatory disease may be selected from the group consisting of rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, and sepsis. The renal disease may be selected from the group consisting of acute kidney injury, chronic kidney disease, and renal fibrosis.

[0046] The gastrointestinal disease may be selected from the group consisting of gastroesophageal reflux disease (GERD), peptic ulcer disease, and irritable bowel syndrome (IBS).

[0047] The metabolic disorder may be selected from the group consisting of diabetes, obesity, and metabolic syndrome.

[0048] The cancer may be selected from the group consisting of glioblastoma, breast cancer, lung cancer, colorectal cancer, and prostate cancer.

[0049] The addiction may be smoking addiction.

[0050] The ASIC-related disease may be acidosis (e.g. metabolic acidosis) or an acidosis-related (e.g. metabolic acidosis-related) condition such as a neurodegenerative disease (e.g. Parkinson’s disease), renal failure, chronic kidney disease (abbreviated as “CKD”), acute kidney injury, Addison’s disease, or diabetes.

[0051] The cyclic polyarginine polypeptide (or pharmaceutically acceptable salt thereof) for use of the invention or the pharmaceutical composition for use of the invention may be administered to the subject via oral, parenteral, subcutaneous, intravenous, intramuscular, intranasal and / or transdermal administration route.

[0052] The invention further provides a method of treatment comprising the administration of a cyclic polyarginine polypeptide (or pharmaceutically acceptable salt thereof) of the invention or a pharmaceutical composition of the invention to a subject. The treatment may be for an ASIC-related disease in a subject.

[0053] The invention further provides the use of a cyclic polyarginine polypeptide (or pharmaceutically acceptable salt thereof) of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament. The medicament may be for the treatment of an ASIC-related disease in a subject.

[0054] Brief Description of Drawings

[0055] The following figures represent the structures and results of the compounds of the invention by way of non-limiting examples.

[0056] Figure 1. Presents the analytical characterization of the R2 (A) and R2-NH2 (B) peptides using high-performance liquid chromatography (HPLC) and mass spectrometry. These figures confirm the successful synthesis, purity, and expected molecular identity of the peptides. Figure 2. Presents the analytical characterization of the R3 (A) and R3-NH2 (B) peptides using HPLC and mass spectrometry. These figures confirm the successful synthesis, purity, and expected molecular identity of the peptides.

[0057] Figure 3. Presents the analytical characterization of the R18 (A) and R18-NH2 (B) peptides using HPLC and mass spectrometry. These figures confirm the successful synthesis, purity, and expected molecular identity of the peptides.

[0058] Figure 4. Presents the analytical characterization of the R9-NH2 peptide using HPLC and mass spectrometry. These figures confirm the successful synthesis, purity, and expected molecular identity of the peptide.

[0059] Figure 5. Provides an analytical characterization of the cR9 peptide through HPLC and mass spectrometry. These results validate the successful synthesis, purity, and anticipated molecular identity of the monocyclic nona-L-arginine peptide outlined in the invention.

[0060] Figure 6. The analytical characterization of the 3cR10ss peptide is presented using HPLC and mass spectrometry. These figures confirm the successful synthesis, purity, and expected molecular identity of the first-in-class ASIC1 inhibitor, the tricyclic arginine-finger peptide described in the invention. Expected mass: 1970.50 Da, trifluoroacetic (TFA) adducts:

[0061] [3cR10ss+6*TFA]4+m / z expected = 664.63, m / z observed = 664.6; [3cR10ss+6*TFA]3+m / z expected = 885.83, m / z observed = 885.6; [3cR10ss+7*TFA]3+m / z expected = 923.83, m / z observed = 923.7.

[0062] Figure 7. The two structures illustrate the monocyclic nona-arginine configuration: on the left is an expanded chemical structure, while the structure on the right features abbreviated labels. The cyclic structures show that arginine residues are in closer proximity and confined to a single conformation, unlike linear nona-arginine, which allows the arginine residues to remain free from any constraints.

[0063] Figure 8. The tricyclic arginine finger peptide (3cR10ss) is represented on the top right and left with abbreviated labels, while the expanded chemical structure is depicted on the bottom.

[0064] Figure 9. Representative patch-clamp current traces demonstrating the inhibitory effect of chronic treatment with 10 mM L-arginine. Pretreatment with L-arginine resulted in the inhibition of currents induced by pH 6.5 (left), pH 5.5 (middle), and pH 4.5 (right) in the endogenous hASIC1a in HEK293 cells. Representative current traces at - 70 mV.

[0065] Figure 10. Representative patch-clamp current traces showing the effects of NG-amino-L-arginine (left) and L-arginine amide (right) on pH 6.5-induced currents in endogenous hASIC1a expressed in HEK 293 cells. In the left panel, four independent biological repeats comparing treated and untreated cells demonstrate that NG-amino-L-arginine produces no inhibition, indicating the necessity of a free (unsubstituted) guanidino group for activity. In the right panel, a representative trace compares L-arginine amide-treated cells (asterisk) with untreated controls (plus), showing greater inhibition than L-arginine and underscoring the contribution of the amide group to potency.

[0066] Figure 11. Representative patch-clamp current traces demonstrating the direct coapplication effect of 5 pM R9 (left) and R18 (right) peptides at pH 6.5 on the endogenous hASIC1a in HEK 293 cells. Neither R9 nor R18 produced any effect when applied directly, (n = 4). Representative current traces at - 70 mV.

[0067] Figure 12. Representative patch-clamp current traces showing the immediate effect of 5 pM tricyclic polyarginine finger peptide (3cR10ss) at pH 6.5 on endogenous hASIC1a in HEK 293 cells. The black trace represents the pH 6.5 application alone. The direct co-application of 3cR10ss with a pH 6.5 solution (light grey trace) resulted in 79 ± 7.6% inhibition (n = 20). After 3 minutes of the first washout (first dark grey trace), currents recovered to 28.5 ± 13.8% of the original amplitude. After 6 minutes of the second washout (second dark grey trace), recovery reached 38.3 ± 17.5%. The slow recovery profile indicates a prolonged inhibitory effect and a wide therapeutic window. Recordings at -70 mV.

[0068] Figure 13. Representative patch-clamp current traces demonstrate the full recovery of the receptors after a 2-minute pre-application with the R9 peptide (3 pM). Only a 2-minute washout was sufficient to restore the receptors, reflecting the narrow impact of R9. Representative current traces at - 70 mV.

[0069] Figure 14. Representative patch-clamp current traces demonstrate the full recovery of the receptors after a 10-minute pre-application with the R18 peptide (1 pM). Three 2-minute washouts were sufficient to restore the receptors, reflecting the narrow impact of R18. Representative current traces at - 70 mV.

[0070] Figure 15. Representative patch-clamp current traces demonstrate the 3-minute pre-application effect of Nerinetide (25 pM) at pH 6.0 on endogenous hASIC1a in HEK 293 cells. The pre-application of Nerinetide inhibited the pH 6.0-induced currents, and the channels quickly recovered after the second washout. Representative current traces were recorded at -70 mV.

[0071] Figure 16. Several repeats of patch-clamp current traces demonstrate the full recovery of the receptors after a 2-minute pre-application with the R18 peptide (1 pM). A 5-minute washout was sufficient to restore the receptors, which reflects the short impact of the R18. Representative current traces at - 70 mV.

[0072] Figure 17. Representative patch-clamp current traces show the effect of a 2-minute pre-application of 2 pM Tricyclic polyarginine finger peptide (3cR10ss) at pH 7.5 on the pH 6.5-induced currents (trace 0). Pre-application of 3cR10ss for 2 minutes at pH 7.5 (trace 1) resulted in 90 ± 8.6 % inhibition (trace 2) of pH 6.5-induced currents (n = 7). After different washouts (every 3 minutes), the channels showed slow receptor recovery (grey traces), indicating the peptide's wide therapeutic window. Representative current traces at - 70 mV.

[0073] Figure 18. Representative patch-clamp current traces illustrating the recovery time of hASIC1a currents following a 2-minute pre-application of 3cR10ss at pH 7.5 (light grey) priorto pH 6.5 stimulation. The gradual increase in current over successive grey traces reflects partial recovery, with full restoration of pH 6.5-induced currents achieved after approximately 40 minutes of repeated washouts at 3-minute intervals. This is compared to the black trace, which shows the pH 6.5-induced currents before treatment with peptide. Recordings at -70 mV.

[0074] Figure 19. Representative patch-clamp current traces showing the immediate effect of 20 pM 3cR10ss at pH 6.0 on endogenous hASIC1a in HEK 293 cells. Co-application of 3cR10ss with pH 6.0 solution (trace 1) produced 73.5 ± 8.8% inhibition (n = 12) relative to the pre-treatment response (black trace). Following 20 minutes of separate washouts (dark grey traces), receptor recovery remained incomplete, indicating a prolonged inhibitory effect and a wide therapeutic window. Recordings at -70 mV.

[0075] Figure 20. Representative patch-clamp current traces showing the 2-minute preapplication effect of the R9 vs R9-NH2 peptides (600 nM) at pH 6.5 on the endogenous hASIC1a in HEK 293 cells. The R9-NH2 peptide exhibits a stronger effect than R9, showing the significance of the amide C-terminal modification of the peptide. Representative current traces at - 70 mV.

[0076] Figure 21. Representative patch-clamp current traces showing the 2-minute preapplication effect of the R9 vs cR9 peptides (600 nM) at pH 6.5 on the endogenous hASIC1a in HEK 293 cells. cR9 peptide shows a stronger effect than the linear R9, showing the significance of the head-to-tail lactam cyclisation of the peptide. Representative current traces at - 70 mV.

[0077] Figure 22. Dose-response for the inhibitory effect of 2 min preincubation with R9, R18, and cR9 on pH 6.5-evoked currents from endogenous hASIC1a in HEK cells. Currents were normalised to control the pH 6.5-evoked response. Data were fitted to the Hill equation, shown as curves with parameters R9JC50 = 0.69 pM (95% Cl: 0.57 - 0.719 pM), R18JC50 = 0.45 pM, (95% Cl: 0.41 - 0.59 pM), and cR9_IC50 = 0.43 pM, (95% Cl: 0.38 - 0.49 pM). All data shown are the mean.

[0078] Figure 23. Column graph with a scatter plot of data forthe comparison of the effect of 800 nM R9, cR9, and R18 on pH 6.5-induced currents on HEK 293 cells, showing cR9 is significantly more potent than R9, while no significant difference between cR9 and R18 on pH 6.5-induced currents (n = 3). One-way ANOVA ***P < 0.0005, ns indicates non-statistically significant (P = 0.5325). Figure 24. Bar graph showing the effect on HEK293 cell viability by MTT assay after treatment of HEK 293 cells with pH 6 compared to pH 7.4 (control). cR9 (30 pM) and PcTX1 (0.5 pg / ml) reduced the cytotoxic effect of pH 6.0, while R18 (30 pM) did not rescue the acid-induced toxicity (n = 3). One-way ANOVA, ****P < 0.0001, ns indicates non-statistically significant (P = 0.13).

[0079] Figure 25. Multiple repetitions of patch-clamp current traces illustrate the effects of a 2-minute pre-application of low-dose (100 nM) R18 peptide on the currents induced at pH 6.5. The application of 100 nM R18 resulted in the potentiation of pH 6.5-induced currents. This effect at low doses highlights undesirable effects of linear polyarginine peptides.

[0080] Figure 26. Multiple repetitions of patch-clamp current traces illustrate the effects of a 2-minute pre-application of low-dose (5 pM) Nerinetide peptide on the currents induced at pH 6.5. The application of 5 pM Nerinetide resulted in the potentiation of pH 6.5-induced currents. This effect at low doses highlights the linear peptide's multimodal nature, producing undesirable effects of the peptide.

[0081] Figure 27. Multiple repetitions of patch-clamp current traces illustrate the effects of direct application of 1 and 2 pM R18 peptide at pH 8.0 on ASIC1a currents. The application of 1 and 2 pM R18 peptide at pH 8.0 led to different activation of ASIC1a currents. This effect at pH 8.0 highlights the peptide's pH-dependent impact, which produced an opposite effect for the peptide. Representative current traces are shown at -70 mV.

[0082] Figure 28. Representative patch-clamp current traces show the effect of a 2-minute pre-application of 2 pM 3cR10ss at pH 8.0 on the pH 6.5 induced currents. Unlike R18, pre-application of 3cR10ss for 2 minutes at pH 8.0 did not affect the inhibition effect of the peptide on pH 6.5-induced currents (n = 6). This effect at pH 8.0 shows the peptide pH-independent effect, and the slow receptor recovery indicates the peptide's wide therapeutic window. Representative current traces at - 70 mV.

[0083] Figure 29. Effects of glutamate on hASIC1a and tissue acidosis: A, a one-minute preapplication of 5mM monosodium glutamate enhances the human ASIC1a current endogenously expressed in HEK cells (n = 8). B, Direct application of Monosodium glutamate (5 mM) at pH 6.5 enhances human ASIC1a currents (n = 12), which are blocked by a one-minute preapplication of PcTX1 (25 nM) (n = 4). C, Direct application of glutamic acid 500 pM and 5 mM at pH 7 enhances human ASIC1a currents (n = 16). D, Direct application of glutamic acid 15 mM at pH 7 enhances human ASIC1a currents as much as pH 6-induced currents (n = 8). E, Direct application of glutamic acid 500 pM at pH 6.8 shows significantly enhanced currents as much as pH (n = 8). F, Bar graph showing pH 6.5 and pH 6 -induced injury of HEK293 cells and significant enhancement of the pH 6.5-induced injury by addition of 5 mM glutamic acid and 10 mM glutamic acid without pH adjustment resulted in low cell viability, while 10 mM glutamic acid at pH 7.4 has no effect on cell viability n= 9 (P<0.001). Figure 30. Left: Representative automated patch-clamp current traces show the effect of nicotine (3 mM) on pH 7 and pH 6.8-induced currents on the endogenous hASIC1a in HEK293 cells. Applications (3 s) of pH 7.0 (top left) and pH 6.8 (bottom left) in the presence of 3 mM nicotine resulted in significant potentiation of pH-induced currents. Right: Column graph with scatter plot data for the effect of Nicotine 3 mM on pH 7.0 (top right) and pH 6.8 (bottom right) on ASIC1 a in HEK 293 cells (n = 4). Unpaired t-test, ***P < 0.0001.

[0084] Figure 31. ASIC1a inhibition by 3cR10ss (SEQ ID NO: 15 - Formula 6) prevents acidosis-induced mitochondrial energy failure, maintaining intracellular ATP levels in a dosedependent manner. ATP content was quantified by a luminescence assay after 24 h treatment under the following conditions: physiological pH 7.5, acidic pH 5.5, pH 5.5 + 3cR10ss 5 pM (P5), and pH 5.5 + 3cR10ss 20 pM (P20). Data represent mean ± SEM from three independent experiments, normalized to the pH 7.5 condition. * denotes P< 0.05 ** denotes P< 0.01; *** denotes P< 0.001; and **** denotes P< 0.0001, calculated using one-way ANOVA with a Tukey post-hoc test.

[0085] Figure 32. 3cR10ss (SEQ ID NO: 15 - Formula 6) selectively reduces mitochondrial and total reactive oxygen species (ROS) in mitoPRS neuronal cultures. A) Mitochondrial ROS levels measured by MitoSOX Red fluorescence in high- and low-mitoPRS neuronal cultures following 24 h treatment with (untreated) vehicle, 3cR10ss (10 μM), exenatide (10 μM) and semaglutide (10 μM). Positive control (pyocyanin at 10 μM) is not shown as data exceeds 5 units of relative fluorescent intensity / μM of protein, hence rendering the compound data indistinguishable; it is therefore not shown, in order to improve the clarity of data from the tested compounds. B) Total ROS levels measured using DCFH-DA in highland low-mitoPRS neuronal cultures following 24 h treatment with (untreated) vehicle and 3cR10ss (10 μM). Positive control (pyocyanin at 10 μM) is not shown for the same reasons as above. Only 3cR10ss significantly decreased mitochondrial ROS and shown statistical reduction of total ROS in mitoPRS neurons, while GLP-1 agonists showed no statistically significant effect. Data represent mean ± SEM from three independent biological replicates. * denotes P< 0.05 ** denotes P< 0.01; *** denotes P< 0.001; and **** denotes P< 0.0001, calculated using one-way ANOVA with a Tukey post-hoc test.

[0086] Detailed Description

[0087] Definitions

[0088] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

[0089] The section headings used herein are for organisational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

[0090] Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.

[0091] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognise. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts described herein to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0092] As used herein, the term "capable of when used with a verb, encompasses or means the action of the corresponding verb. For example, "capable of interacting" also means interacting, "capable of inhibiting" also means inhibits, "capable of binding" also means binds and "capable of specifically targeting..." also means specifically targets.

[0093] Numeric ranges are inclusive of the numbers defining the range. Thus, for example, ranges identified as “between” two end points or “from... to...”, i.e. ranges in the form “between x and y" or “from x to y”, should be understood to include those end points x and y. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0094] As used herein, the articles "a" and “an” may refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including", as well as otherforms, such as "includes" and "included", is not limiting.

[0095] “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Optionally, the term “about” shall be understood herein as plus or minus (±) 5%, further optionally ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, of the numerical value of the number with which it is being used.

[0096] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the disclosure.

[0097] As used herein the term "consisting essentially of refers to those elements required for a given disclosure. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that disclosure (i.e., inactive or non-immunogenic ingredients).

[0098] Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of and / or “consisting essentially of such features.

[0099] Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation, or the single letter abbreviation. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0100] The term “polypeptide", as used herein, includes polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and / or the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. The terms " peptide " and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0101] As used herein, the terms “natural amino acid” and “native amino acid” are used interchangeably to refer to any proteinogenic amino acid, i.e. to any amino acid that can be incorporated into protein during translation of mRNA. In eukaryotes there are 21 proteinogenic amino acids: Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Vai, and Sec.

[0102] As used herein, the term “unnatural amino acid” refers to a non-proteinogenic amino acid, i.e. to an amino acid that is not naturally encoded by the genome or incorporated into a protein during translation by cellular machinery. An unnatural amino acid may be any amino acid other than the 21 proteinogenic amino acids present in eukaryotes.

[0103] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0104] The polypeptides provided herein may be prepared by any means known in the art. For example, the polypeptides may be produced by standard solid-phase peptide synthesis (SPPS). Alternatively liquid-phase peptide synthesis, native chemical ligation, or recombinant protein expression may be employed.

[0105] The terms “decrease” "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. The terms "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" encompasses a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition (i.e., abrogation) as compared to a reference level. The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. The terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 25%, at least 50% as compared to a reference level, for example an increase of at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, at least about 95%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 250% or more compared with a reference level, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 2.5-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 1.5-fold and 10-fold or greater as compared to a reference level.

[0106] As used herein, polypeptides of the disclosure may be used to inhibit ASIC, e.g. ASIC1, and in particular ASIC1 a. As used herein, polypeptides of the disclosure may also be used to monitor the presence and activity of ASIC ex vivo and / or in vivo; e.g. ASIC1, and in particular ASIC1a. As used herein, polypeptides of the disclosure may also be used to treat and / or prevent ASIC-related disorders in patients in need thereof, e.g. ASIC1 -related disorders, and in particular ASIC1a-related disorders. As used herein, polypeptides of the disclosure may also be used to treat ASIC-related disorders in patients in need thereof, e.g. ASIC1 -related disorders and in particular ASIC1a-related disorders. As used herein, polypeptides of the disclosure may also be used to prevent ASIC-related disorders in patients in need thereof, e.g. ASIC1 -related disorders and in particular ASIC1a-related disorders.

[0107] As used herein “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds. Pharmaceutically acceptable salts retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For example, the term “pharmaceutically acceptable” may refer to salts, excipients, carriers, diluents, etc. approved by a regulatory agency of the Federal or a state government, or listed in the U. S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia.

[0108] As used herein “pharmaceutical composition” means a mixture of substances suitable for administering to a subject. For example, a pharmaceutical composition may comprise an oligomeric compound and a sterile aqueous solution. In certain embodiments, a pharmaceutical composition shows activity in free uptake assay in certain cell lines.

[0109] As used herein, “treating” means improving a subject’s disease or condition by administering a polypeptide described herein. In certain embodiments, treating a subject improves a symptom relative to the same symptom in the absence of the treatment. In certain embodiments, treatment reduces in the severity or frequency of a symptom, or delays the onset of a symptom, slows the progression of a symptom, or slows the severity or frequency of a symptom.

[0110] As used herein, “preventing” means inhibiting or delaying the onset or reoccurrence of a subject’s disease or condition (or a symptom thereof) by administering a polypeptide described herein. In some embodiments, “preventing” means preventing the onset or reoccurrence of subject’s disease or condition (or a symptom thereof) entirely. In some embodiments, “preventing” means either lessening the likelihood of the onset of a subject’s disease or condition (or a symptom thereof).

[0111] As used herein, “therapeutically effective amount” means an amount of a pharmaceutical agent or composition that has been observed to provide a therapeutic benefit to an animal. For example, a therapeutically effective amount may be observed to improve a symptom of a disease in an animal. Typically, a “therapeutically effective amount” means an amount of a pharmaceutical agent or composition that has been observed to provide a therapeutic benefit to a human. For example, a therapeutically effective amount may be observed to improve a symptom of a disease in a human.

[0112] The terms "individual,” "subject,” and "patient,” are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and / or therapy optimisation is desired. The mammal can be (without limitation) a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The individual, subject, or patient may be a human. An “individual” may be an adult, juvenile or infant. An “individual” may be male orfemale.

[0113] A "subject in need" of treatment for a particular condition can be an individual having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0114] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications or symptoms related to such a condition, and optionally, have already undergone treatment for a condition as defined herein or the one or more complications or symptoms related to said condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition as defined herein or one or more or symptoms or complications related to said condition. For example, a subject can be one who exhibits one or more risk factors for a condition, or one or more or symptoms or complications related to said condition or a subject who does not exhibit risk factors.

[0115] As used herein, the term “healthy individual” refers to an individual or group of individuals who are in a healthy state, e.g., individuals who have not shown any symptoms of the disease, have not been diagnosed with the disease and / or are not likely to develop the disease e.g., a thrombotic event. Optionally said healthy individual(s) is not on medication affecting haemostasis and has not been diagnosed with any other disease. The one or more healthy individuals may have a similar sex, age, and / or body mass index (BMI) as compared with the test individual. Application of standard statistical methods used in medicine permits determination of normal levels of expression in healthy individuals, and significant deviations from such normal levels.

[0116] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. All documents cited herein are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.

[0117] Acid-sensing ion channels (ASICs)

[0118] Acid-sensing ion channels (ASICs) are ligand-gated cation channels and members of the vast degenerin / epithelial sodium channel (DEG / ENaC) family. ASICs are activated by extracellular acidosis and are involved in many physiological and pathophysiological processes. Inhibiting these channels has shown significant efficacy in alleviating nociception, cancer, and ischemic stroke.

[0119] There are four genes of ASICs, namely ASIC1, ASIC2, ASIC3, and ASIC4, and these four genes encode at least six different subunits, ASIC1 a, ASIC1 b, ASIC2a, ASIC2b, ASIC3, and ASIC4. Functional ASICs can be homo-trimeric or hetero-trimeric. The functional homotrimeric channels can be formed by ASIC1 a, ASIC1 b, ASIC2a, and ASIC3, and these channels can be activated with different ranges of acidic pH, with different pH5o (pH of half-maximal activation). The pHso of ASIC1a is 6.45, then ASIC3 (pHso ~ 6.5), while ASIC2a is the least sensitive one with pH5o ~ 4.7. But this pH5o (4.7) for ASIC2a is unlikely to be achieved (not in the physiological pH range) in animal models. (Price, et al., 1996, Journal of Biological Chemistry, 271(14), pp.7879-7882; Waldmann, et al., 1997, Journal of Biological Chemistry, 272(34), pp.20975-20978; Waldmann, et al., 1997, Nature, 386(6621), pp.173-177; Chen, et al., 1998, Proceedings of the National Academy of Sciences, 95(17), pp.10240-10245; de Weille, et al., 1998, FEBS letters, 433(3), pp.257-260; Babinski, and Seguela, 1999, Journal of neurochemistry, 72(1), pp.51-57; Akopian, etal., 2000, Neuroreport, 11 (10), pp.2217-2222; Grunder, et al., 2000, Neuroreport, 11(8), pp.1607-1611; Bassler, et al., 2001, Journal of Biological Chemistry, 276(36), pp.33782-33787; Benson, et al., 2002, Proceedings of the National Academy of Sciences, 99(4), pp.2338-2343).

[0120] ASICs in pathology

[0121] ASICs are essential in chemo-sensing and mechano-sensing and are expressed in different body systems, including the cardiovascular system. ASICs are expressed in the skin, muscles, and gastrointestinal tract (GIT). ASIC1a is the most abundant in the CNS but is also expressed in other tissues, including the tongue, intestine, peripheral tissues, arteries, urinary bladder, and kidney. ASIC1a has been reported to modulate both the colonic mechanoreceptor and gastroesophageal mechanoreceptor. Knocking out the ASIC1a gene increased the activity of both mesenteric and Serosal afferents, as well as increasing the activity of both Mucosal and tension receptors (Price, etal., 2000, Nature, 407, pp.1007-1011; Page, etal., 2004, Gastroenterology, 127(6), pp.1739-1747; Page, etal., 2005, Gut, 54(10), pp.1408-1415).

[0122] Zha, et al., 2006, demonstrated that ASIC1 a maintains the density of dendritic spines in the postsynaptic terminals. Three years later, Zha, et al., 2009, Journal of Neuroscience, 29(26), pp.8438-8446 showed that ASIC2a channels could increase the localization of ASIC1a channels in the dendritic spines. They found this mechanism was achieved through an interaction between the ASIC2a channels and the PSD-95 (postsynaptic density-95) protein. Moreover, ASIC1a-dependent acidosis has been shown to significantly decrease the length and density of dendritic spines (Jing, et al., 2012, Journal of Neuroscience, 32(12), pp.4080-4091).

[0123] Recently, ASICs became a receptor of interest in nociception. ASICs are expressed in the nociceptive sensory neurons, and ASIC1a, ASIC2b, and ASIC3 are overexpressed in inflammation. Several studies demonstrated that nociception could be achieved by activating ASICs, specifically ASIC1a and ASIC3. This activation has been achieved using selective ASICs activators like GMQ and MitTx (Bohlen, et al., 2011, Nature, 479(7373), pp.410-414). Moreover, several studies showed that blocking ASICs by different selective inhibitors can result in anti-nociception. Several studies showed the implication of ASIC3 in pain, allodynia and fibromyalgia (Wemmie, et al., 2013, Nature Reviews Neuroscience, 14(7), pp.461-471; Liu, etal., 2015, Translational Neurodegeneration, 4(1), pp.1-8).

[0124] The involvement of membrane proteins in cancer metastases has recently become an important topic. ASIC1a is involved in maintaining the acidic tumour microenvironment to pH 6.5. In 2017, Jin, etal., showed that ASIC1a is significantly involved in liver cancer proliferation and tumorigenicity in both in vitro and in vivo models. They also showed that knocking out the ASIC1a gene in liver cancer cells inhibits LEF-TCF (lymphoid enhancer factor / T cell factor), resulting in cell cycle arrest. Moreover, the ASIC1a gene knockout significantly suppressed the / 7-catenin / LEF-TCF, one of the critical pathways to cancer cell migration, adhesion, and proliferation. A recent study by Yang, et al., 2020, showed that ASIC1a is overexpressed in breast cancer and enhance cancer cell migration, invasion, and proliferation. They also showed that either inhibition of the ASIC1a by the selective inhibitor “PcTx1” or downregulation of the ASIC1a gene could significantly decrease tumour migration, invasion, and proliferation. Another recent study showed that inhibiting ASIC1a in glioma cells by mambalgin-2 toxin can possess an antitumor effect via cell cycle arrest and apoptosis mechanisms (Bychkov, etal., 2020, Cancers, 12(7), p.1837). Tissue acidosis is a significant challenge in ischemic stroke and one of the leading causes of brain neuronal injury. Tissue acidosis results from substantial elevation in brain lactate concentrations, at which extracellular pH has been reported to drop to pH 6. This drop in the extracellular pH is enough to activate ASICs, leading to lower Ca2+permeability and neuronal injury (Toth, et al., 2020, Biology, 9(12), p.460). Moreover, Xiong et al., 2004, Cell, 118, pp.687-698, showed that acidosis can induce Ca2+-dependent neuronal injury, which ASIC blockers inhibit.

[0125] ASIC1 structure

[0126] The structure of ASIC1 has been determined using X-ray crystallography. The X-ray crystallography structure of chicken ASIC1 was determined at a resolution of 1.9 A (PDB ID: 2QTS) (Jasti et al., 2007, Nature, 449(7160), pp.316-323), and at a resolution of 3.0 A (PDB ID: 3S3X) (Dawson et al., 2018, Nature communications, 3(1), pp.1-8). Both structures are homotrimers, with each subunit consisting of two transmembrane helices (TM1 and TM2) and a large extracellular domain (ECD) that forms the ion-conducting pore.

[0127] ASIC inhibitors

[0128] The present invention disclosure relates to polypeptides which specifically target ASICs, e.g. ASIC1 and in particular ASIC1 a. The polypeptides of the disclosure may inhibit specifically ASICs, e.g. ASIC1 and in particular ASIC1 a.

[0129] Polypeptides of the disclosure typically bind to the extracellular domain of ASICs (e.g. ASIC1 and in particular ASIC1a), or a subdomain thereof. Optionally, polypeptides of the disclosure typically bind to the acidic pocket as described in the PcTx1 (SEQ ID NO: 17) interactions with the ASIC1 a acidic pocket (Baconguis, and Gouaux, Nature, vol. 489, pp. 400-405, 2012; PDB IDs: 4FZ0 and 4FZ1).

[0130] Polypeptides

[0131] Provided herein are polypeptides which bind to and inhibits ASICs, e.g. ASIC1 and in particular ASIC1a. All references herein to a polypeptide also refer equally and without reservation to pharmaceutically acceptable salts of said polypeptide. The inhibitory activity of the polypeptides of the disclosure may be assessed through methods as described herein, for example in the Example section.

[0132] As described herein, it is the first time that a specific polypeptide motif has been demonstrated to bind specifically to ASICs, e.g. ASIC1 and in particular ASIC1a. As exemplified herein, it has been demonstrated that a polypeptide of the disclosure, specifically binds and inhibit ASICs, e.g. ASIC1 and in particular ASIC1a. The present disclosure provides polypeptides which specifically bind to a defined acidic pocket in ASICs (Baconguis, and Gouaux, Nature, vol. 489, pp. 400-405, 2012; PDB IDs: 4FZ0 and 4FZ1), e.g. ASIC1 and in particular ASIC1a, such that the polypeptide motif of the present disclosure would be applicable to larger structures containing several of the polypeptide motifs of the disclosure.

[0133] Binding of a polypeptide of the disclosure to ASICs, e.g. ASIC1 and in particular ASIC1a, and identification of contact residues may be determined using any appropriate technique, such as X-ray crystallography and / or surface plasmon resonance (SPR). Suitable techniques and protocols are known in the art and may be selected by one of ordinary skill without undue burden.

[0134] Patch-clamp current traces may be utilised to show the effect of the cyclic polyarginine peptide on ASICs, e.g. ASIC1 and in particular ASIC1a, such as hASIC1a (human ASIC1a) using methods described herein or found in the common general knowledge of the person skilled in the art, which may demonstrate the inhibition efficacy of the peptide.

[0135] The polypeptides of the disclosure may comprise unnatural amino acids such as D-amino acids, N-methyl amino acid, alpha-methyl amino acids, beta-amino acids or any combination therein. Othe unnatural amino acids are contemplated by the disclosure including modified side chain amino acids. Optionally an unnatural amino acid may be a cysteine derivative amino acid selected from homocysteine, N-methyl cysteine, D-cysteine, alphamethyl cysteine, or penicillamine.

[0136] The “arginine content” of the polypeptide of the disclosure as defined herein refers to the mass of the arginine residues, at physiological pH, comprised in the polypeptide divided by the mass of the polypeptide. It is referred herein in % w / w, which is a term understood by the skilled person in the art. For the avoidance of doubt, it is noted that in the context of the “arginine content” the mass of the polypeptide does not include the mass of any counterions that may be present in a pharmaceutically acceptable salt thereof. The polyarginine polypeptide of the disclosure comprises at least 3 arginine residues as defined in the SEQ ID NO:1 motif. The polyarginine polypeptide of the disclosure may comprise at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39 or at least 40 arginine residues. The polyarginine polypeptide of the disclosure may comprise 50 or fewer, 45 or fewer, 40 or fewer, 39 or fewer, 38 or fewer, 37 or fewer, 36 or fewer, 35 or fewer, 34 or fewer, 33 or fewer, 32 or fewer, 31 or fewer, 30 or fewer, 29 or fewer, 28 or fewer, 27 or fewer, 26 or fewer, 25 or fewer, 24 or fewer, 23 or fewer, 22 or fewer, 21 or fewer, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, or 9 or fewer arginine residues. In embodiments the polyarginine polypeptide of the disclosure may comprise 50 or fewer arginine residues. In embodiments the polyarginine polypeptide of the disclosure may comprise 30 or fewer arginine residues. Accordingly, the polyarginine polypeptide of the disclosure may comprise between 3 and 9, between 3 and 10, between 3 and 12, between 3 and 15, between 3 and 18, between 3 and 20, between 3 and 21, between 3 and 24, between 3 and 25, between 3 and 27, between 3 and 30, between 3 and 33, between 3 and 35, between 3 and 36, between 3 and 39, between 3 and 40, between 3 and 42, between 3 and 45, between 3 and 48, or between 3 and 50 arginine residues; optionally the polyarginine polypeptide of the disclosure may comprise between 3 and 50 arginine residues; further optionally the polyarginine polypeptide of the disclosure may comprise between 3 and 30 arginine residues.

[0137] Optionally, the polyarginine polypeptide of the disclosure may comprise between 4 and 10, between 4 and 15, between 4 and 20, between 4 and 25, between 4 and 30, between 4 and 35, between 4 and 40 or between 4 and 50 arginine residues. Optionally, the polyarginine polypeptide of the disclosure may comprise between 5 and 10, between 5 and 15, between 5 and 20, between 5 and 25, between 5 and 30, between 5 and 35, between 5 and 40 or between 5 and 50 arginine residues. Optionally, the polyarginine polypeptide of the disclosure may comprise between 6 and 10, between 6 and 15, between 6 and 20, between 6 and 25, between 6 and 30, between 6 and 35, between 6 and 40 or between 6 and 50 arginine residues. Optionally, the polyarginine polypeptide of the disclosure may comprise between 7 and 10, between 7 and 15, between 7 and 20, between 7 and 25, between 7 and 30, between 7 and 35, between 7 and 40 or between 7 and 50 arginine residues. Optionally, the polyarginine polypeptide of the disclosure may comprise between 8 and 10, between 8 and 15, between 8 and 20, between 8 and 25, between 8 and 30, between 8 and 35, between 8 and 40 or between 8 and 50 arginine residues. Optionally, the polyarginine polypeptide of the disclosure may comprise between 9 and 10, between 9 and 15, between 9 and 20, between 9 and 25, between 9 and 30, between 9 and 35, between 9 and 40 or between 9 and 50 arginine residues. Optionally, the polyarginine polypeptide of the disclosure may comprise between 10 and 15, between 10 and 20, between 10 and 25, between 10 and 30, between 10 and 35, between 10 and 40 or between 10 and 50 arginine residues.

[0138] The arginine content of the polyarginine polypeptide of the disclosure may therefore be at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w or at least 95% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 40% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 50% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 60% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 70% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 75% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 80% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 85% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 90% w / w. In embodiments the arginine content of the polyarginine polypeptide of the disclosure may be at least 95% w / w.

[0139] Arginine finger

[0140] The crystal structures of chicken ASIC1a in complex with each of two potent toxins (PcTx1 and Mamba-1) that inhibit ASIC1a have previously been identified (Baconguis, and Gouaux, Nature, vol. 489, pp. 400-405, 2012; PDB IDs: 4FZ0 and 4FZ1). The present inventor has now surprisingly identified that enhanced ASIC inhibitory activity compared to PcTx1 can be achieved by the provision of a polypeptide comprising an “arginine finger” motif ( / .e. a motif as defined herein according to any of SEQ ID NOs: 1 to 4, 15 and 18). This motif is smaller, more rigid and more constrained than the PcTx1 motif (SEQ ID NO: 17).

[0141] The disclosure therefore provides polyarginine polypeptides comprising at least one motif X(1) - X(2) - X(3) - RRR - X(4) - X(5) - X(6) (SEQ ID NO: 1) wherein (a) X(1) and X(6) are amino acid residues other than arginine and X(1) is stapled to X(6); (b) X(2), X(3), X(4) and X(5) are, independently, either absent or amino acid residues other than arginine; and (c) the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof. The polyarginine polypeptide of the disclosure may have one, two, three or four of X(2), X(3), X(4) and X(5) present in SEQ ID NO: 1. By way of non-limiting examples, the polyarginine polypeptide of the disclosure may be X(1) - X(2) -X(3) - RRR - X(4) - X(5) - X(6), X(1) - X(3) - RRR - X(4) - X(5) - X(6), X(1) - X(2) - RRR - X(4) - X(5) - X(6), X(1) - X(2) - X(3) - RRR - X(5) - X(6), X(1) - X(2) - X(3) - RRR - X(4) - X(6), X(1) - RRR - X(4) - X(5) - X(6), X(1) - X(3) - RRR - X(5) - X(6), X(1) - X(3) - RRR - X(4) - X(6), X(1) - X(2) - RRR - X(5) - X(6), X(1) - X(2) - RRR - X(4) - X(6), X(1) - X(2) - X(3) - RRR - X(6), X(1 ) - RRR - X(5) - X(6), X(1 ) - RRR - X(4) - X(6), X(1 ) - X(2) - RRR - X(6), X(1)- X(3) - RRR - X(6), or X(1) - RRR - X(6); wherein (a) X(1) and X(6) are amino acid residues other than arginine and X(1) is stapled to X(6); (b) X(2), X(3), X(4) and X(5) are, independently amino acid residues other than arginine; and (c) the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof; optionally the polyarginine peptide of the disclosure comprises X(1) - RRR - X(6); wherein (a) X(1) and X(6) are amino acid residues other than arginine and X(1) is stapled to X(6); and (b) the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof. X(1) and X(6) may be cysteines.

[0142] Stapling

[0143] The terms “staple”, “stapling” and “stapled” as used herein refer to the covalent bonding of 2 or more residues either directly or indirectly. In the context of amino acids of the polypeptide of the disclosure, the stapling may be direct or indirect between 2 or more amino acid side-chains or backbone; optionally the stapling may be direct or indirect between 2 or more amino acid side-chains.

[0144] Direct stapling refers to the covalent bonding of an atom comprised in one of the two residues to an atom comprised in the other residue; optionally between an atom comprised in the side chain of an amino acid to an atom comprised in the side chain of another amino acid. For instance, direct stapling may refer to thiol oxidation (disulfide bridge formation) of two cysteine (C) residues, lactam formation (either by amide condensation of two amino acid side chains or by head-to-tail cyclisation, i.e. formation of an amide bond between the N-terminal and the C-terminal residues of the peptide defined with respect to the peptide sequence prior to cyclization), backbone cyclisation, or thioether formation. By way of a non-limiting example, direct stapling may be the formation of an amide bond between a glutamate side chain and a lysine side chain or the formation of disulfide bridge between two cysteines. In the present disclosure the stapling of two amino acids may in some embodiments be the formation of a disulfide bridge between two cysteines (i.e. the stapling moiety is a disulfide bridge).

[0145] Indirect stapling refers to the covalent bonding of an atom comprised in each of the two or more residues to atoms present in a stapling moiety. A stapling moiety as used herein may have several reactive sites, such as 2 (bidentate), 3 (tridentate), 4 (tetradentate) or 5 (pentadentate). Indirect stapling in the present disclosure also contemplates the use of several stapling moieties reacting between each other to form a covalent bond between an atom comprised in one of the two or more residues to an atom comprised in the other residue. In the present disclosure indirect stapling may be between two cysteines. By way of non-limiting example, any stapling moiety known in the art may be used, such as:

[0146]

[0147] Examples of stapling chemistry are present in the art and at the disposal of the person skilled in the art. Examples of peptide stapling techniques known to the person skilled in the art include, but are not limited to, those described in Lau et al., Chemical Society Reviews, vol. 44, pp. 91-102, 2015, “Peptide stapling techniques based on different macrocyclization chemistries”. Byway of non-limiting examples, stapling of the polyarginine polypeptides of the disclosure may be achieved through any chemical structural constraints, such as disulfide bridge, backbone cyclization, lactam bridges (amide bond), lactone bridges (ester bond), click chemistry (such as copper-catalyzed azide-alkyne cycloaddition), olefin synthesis (such as Grubbs reaction), Diels-alder 4+2 cycloaddition, thioether formation, Micheal addition, reductive amination, or synthetic stapling moieties as described herein.

[0148] The cyclic polyarginine polypeptides of the disclosure may therefore comprise C(1) -X(2) - X(3) - RRR - X(4) - X(5) - C(6) (SEQ ID NO: 2) wherein (a) C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof; optionally wherein the stapling moiety is a disulfide bridge; (b) X(2), X(3), X(4) and X(5) are, independently, either absent or amino acid residues other than arginine; and (c) the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof. The cyclic polyarginine polypeptides of the disclosure may have one, two, three or four of X(2), X(3), X(4) and X(5) present in SEQ ID NO: 2. Byway of non-limiting examples, the cyclic polyarginine polypeptide of the disclosure may be C(1) - X(2) - X(3) - RRR - X(4) - X(5) - C(6), C(1) - X(3) - RRR -X(4) - X(5) - C(6), C(1) - X(2) - RRR - X(4) - X(5) - C(6), C(1) - X(2) - X(3) - RRR - X(5) - C(6), C(1) - X(2) - X(3) - RRR - X(4) - C(6), C(1) - RRR - X(4) - X(5) - C(6), C(1) - X(3) - RRR - X(5) - C(6), C(1) - X(3) - RRR - X(4) - C(6), C(1) - X(2) - RRR - X(5) - C(6), C(1) - X(2) - RRR - X(4) - C(6), C(1) - X(2) - X(3) - RRR - C(6), C(1) - RRR - X(5) - C(6), C(1) - RRR - X(4) - C(6), C(1) - X(2) - RRR - C(6), C(1)- X(3) - RRR - C(6), or C(1) - RRR -C(6); wherein (a) C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof; optionally wherein the stapling moiety is a disulfide bridge; (b) X(2), X(3), X(4) and X(5) are, independently, amino acid residues other than arginine; and (c) the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof.

[0149] In particular, the cyclic polyarginine polypeptides of the disclosure may comprise C(1) - RRR - C(6) (SEQ ID NO: 3); (a) wherein C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof; optionally wherein the stapling moiety is a disulfide bridge and (b) the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof.

[0150] Further, the cyclic polyarginine polypeptides of the disclosure may comprise C(1) -RRR - C(6) (SEQ ID NO: 4 - Formula 5); (a) wherein C(1) and C(6) form a disulfide bridge and (b) the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof.

[0151]

[0152] Formula 5

[0153] The cyclic polyarginine polypeptides of the present disclosure may comprise a plurality of copies of SEQ ID NO: 1, 2, 3 or 4 or any combination thereof. The cyclic polyarginine polypeptides of the disclosure may comprise at least 2, at least 3, at least 4, at least 5 or at least 6 copies of SEQ ID NO: 1, 2, 3 or 4 or any combination thereof. Typically, the cyclic polyarginine polypeptides of the disclosure comprises at least 2 copies of SEQ ID NO: 1, 2, 3 or 4 or any combination thereof.

[0154] The cyclic polyarginine polypeptides of the present disclosure may comprise a plurality of copies of SEQ ID NO: 1 or any combination thereof. The cyclic polyarginine polypeptides of the disclosure may comprise at least 2, at least 3, at least 4, at least 5 or at least 6 copies of SEQ ID NO: 1 or any combination thereof. Typically, the cyclic polyarginine polypeptide of the disclosure comprises at least 2 copies of SEQ ID NO: 1 or any combination thereof.

[0155] The cyclic polyarginine polypeptides of the present disclosure may comprise a plurality of copies of SEQ ID NO: 2 or any combination thereof. The cyclic polyarginine polypeptides of the disclosure may comprise at least 2, at least 3, at least 4, at least 5 or at least 6 copies of SEQ ID NO: 2 or any combination thereof. Typically, the cyclic polyarginine polypeptide of the disclosure comprises at least 2 copies of SEQ ID NO: 2 or any combination thereof.

[0156] The cyclic polyarginine polypeptides of the present disclosure may comprise a plurality of copies of SEQ ID NO: 3 or any combination thereof. The cyclic polyarginine polypeptides of the disclosure may comprise at least 2, at least 3, at least 4, at least 5 or at least 6 copies of SEQ ID NO: 3 or any combination thereof. Typically, the cyclic polyarginine polypeptide of the disclosure comprises at least 2 copies of SEQ ID NO: 3 or any combination thereof.

[0157] The cyclic polyarginine polypeptides of the present disclosure may comprise a plurality of copies of SEQ ID NO: 4 or any combination thereof. The cyclic polyarginine polypeptides of the disclosure may comprise at least 2, at least 3, at least 4, at least 5 or at least 6 copies of SEQ ID NO: 4 or any combination thereof. Typically, the cyclic polyarginine polypeptide of the disclosure comprises at least 2 copies of SEQ ID NO: 4 or any combination thereof. Optionally the cyclic polyarginine polypeptide of the disclosure consists of the polypeptide sequence SEQ ID NO: 15 (Figure 8 - Formula 6). In some aspects, the cyclic polyarginine polypeptide of the disclosure consists of the polypeptide sequence SEQ ID NO: 19 (Formula 7).

[0158]

[0159]

[0160] Formula 7

[0161] Cyclisation

[0162] The polyarginine polypeptides of the disclosure are cyclic and as such are referred to as cyclic polyarginine polypeptides.

[0163] As described herein, cyclic polyarginine polypeptides of the disclosure may be monocyclic, bicyclic, tricyclic, tetra cyclic, pentacyclic, hexacyclic, heptacyclic, octacyclic, nonacyclic or decacyclic, further optionally monocyclic, bicyclic or tricyclic. The disclosure of a cyclic polypeptide herein further discloses the unreacted linear polypeptide prior to its cyclisation. It is to be understood, for example, that the disclosure of a cyclic peptide of the disclosure cyclised through a disulfide bridge also discloses the linear peptide with reduced cysteines. Similarly, it is to be understood, for example, that the disclosure of a cyclic peptide of the disclosure cyclised through head-to-tail cyclisation also discloses the linear peptide with unreacted N- and C-termini.

[0164] As described herein, polypeptides of the disclosure typically cyclise by the formation of a covalent bond between X (1) and X (6), wherein X (1) and X (6) are amino acid residues other than arginine. The covalent bond between X (1) and X (6) may be done through stapling as described herein. Typically, X (1) and X (6) are each independently cysteine or cysteine derivatives and either form a disulfide bridge or are stapled via a bidentate or tridentate stapling moiety as described herein. This forms a monocyclic structure, reducing the flexibility of the arginine finger motif and hence typically increasing the stability of the peptide and its ASICs activity.

[0165] As described herein, polypeptides of the disclosure typically cyclise by the formation of a disulphide bond between X (1) and X (6), wherein X (1) and X (6) are each independently cysteine or cysteine derivatives, as described herein. Optionally, polypeptides of the disclosure cyclise by the formation of a disulphide bond between C(1) and C(6). This forms a monocyclic structure, reducing the flexibility of the arginine finger motif and hence typically increasing the stability of the peptide and its ASICs activity.

[0166] Any one of polyarginine polypeptides of the present disclosure may be head-to-tail cyclised. Head-to-tail cyclisation typically involves the formation of a lactam bond (cyclic amide bond) between the N-terminal amino acid residue of the polypeptide and the C-terminal amino acid residue of the polypeptide. Thus, a head-to-tail cyclised polypeptide may be referred to interchangeably as a cyclised polypeptide comprising a lactam bond between the N-terminal amino acid residue of the polypeptide and the C-terminal amino acid residue of the polypeptide.

[0167] As described herein, polypeptides of the disclosure are cyclic, optionally monocyclic or bicyclic. The disclosure of a cyclic polypeptide herein further discloses the unreacted linear polypeptide prior to its cyclisation. It is to be understood, for example, that the disclosure of a cyclic peptide of the disclosure cyclised through head-to-tail cyclisation also discloses the linear peptide with unreacted N- and C-termini.

[0168] Head-to-tail cyclisation is a known technique in the field of polypeptide therapeutics, and appropriate protocols and reagents are well-known in the art and exemplified herein.

[0169] To facilitate head-to-tail cyclisation, a polypeptide of the disclosure may have a spacer (referred herein interchangeably as linker) added to the N- and / or C-terminus of the polypeptide, typically to the C-terminus. The spacer may be selected from Serine, Glycine, Serine-Glycine, Alanine, aminocaproic acid (Ahx), sarcosine (Sar), gamma glutamic acid (GammaE), [2-(2-aminoethoxy)ethoxy]acetic acid (Ado), Polyethylene glycol (PEG) repetition and / or combination thereof. By way of non-limiting example, the spacer may be a GS based linker such as GSG, GSSS, SG or a tri-Ala linker AAA. The resulting peptides may undergo head-to-tail cyclisation.

[0170] In stark contrast to prior research on linear peptides such as those described above, the present invention relates to cyclic polyarginine peptides that are structurally complex and possess a rigid, finger-like architecture, which selectively and potently inhibits ASIC ion channels (e.g. ASIC1, such as ASIC1 a) in the extracellular space. This novel topology affords receptor specificity and selectivity for ASIC (e.g. ASIC1, such as ASIC1a), enhanced pharmacodynamic stability, rapid onset of action (within a millisecond timeframe, e.g. at acidic pH), and sustained inhibitory action. These are features that were entirely absent in the simple, linear peptides outlined in previous research, which exert their neuroprotective effects through intracellular mechanisms. The cyclic polyarginine peptides of the present invention therefore represent a significant advancement in mechanism and design compared to these earlier studies.

[0171] Furthermore, in contrast to e.g. Meloni etal., who concluded that there is no significant difference in the activity of short linear versus cyclic peptides, the present inventor has found that the pharmacodynamics of the cyclic polyarginine peptides of the invention exhibit entirely distinct properties compared to short linear peptides. In addition, the cyclic polyarginine peptides of the present invention also possess features similar to larger toxins that facilitate a gradual recovery of the receptor. This phenomenon illustrates a rapid effect coupled with sustained action, culminating in a wide therapeutic window, in contrast to long-chain linear peptides such as R18 and Nerinetide which exhibit greater sensitivity to conditions such as concentration and pH.

[0172] The peptides of the present invention also differ fundamentally to cyclic structures described in previous studies such as those of Lian etal. and Xu etal, which were specifically designed for intracellular enzyme inhibition. In contrast the cyclic polyarginine peptides of the present invention are single-domain, highly arginine-dense structures with finger-shaped macrostructures specifically designed to directly and swiftly inhibit extracellular ASIC (e.g. ASIC1, such as ASIC1 a) ion channels and the binding site located in the acidic pocket of these channels. The peptides of the present invention do not depend on CPP motifs or chemical linkers for constraint, delivery or cellular uptake, as their polycationic, novel, and unique fingerlike structure inherently facilitates both cell penetration and channel blockade and merges target engagement, proteolytic stability, and cell-penetrating capacity, attributable to their rigid, arginine-dense topology, eliminating the need for non-natural amino acids or auxiliary transport motifs.

[0173] Thus, the ASIC-targeting (e.g. ASIC1, such as ASIC1a) peptides of the present invention a first-in-class therapeutic modality that is distinct from strategies previously employed.

[0174] Labelling

[0175] Cyclic polyarginine polypeptides of the disclosure may be labelled with a detectable or functional label. A detectable label as referred to herein may be any label which produces or can be induced to produce a signal, including but not limited to fluorescers, chemiluminescers (e.g., horseradish peroxidase), coloured labels (e.g. latex [blue] or colloidal gold [red]), radiolabels, enzymes, photosensitisers and magnetic labels. The amount of label bound at a surface, e.g., a surface of a capillary bore, may therefore be detected and / or measured by detecting fluorescence or luminescence, colour, radioactivity, enzyme activity, light absorbance or changes in magnetic field. Detectable labels may be attached to polypeptides of the disclosure using conventional chemistry. Optionally, a detectable label is a label detectable by optical interrogation, e.g., with a digital camera or flatbed scanner. Labels that can be detected by optical interrogation include fluorescers, chemiluminescers and coloured labels. The mechanism by which a signal can be generated for optical detection includes (but is not necessarily limited to): light absorption, light scattering, light diffraction, light reflection, fluorescence or luminescence.

[0176] Suitable labels include, by way of illustration and not limitation:

[0177] a. enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase, e.g., horseradish peroxidase;

[0178] b. dyes;

[0179] c. fluorescent labels, such as fluorescein and its derivatives, fluorochrome, rhodamine compounds and derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine;

[0180] d. fluorophores such as lanthanide cryptates and chelates;

[0181] e. chemoluminescent labels, such as isoluminol, luminol and the dioxetanes;

[0182] f. bio-luminescent labels, such as luciferase and luciferin;

[0183] g. sensitizers;

[0184] h. coenzymes;

[0185] i. enzyme substrates;

[0186] j. radiolabels, such as bromine77, carbon14, cobalt57, fluorine8, gallium67, gallium68, hydrogen3(tritium), indium111, indium113, iodine123, iodine125, iodine126, iodine131, iodine133, mercury107, mercury203, phosphorous32, rhenium99, rhenium101, rhenium105, ruthenium95, ruthenium97, ruthenium103, ruthenium105, scandium47, selenium75, sulphur35, technetium99, technetium99, tellurium121, tellurium122, tellurium125, thulium165, thulium167, thulium168, yttrium199;

[0187] k. paramagnetic labels, such as nitroxide spin labels, e.g. methanethiosulfonate spin label (MTSSL), or lanthanide-chelating tags;

[0188] l. particles, such as latex or carbon particles;

[0189] m. contrast agents.

[0190] Examples of paramagnetic labels suitable for use with the cyclic polyarginine polypeptides of the disclosure include, but are not limited to, those described in e.g. Shah et al., Current Opinion in Chemical Biology, vol. 84, February 2025, p. 102564. Other suitable paramagnetic labels are known to those skilled in the art.

[0191] The detectable label may be covalently attached to the cyclic polyarginine polypeptide of the disclosure via a linker, spacer and / or anchor group, e.g. a cleavable linker, as described herein. Linker, spacer and / or anchor group as described herein for the purpose of labelling the cyclic polyarginine polypeptide of the disclosure may be attached at the N-terminus, the C-terminus and / or to any amino acid of the polypeptide.

[0192] Anchor group or cleavable linkers as used herein refer to group covalently linked to the cyclic polyarginine polypeptide of the disclosure at the N-terminus, the C-terminus and / or to any amino acid of the polypeptide. Anchor group or cleavable linkers may further be susceptible to cleavage under specific conditions. Such chemical groups susceptible to cleavage under specific conditions are well known in the art and are at the disposal of the skilled person in the art.

[0193] This labelling may facilitate imaging, diagnosis, or monitoring of the treatment of ASIC-related diseases.

[0194] Pharmaceutical compositions

[0195] Also provided by the present disclosure are pharmaceutical compositions comprising a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein.

[0196] Typically, the pharmaceutical composition further comprises a pharmaceutically acceptable diluent or carrier. The pharmaceutically acceptable diluent may be water or saline. A pharmaceutical composition may comprise a sterile saline solution and one or more polypeptide or pharmaceutically acceptable salt thereof as described herein. The sterile saline is optionally pharmaceutical grade saline. The saline may be phosphate-buffered saline (PBS), optionally sterile PBS.

[0197] A pharmaceutical composition may comprise or consist of one or more cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein and sterile water. Optionally the sterile water is pharmaceutical grade water, e.g., water for injection.

[0198] A pharmaceutical composition may comprise one or more cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein and one or more excipients. Excipients may be selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

[0199] In certain instances, a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein may be admixed with pharmaceutically acceptable active and / or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

[0200] Pharmaceutical compositions comprising a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein encompass any pharmaceutically acceptable salts of the cyclic polyarginine polypeptide, esters of the cyclic polyarginine polypeptide, or salts of such esters. Pharmaceutical compositions comprising one or more cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein are typically capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of cyclic polyarginine polypeptides, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0201] A pharmaceutical composition may comprise a co-solvent system. Such co-solvent systems may comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.

[0202] A pharmaceutical composition may be prepared for administration via a route selected from the group consisting of oral, parenteral, subcutaneous, intravenous, intramuscular, intranasal, transdermal, and a combination thereof. In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain instances, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.

[0203] A cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein may be in aqueous solution with sodium. A cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein may be in aqueous solution with potassium. A cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein may be in PBS. A cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof as described herein may be in water. The pH of the solution may be adjusted with NaOH and / or HCI to achieve a desired pH.

[0204] The pharmaceutical compositions of the disclosure may be formulated as a tablet, a capsule, a solution, a suspension, an emulsion, a gel or a combination thereof and comprising appropriate formulation agents as described herein.

[0205] The pharmaceutical compositions of the disclosure may be sterile. The pharmaceutical compositions of the disclosure may be pyrogen-free. The pH of the pharmaceutical composition may be particularly relevant in the context of arginine rich polypeptides like the polypeptides of the disclosure. Typically, the pharmaceutical compositions of the disclosure may have a pH between about 4.0 and about 8.0. The pharmaceutical compositions of the disclosure may have a pH of at least about 4.0, at least about 5.0, at least about 6.0 or at least about 7.0, optionally the pharmaceutical compositions of the disclosure may have a pH of at least about 4.0. The pharmaceutical compositions of the disclosure may have a pH of about 5.0 or less, about 6.0 or less, about 7.0 or less or about 8.0 or less, optionally the pharmaceutical compositions of the disclosure may have a pH of about 8.0 or less. Typically, the pharmaceutical compositions of the disclosure may have a pH of about 7.4.

[0206] Methods of treatment and therapeutic uses

[0207] Any references of method of treatment, method of therapy, compound for use in therapy or use of compound in the manufacture of a medicament for therapy are to be used interchangeably.

[0208] The disclosure provides a method of treatment comprising administering to a subject, an effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.

[0209] The disclosure provides a method of treatment comprising administering to a subject suffering from an ASICs-related disease, an effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.

[0210] The disclosure provides a method of treatment comprising administering to a subject suffering from an ASIC1a-related disease, an effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.

[0211] The disclosure also provides a method of treating or preventing an ASICs-related disease, comprising administering to a subject having an ASICs-related disease a therapeutically effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, ora pharmaceutical composition as described herein; thereby treating or preventing the ASICs-related disease. Thus, the disclosure provides a method of treating or preventing neurological disorders, neurodegenerative diseases, psychological disorders, sexual disorders, cardiovascular diseases, respiratory diseases, inflammatory diseases, renal diseases, gastrointestinal diseases, metabolic disorders, cancer, and addiction, e.g. smoking addiction. The neurological disorder may be selected from the group consisting of stroke, ischemia, traumatic brain injury, spinal cord injury, neuropathic pain, and epilepsy. The neurodegenerative disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis. The psychological disorder is selected from the group consisting of anxiety, depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), and addiction. The cardiovascular disease may be selected from the group consisting of myocardial infarction, heart failure, arrhythmia, and hypertension. The respiratory disease may be selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS). The inflammatory disease may be selected from the group consisting of rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, and sepsis. The renal disease may be selected from the group consisting of acute kidney injury, chronic kidney disease, and renal fibrosis. The gastrointestinal disease may be selected from the group consisting of gastroesophageal reflux disease (GERD), peptic ulcer disease, and irritable bowel syndrome (IBS). The metabolic disorder may be selected from the group consisting of diabetes, obesity, and metabolic syndrome. The cancer may be selected from the group consisting of glioblastoma, breast cancer, lung cancer, colorectal cancer, and prostate cancer. The addiction may be smoking addiction. Said method may prevent or slow disease progression. The administering may comprise or consist of oral, parenteral, subcutaneous, intravenous, intramuscular, intranasal and / or transdermal administration.

[0212] The disclosure also provides a method of treating or preventing an ASICs-related disease, comprising administering to a subject having an ASICs-related disease a therapeutically effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, ora pharmaceutical composition as described herein; thereby treating or preventing the ASICs-related disease. Thus, the disclosure provides a method of treating or preventing acidosis, optionally ASICs-related acidosis (e.g. metabolic acidosis) or an acidosis-related (e.g. metabolic acidosis-related) condition such as a neurodegenerative disease (e.g. Parkinson’s disease), renal failure, chronic kidney disease (abbreviated as “CKD”), acute kidney injury, Addison’s disease, or diabetes.

[0213] The disclosure also provides a method of treating an ASICs-related disease, comprising administering to a subject having an ASICs-related disease a therapeutically effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein; thereby treating the ASICs-related disease. Thus, the disclosure provides a method of treating neurological disorders, neurodegenerative diseases, psychological disorders, sexual disorders, cardiovascular diseases, respiratory diseases, inflammatory diseases, renal diseases, gastrointestinal diseases, metabolic disorders, cancer, and addiction, e.g. smoking addiction. The neurological disorder may be selected from the group consisting of stroke, ischemia, traumatic brain injury, spinal cord injury, neuropathic pain, and epilepsy. The neurodegenerative disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis. The psychological disorder is selected from the group consisting of anxiety, depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), and addiction. The cardiovascular disease may be selected from the group consisting of myocardial infarction, heart failure, arrhythmia, and hypertension. The respiratory disease may be selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS). The inflammatory disease may be selected from the group consisting of rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, and sepsis. The renal disease may be selected from the group consisting of acute kidney injury, chronic kidney disease, and renal fibrosis. The gastrointestinal disease may be selected from the group consisting of gastroesophageal reflux disease (GERD), peptic ulcer disease, and irritable bowel syndrome (IBS). The metabolic disorder may be selected from the group consisting of diabetes, obesity, and metabolic syndrome. The cancer may be selected from the group consisting of glioblastoma, breast cancer, lung cancer, colorectal cancer, and prostate cancer. The addiction may be smoking addiction. Said method may prevent or slow disease progression. The administering may comprise or consist of oral, parenteral, subcutaneous, intravenous, intramuscular, intranasal and / or transdermal administration.

[0214] The disclosure also provides a method of treating an ASICs-related disease, comprising administering to a subject having an ASICs-related disease a therapeutically effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein; thereby treating the ASICs-related disease. Thus, the disclosure provides a method of treating acidosis, optionally ASICs-related acidosis (e.g. metabolic acidosis) or an acidosis-related (e.g. metabolic acidosis-related) condition such as a neurodegenerative disease (e.g. Parkinson’s disease), renal failure, chronic kidney disease (abbreviated as “CKD”), acute kidney injury, Addison’s disease, or diabetes.

[0215] The disclosure also provides a method of preventing an ASICs-related disease, comprising administering to a subject having an ASICs-related disease a therapeutically effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein; thereby preventing the ASICs-related disease. Thus, the disclosure provides a method of preventing neurological disorders, neurodegenerative diseases, psychological disorders, sexual disorders, cardiovascular diseases, respiratory diseases, inflammatory diseases, renal diseases, gastrointestinal diseases, metabolic disorders, cancer, and addiction, e.g. smoking addiction. The neurological disorder may be selected from the group consisting of stroke, ischemia, traumatic brain injury, spinal cord injury, neuropathic pain, and epilepsy. The neurodegenerative disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis. The psychological disorder is selected from the group consisting of anxiety, depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), and addiction. The cardiovascular disease may be selected from the group consisting of myocardial infarction, heart failure, arrhythmia, and hypertension. The respiratory disease may be selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS). The inflammatory disease may be selected from the group consisting of rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, and sepsis. The renal disease may be selected from the group consisting of acute kidney injury, chronic kidney disease, and renal fibrosis. The gastrointestinal disease may be selected from the group consisting of gastroesophageal reflux disease (GERD), peptic ulcer disease, and irritable bowel syndrome (IBS). The metabolic disorder may be selected from the group consisting of diabetes, obesity, and metabolic syndrome. The cancer may be selected from the group consisting of glioblastoma, breast cancer, lung cancer, colorectal cancer, and prostate cancer. The addiction may be smoking addiction. Said method may prevent or slow disease progression. The administering may comprise or consist of oral, parenteral, subcutaneous, intravenous, intramuscular, intranasal and / or transdermal administration.

[0216] The disclosure also provides a method of preventing an ASICs-related disease, comprising administering to a subject having an ASICs-related disease a therapeutically effective amount of a cyclic polyarginine polypeptide or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein; thereby preventing the ASICs-related disease. Thus, the disclosure provides a method of preventing acidosis, optionally ASICs-related acidosis (e.g. metabolic acidosis) or an acidosis-related (e.g. metabolic acidosis-related) condition such as a neurodegenerative disease (e.g. Parkinson’s disease), renal failure, chronic kidney disease (abbreviated as “CKD”), acute kidney injury, Addison’s disease, or diabetes.

[0217] In embodiments of any of the methods of treating or preventing an ASICs-related disease described herein, the ASICs-related disease is an ASIC1 -related disease, optionally an ASIC1a-related disease.

[0218] In embodiments, the disclosure provides the use of a cyclic polyarginine polypeptide or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein, as an inhibitor of acid-sensing ion channels (ASICs), particularly acid-sensing ion channel 1 (ASIC1), optionally acid-sensing ion channel 1a (ASIC1a).

[0219] In embodiments, the disclosure provides a cyclic polyarginine polypeptide or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein, for use as an inhibitor of acid-sensing ion channels (ASICs), particularly acid-sensing ion channel 1 (ASIC1), optionally acid-sensing ion channel 1a (ASIC1a).

[0220] Additional embodiments include, but are not limited to, those described in clauses 1 to 27 below:

[0221] [Clause 1] A cyclic polyarginine peptide comprising 3 to 50 arginine residues or a pharmaceutically acceptable salt thereof, wherein the peptide is synthesized by a method selected from the group consisting of solid-phase peptide synthesis, liquid-phase peptide synthesis, native chemical ligation, and recombinant protein expression, and wherein the peptide is an inhibitor of acid-sensing ion channels (ASICs), particularly acid-sensing ion channel 1a (ASIC1a), for treating ASIC-related diseases.

[0222] [Clause 2] The cyclic polyarginine peptide of clause 1, wherein the ASIC-related disease is selected from the group consisting of neurological disorders, neurodegenerative diseases, psychological disorders, sexual disorders, cardiovascular diseases, respiratory diseases, inflammatory diseases, renal diseases, gastrointestinal diseases, metabolic disorders, and cancer.

[0223] [Clause 3] The cyclic polyarginine peptide of clause 2, wherein the neurological disorder is selected from the group consisting of stroke, ischemia, traumatic brain injury, spinal cord injury, neuropathic pain, and epilepsy.

[0224] [Clause 4] The cyclic polyarginine peptide of clause 2, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis.

[0225] [Clause 5] The cyclic polyarginine peptide of clause 2, wherein the psychological disorder is selected from the group consisting of anxiety, depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), and addiction.

[0226] [Clause 6] The cyclic polyarginine peptide of clause 2, wherein the cardiovascular disease is selected from the group consisting of myocardial infarction, heart failure, arrhythmia, and hypertension.

[0227] [Clause 7] The cyclic polyarginine peptide of clause 2, wherein the respiratory disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS). [Clause 8] The cyclic polyarginine peptide of clause 2, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, and sepsis.

[0228] [Clause 9] The cyclic polyarginine peptide of clause 2, wherein the renal disease is selected from the group consisting of acute kidney injury, chronic kidney disease, and renal fibrosis.

[0229] [Clause 10] The cyclic polyarginine peptide of clause 2, wherein the gastrointestinal disease is selected from the group consisting of gastroesophageal reflux disease (GERD), peptic ulcer disease, and irritable bowel syndrome (IBS).

[0230] [Clause 11] The cyclic polyarginine peptide of clause 2, wherein the metabolic disorder is selected from the group consisting of diabetes, obesity, and metabolic syndrome.

[0231] [Clause 12] The cyclic polyarginine peptide of clause 2, wherein the cancer is selected from the group consisting of glioblastoma, breast cancer, lung cancer, colorectal cancer, and prostate cancer.

[0232] [Clause 13] The cyclic polyarginine peptide of clause 1, wherein the peptide is monocyclic, bicyclic, tricyclic, tetracyclic, pentacyclic, hexacyclic, heptacyclic, octacyclic, or decacyclic.

[0233] [Clause 14] The cyclic polyarginine peptide of clause 13, wherein the monocyclic peptide offers higher potency than a linear peptide in inhibiting ASICs, particularly ASIC1 a.

[0234] [Clause 15] The cyclic polyarginine peptide of clause 13, wherein the polycyclic peptide is a novel first-in-class molecule that rapidly inhibits ASIC1a within milliseconds and induces slow receptor recovery of ASIC1a, thereby offering a wide therapeutic window for treating ASIC-related diseases.

[0235] [Clause 16] The cyclic polyarginine peptide of clause 15, wherein the polycyclic peptide is bridged directly by peptide bonds or indirectly by disulfide bridges.

[0236] [Clause 17] The cyclic polyarginine peptide of clause 1, wherein the arginine content of the peptide is between 30% and 100%, preferably 85%.

[0237] [Clause 18] A pharmaceutical composition comprising the cyclic polyarginine peptide of clause 1 and a pharmaceutically acceptable carrier, wherein the composition is formulated for administration via a route selected from the group consisting of oral, parenteral, subcutaneous, intravenous, intramuscular, intranasal, transdermal, and a combination thereof.

[0238] [Clause 19] The pharmaceutical composition of clause 18, wherein the composition is formulated as a dosage form selected from the group consisting of a tablet, a capsule, a solution, a suspension, an emulsion, a gel, and a combination thereof.

[0239] [Clause 20] The pharmaceutical composition of clause 18, wherein the composition is sterile, pyrogen-free, and has a pH between 4.0 and 8.0. [Clause 21] A method of treating an ASIC-related disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the cyclic polyarginine peptide of clause 1.

[0240] [Clause 22] The method of clause 21, wherein the cyclic polyarginine peptide is administered in combination with an additional therapeutic agent selected based on the ASIC-related disease being treated.

[0241] [Clause 23] The method of clause 23, wherein the additional therapeutic agent is administered simultaneously, sequentially, or separately from the cyclic polyarginine peptide.

[0242] [Clause 24] The cyclic polyarginine peptide of clause 1, wherein the peptide is labelled with a detectable moiety selected from the group consisting of a fluorophore, a chromophore, a radioisotope, a paramagnetic label, and a combination thereof.

[0243] [Clause 25] The cyclic polyarginine peptide of clause 25, wherein the detectable moiety is used for imaging, diagnosis, or monitoring the treatment of an ASIC-related disease in a subject.

[0244] [Clause 26] A kit comprising the cyclic polyarginine peptide of clause 1 or the pharmaceutical composition of clause 18, and instructions for use in treating an ASIC-related disease in a subject.

[0245] [Clause 27] The kit of clause 27, further comprising an additional therapeutic agent selected based on the ASIC-related disease being treated, and instructions for administering the additional therapeutic agent simultaneously, sequentially, or separately from the cyclic polyarginine peptide or the pharmaceutical composition.

[0246] [Clause 28] A cyclic polyarginine polypeptide comprising at least one SEQ ID NO: 1 motif

[0247] X(1) - X(2) - X(3) - RRR - X(4) - X(5) - X(6) (SEQ ID NO: 1)

[0248] wherein:

[0249] a. X(1) and X(6) are amino acid residues other than arginine and X(1) is stapled to X(6);

[0250] b. X(2), X(3), X(4) and X(5) are, independently, either absent or amino acid residues other than arginine; and

[0251] c. the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w;

[0252] or a pharmaceutically acceptable salt thereof.

[0253] [Clause 29] The cyclic polyarginine polypeptide according to clause 28, wherein the cyclic polyarginine polypeptide comprises at least one SEQ ID NO: 2 motif

[0254] C(1) - X(2) - X(3) - RRR - X(4) - X(5) - C(6) (SEQ ID NO: 2) wherein C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof; optionally wherein the stapling moiety is a disulfide bridge.

[0255] Sequence Information

[0256] SEQ ID NO: 1

[0257] X(1) - X(2) - X(3) - RRR - X(4) - X(5) - X(6)

[0258] X(1) and X(6) are amino acid residues other than arginine and X(1) is stapled to X(6). X(2), X(3), X(4) and X(5) are, independently, either absent or amino acid residues other than arginine.

[0259] SEQ ID NO: 2

[0260] C(1) - X(2) - X(3) - RRR - X(4) - X(5) - C(6)

[0261] C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof. X(2), X(3), X(4) and X(5) are, independently, either absent or amino acid residues other than arginine.

[0262] SEQ ID NO: 3

[0263] C(1) - RRR - C(6)

[0264] C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof.

[0265] SEQ ID NO: 4

[0266] C(1) - RRR - C(6)

[0267] C(1) is stapled to C(6) by a disulfide bridge.

[0268] SEQ ID NO: 5 (R2)

[0269] RR SEQ ID NO: 6 (R2-amide)

[0270] RR-NH2

[0271] C-terminus amide

[0272] SEQ ID NO: 7 (R3)

[0273] RRR SEQ ID NO: 8 (R3-amide)

[0274] RRR-NH2

[0275] C-terminus amide

[0276] SEQ ID NO: 9 (R9)

[0277] RRRRRRRRR SEQ ID NO: 10 (R9-amide)

[0278] RRRRRRRRR-NH2

[0279] C-terminus amide

[0280] SEQ ID NO: 11 (R18)

[0281] RRRRRRRRRRRRRRRRRR SEQ ID NO: 12 (R18-amide)

[0282] RRRRRRRRRRRRRRRRRR-NH2

[0283] C-terminus amide

[0284] SEQ ID NO: 13 (Nerinetide)

[0285] YGRKKRRQRRRKLSSIESDV SEQ ID NO: 14 (cR9)

[0286] RRRRRRRRR

[0287] Head-to-tail lactam cyclised

[0288] SEQ ID NO: 15 (3cR10ss)

[0289] RCRRRCRRCRRRCR

[0290] Head-to-tail lactam cyclised

[0291] C and C form a disulfide bridge

[0292] C and £ form a disulfide bridge

[0293] SEQ ID NO: 16 (Mamba-1 toxin) SLKCYQHGKVVTCHRDMKFCYHNTGMPFRNLKLILQGCSSSCSETENNKCCSTDRGNK

[0294] C and C form a disulfide bridge

[0295] C and £ form a disulfide bridge

[0296] G and G form a disulfide bridge

[0297] and G form a disulfide bridge

[0298] SEQ ID NO: 17 (PcTx1 toxin) EDCIPKWKGCVNRHGDCCEGLECHKRRRSFEVCVPKTPKT

[0299] C and C form a disulfide bridge C and £ form a disulfide bridge

[0300] G and G form a disulfide bridge

[0301] SEQ ID NO: 18 (Formula la)

[0302] cyclo[B-(J)m-RRR-(U)n-Z]

[0303] B, J, U and Z each represent amino acid residues other than arginine;

[0304] m and n are each independently integers from 0 to 6, wherein when m is nonzero, each instance of J may independently be the same or different, and wherein n is nonzero, each instance of U may independently be the same or different;

[0305] B and Z may independently be the same as, or different from, one another and may independently be the same as, or different from, any instance of J or U;

[0306] and B is stapled to Z.

[0307] SEQ ID NO: 19

[0308] RCRRRCRRRCRRRCRR

[0309] Head-to-tail lactam cyclised

[0310] C and C form a disulfide bridge

[0311] C and C form a disulfide bridge

[0312] Examples

[0313] General Methods

[0314] Mammalian cell culture

[0315] Wild-type Human Embryonic Kidney (HEK 293) cells were obtained from Abeam. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v / v) foetal bovine serum, 2 mM glutamine, 100 U / ml penicillin, and 0.1 mg / ml streptomycin at 37°C and 5% CO2 in a humidified incubator.

[0316] Peptide Synthesis and Purification

[0317] Peptides were synthesised using standard Fmoc solid-phase peptide synthesis (SPPS) techniques on Rink amide resin. Fmoc deprotection was carried out with 20% piperidine in DMF, and coupling reactions involved HBTU / HOBt activation in the presence of DIPEA. After completing chain assembly, the peptides were cleaved from the resin using a trifluoroacetic acid (TFA)-based cocktail along with appropriate scavengers. Cyclization, when applicable, was achieved through head-to-tail cyclization via lactam bridge formation under mild conditions, whereas polycyclization was accomplished through intramolecular disulfide formation. Crude peptides were purified by reverse-phase high-performance liquid chromatography (HPLC) and characterised using electrospray ionization mass spectrometry (ESI-MS). All peptides were obtained with a purity greater than 98%, except for cR9, which had a purity greater than 95%. Linear peptide synthesis and purification were conducted by Biomatic (Canada), whereas cyclic peptides (cR9 and 3cR10ss) were synthesised by Mimotopes Pty Ltd (Australia).

[0318] Electrophysiological recording from HEK 293 cells endogenously expressing ASIC1a by Automated patch clamp

[0319] Electrophysiological recordings were performed utilizing ensemble microfluidic plates on the lonFlux-16TM System (Fluxion Biosciences, USA) (Golden etal., 2011; Spencer et al., 2012; Chen et al., 2012), allowing simultaneous measurement of 16 currents as duplicates from 2 ‘traps’ under varying experimental conditions. Using ‘ensemble’ plates, recording total whole-cell current from up to 20 cells for each recording. Precise solution changes were achieved via pneumatically controlled microfluidics, resulting in exchange times of under 50 ms. A continuous pH 7.5 extracellular solution (ECS) flowed over patch-clamped cells during experiments. HEK 293 cells were washed with PBS, detached with TrypLE or Cell-Stripper for 3 mins, centrifuged at 1000 rpm for 2 min, and resuspended in ECS (in mM: NaC1 138, KCI 4, MgCI2 1, CaCI2 1.8, HEPES 10, glucose 5.6) at 1.5x106 cells / mL for experiments. For ECS with pH <6.5, MES buffer replaced HEPES, resulting in ECS-MES at pH 4.03. ECS pH 7.5 aliquots were adjusted to pH 6.5, 6.0, 5.5, and 5.0 by blending ECS (MES) and ECS (HEPES). The pH of the test compound was verified post-reconstitution in control solutions, and adjustments were made as necessary with NaOH or HCI. A 250 pL volume of resuspended cells was transferred to each “Inlet’ well of 8 experimental patterns on lonFlux plates, preloaded with these solutions: Trap wells 1 and 2 with 250 pL intracellular solution (ICS) (in mM: NaCI 15, KCI 60, KF 70, EGTA 5, HEPES 5, pH 7.25), Compound well 8 (C8) with ECS. Other solutions, including ECS with pH 6 or 6.5 for C1, and test compounds with pH 6 or 6.5, were added to wells C2-C7 for specified period applications. In pre-incubation protocols, compounds were added at pH 7.5, followed by direct application of acidic pH. Whole-cell currents from populations of up to 20 cells, voltage-clamped at -70 mV, were recorded. pH alterations lasted 3 s, with 2- to 3-minute ECS (pH 7.5) washes between applications. All recordings were carried out at room temperature (22-25°C).

[0320] Electrophysiological recording from HEK 293 cells endogenously expressing ASIC1a by Manual patch clamp

[0321] Recordings were performed using a HEKA EPC 10 USB amplifier with built-in LIH 8+8 data acquisition, controlled via PatchMaster NEXT software. The setup included an Olympus IX73 inverted microscope, a SENSAPEX uMp micromanipulator for precision pipette positioning, and a TMC CleanBench, anti-vibration table enclosed in a Faraday cage to minimize electrical noise. Patch pipettes were fabricated using a Sutter P-1000 pipette puller and polished with a Warner MFG-5 microforge. Cells were recorded in a Warner RC-26G recording chamber mounted on a Warner PM-1 platform and maintained at room temperature (22-25 °C). HEK 293 cells were cultured under standard conditions and maintained as adherent monolayers on glass-bottom dishes or 12-mm poly-D-lysine-coated coverslips, placed in Petri dishes. On the day of recording, culture media were gently aspirated, and cells were washed twice and then continuously perfused with extracellular solution (ECS) at room temperature (22-25 °C) during the experiment. Both ECS and ICS (Pipette solution) compositions were the same as for the automated patch clamp. Patch pipettes with a resistance of 2-4 MO were filled with ICS and used to obtain whole-cell configuration under visual control via the Olympus IX73 microscope. Cells were continuously perfused with ECS (pH 7.5) using a Warner VCS-6 perfusion system to maintain baseline conditions. Acidic ECS (pH 6.5 or 6.0) or compound-containing solutions were rapidly applied using a Warner SH-27B in-line heater with perfusion lines connected to a VC3-8 valve controller, allowing for fast and reproducible exchange (<50 ms). Each acidic application was applied for 3 seconds, followed by a 2-10 minutes washout with ECS (pH 7.5). Where indicated, test compounds were pre-applied in ECS pH 7.5 for a designated time before acid application.

[0322] MTT assay

[0323] Diluted HEK 293 cells were prepared, and 150 pL were seeded into each well of a 96-well plate, incubated overnight at 37 °C with 5% CO2. Compounds included 0.5 pg / ml PcTx1, 30 pM R18, and 30 pM cR9 in pH 6.0 solution, with final pH adjusted. The medium over the cells was aspirated, and 150 pL of drug solutions were added and incubated for 12h at 37 °C with 5% CO2. After 12 hours, plates were taken out, solutions aspirated, and replaced with 70 pL fresh medium. Next, 50 pL of MTT dye solution (10 mg of MTT in 2 mL PBS diluted with 5 mL ECS) was added to each well. The plate was wrapped in foil and incubated for 3 hours at 37°C with 5% CO2. After incubation, the medium was aspirated and replaced with 150 pL DMSO to dissolve formazan crystals. Plates were incubated for 30 minutes at room temperature, and then the absorbance was measured at 550 nm using the FLUOstar Omega microplate reader.

[0324] Data analysis and quantification

[0325] For automated patch clamp, whole-cell currents were analysed using lonFlux Data Analyzer software. Further analysis was performed using GraphPad Prism for EC50 calculations, employing nonlinear regression curve-fitting to the Hill equation. To present representative time-course data, sweeps were filtered using the data analyzer and then exported to Clampfit software. Using Clampfit, baselines were adjusted and annotated using Microsoft PowerPoint. On the other hand, Clampfit software was used for the manual patch clamp. Peak currents were exported to Microsoft Excel for averaging and calculation purposes.

[0326] In the analysis of data derived from the MTT assay, the absorbance (A) readings obtained from biological triplicate samples were normalised by subtracting the blank absorbance value from all recorded absorbance values. Statistical significance was assessed utilizing Student’s t-test in GraphPad Prism, with all data being presented as means ± SEM.

[0327] Example 1: L-arqinine and its derivatives (-amino-arginine, and L-arginine amide)

[0328] Mamba 1 toxin (SEQ ID NO: 14) and the PcTx1 (SEQ ID NO: 13) have an arginine residue at position 28 which is one of the most important interacting toxin residues with ASIC1a, as previously reported in site-directed mutagenesis studies. These toxins are potent inhibitors of ASIC1 a, with IC50values of approximately 5 nM for PcTx1 and 130 nM for mamba-1 toxin. Although these toxins come from different families and their amino acid sequences are entirely distinct, with sequence similarity less than 3%, point mutations of the arginine residue at position 28 (R28A) in both toxins resulted in a significant decrease in their binding affinities to the ASIC1a receptor (Saez, et al., 2015, British journal of pharmacology, 172(20), pp.4985-4995; Mourier, et al., 2016, Journal of Biological Chemistry, 291(6), pp.2616-2629; Sun, et al., 2018, Cell discovery, 4(1), pp.1-11). In addition, several ASIC1 modulators, such as amiloride, GMQ, Agmatine, and Arcaine, are based on the guanidine moiety, the functional moiety of L-Arginine.

[0329] L-arginine (hydrochloride) and L-Arginine amide (dihydrochloride) were purchased from Sigma Aldrich (NSW, Australia) with purity > 98.0%. NG-amino-L-arginine (hydrochloride) was purchased from Cayman Chemical with a purity of greater than 97.0%.

[0330] Experimental example:

[0331] HEK-293 cells were pre-incubated with 10 mM l-arginine in ECS for 90 minutes, while the control (untreated) cells were incubated in ECS. After 90 minutes, the cells were washed with ECS and centrifuged twice before the experiment. As shown in Figure 9, incubation of HEK 293 cells with 10 mM l-arginine resulted in a significant 80 ± 4% inhibition of currents induced by pH levels of 6.5, 5.5, and 4.5 compared to the control. The same experimental procedures were performed for NG-amino-arginine and L-arginine amide, where NG-amino-arginine showed no effect (loss of activity), while L-arginine amide exhibited a more potent effect (enhanced activity) (Figure 10). Conclusions:

[0332] Contrary to prior literature that described the neuroprotective effect of L-arginine attributable to its function as a biological precursor for Nitric Oxide (NO), the present results showed a new role for L-arginine as an ASIC1 inhibitor.

[0333] These findings highlighted the high importance of the free guanidine moiety and the significance of having an amide C-terminal end instead of a carboxylic C-terminal end.

[0334] Example 2: Linear peptides (R2, R2-amide, R3, R3-amide, R9, R9-amide, R18, R18-amide, and Nerinetide)

[0335] Nona-L-arginine (R9 - SEQ ID NO: 9) were purchased with a purity of > 98%. Nerinetide (SEQ ID NO: 12) was purchased with a purity of > 98%. R2 (SEQ ID NO: 5), R2-amide (SEQ ID NO: 6), R3 (SEQ ID NO: 7), R3-amide (SEQ ID NO: 8), R9-amide (SEQ ID NO: 10), R18 (SEQ ID NO: 11), and R18-amide (SEQ ID NO: 12) were custom-synthesised using standard Fmoc solid-phase peptide synthesis (SPPS) techniques. The peptides were cleaved from the resin and purified using reverse-phase high-performance liquid chromatography (RP-HPLC) to a purity of > 98%. The identity and molecular weight of the peptide were confirmed by electrospray ionization mass spectrometry (ESI-MS) (Figures 1-4). Peptides were stored as lyophilised powder and dissolved in aqueous buffer for biological assays.

[0336] Experimental example:

[0337] HEK-293 cells were voltage-clamped at -70 mV, and acid-evoked currents were recorded in response to extracellular pH changes from 7.4 to 6.5 or 6.0. The linear peptides R2 (SEQ ID NO: 5), R2-amide (SEQ ID NO: 6), R3 (SEQ ID NO: 7), and R3-amide (SEQ ID NO: 8) were initially applied at acidic pH to determine whether they can act immediately or require pre-incubation. Then, all peptides were pre-applied to the cells at varying concentrations (100 pM to 1000 pM) before the experiment. On the other hand, R9 (SEQ ID NO: 9), R9-amide (SEQ ID NO: 10), R18 (SEQ ID NO: 11), R18-amide (SEQ ID NO: 12), and Nerinetide (SEQ ID NO: 13) were applied extracellularly at concentrations ranging from 1 pM to 25 pM, 2-3 minutes prior to the pH drop.

[0338] None of these linear peptides showed any modulatory effect when co-applied at acidic pH (Figure 11). This indicates that these peptides are not fast-acting and have at least low activity in acidic conditions.

[0339] R2, R2-amide, R3, and R3-amide showed partial inhibition at pH 6.5, but only with chronic application for at least 90 minutes. R9 demonstrated partial inhibition at pH 6.5 (Figure 13), while R9-amide exhibited stronger inhibition than R9 (Figure 20). This indicates that peptides with amide ends are more potent than those with carboxylic ends, indicating potential for cyclic peptides.

[0340] R18 and R18-amide (0.3-5 pM) showed partial inhibition at pH 6.5, and the inhibition was fully recovered within 5-6 minutes after washout (Figures 14 and 16), indicating a narrow therapeutic window for these peptides. However, they paradoxically activated currents in a dose-dependent manner when the pH shifted from 7.4 to 8.0 (Figure 27) and enhanced the currents at low concentrations (100 nM) (Figure 25). This suggests that linear long arginine peptides lack consistent ASIC1a blocking activity and may have pH-dependent off-target effects.

[0341] Nerinetide (25-100 pM) exhibited partial inhibition at pH 6.0 (Figure 15), but enhanced the currents when applied at low concentrations (5 pM) (Figure 26). This confirms that linear long arginine peptides lack consistent ASIC1a blocking activity and may display pH-dependent off-target effects, as seen with R18. Additionally, the inhibition fully recovered after 5 to 6 minutes post-washout, which further indicates the narrow therapeutic window of these peptides, as observed with R18.

[0342] Conclusions:

[0343] These results highlight the limited effect associated with short arginine polypeptides and the disadvantageous effects associated with extended arginine-rich peptides on ASIC1 receptors, specifically R18 and Nerinetide. Both R18 and Nerinetide showed the capacity to enhance the ASIC1 receptors at reduced concentrations and their ability to activate the receptors at diverse pH levels (Figures 25, 26, and 27). The distinct unfavorable dynamics associated with the activation and potentiation of ASIC1 receptors by these peptides may result in divergent clinical outcomes.

[0344] Example 3: Cyclic nona-L-arginine peptide (cR9)

[0345] The monocyclic peptide cR9 (Figure 7 - SEQ ID NO: 14), composed of nine arginine residues with a head-to-tail cyclization, was synthesised using standard Fmoc solid-phase peptide synthesis (SPPS) chemistry. Following chain assembly on a Rink amide resin, the terminal amine and carboxylic acid were cyclised via lactam formation while the peptide was still resin-bound. After cleavage and side-chain deprotection with a TFA cocktail, the crude product was purified by RP-HPLC, and the purified cyclic peptide was verified by ESI-MS (Figure 5). The peptide was stored as a lyophilised powder and then dissolved in an aqueous buffer for biological assays, which were used in subsequent electrophysiology experiments. Experimental example:

[0346] HEK-293 cells were voltage-clamped at -70 mV, and acid-evoked currents were recorded in response to extracellular pH changes from 7.4 to 6.5. The cR9 was initially applied at acidic pH to determine whether it can act immediately or require pre-incubation. Then, the compound was applied extracellularly at concentrations ranging from 0.1 pM to 10 pM, 2-3 minutes prior to the pH drop.

[0347] cR9 did not work immediately at acidic pH, but showed strong inhibition of pH 6.5-induced currents when pre-applied for 2 minutes, exceeding that of the linear R9 (Figure 21). A comparative study showed that cR9 inhibited with an EC50 significantly lowerthan the linear R9 and was as potent as the twice-longer linear peptide, R18 (Figures 22 and 23). Additionally, cR9 demonstrated the ability to protect HEK293 cells from acid-mediated cell death, which R18 did not exhibit. This indicates that cyclic peptides are significantly more potent than their linear counterparts, suggesting that the conformation of the peptide may be important.

[0348] Conclusions:

[0349] These results illustrated the benefits of cyclisation regarding potency and stability in response to pH fluctuations. Nevertheless, cR9 still lack the capacity to exert effects upon immediate application.

[0350] Example 4: Tricyclic polyarginine peptide (c3R10ss)

[0351] The tricyclic polyarginine peptide (Figure 8) consists of 10 arginine residues (R10) separated by four cysteine residues and was cyclised head-to-tail. The two disulfide bridges (ss) between the cysteine residues then formed the tricyclic structure (3c). This peptide was designed in view of the previous results and taking into account the conformation of PcTx1 (SEQ ID NO: 17) interactions with the ASIC1 a acidic pocket (Baconguis, and Gouaux, Nature, vol. 489, pp. 400-405, 2012; PDB IDs: 4FZ0 and 4FZ1) to mimic the conformation of the three arginines in this acidic pocket.

[0352] The 3cR10ss peptide (Figure 8 - SEQ ID NO: 15 - Formula 6) was synthesised using Fmoc-SPPS with orthogonally protected cysteine residues (e.g., Acm, Trt) incorporated at three positions along the peptide sequence. After cleavage from the resin and global deprotection, disulfide bridges were formed by controlled air oxidation or iodine-mediated oxidation, resulting in a rigid tricyclic structure. The final product was purified by preparative RP-HPLC with >98% purity and verified by high-resolution ESI-MS (Figure 6). The peptide was stored as a lyophilised powder and then dissolved in an aqueous buffer for biological assays, which were used in subsequent electrophysiology experiments. Experimental example:

[0353] HEK-293 cells were voltage-clamped at -70 mV, and acid-evoked currents were recorded in response to changes in extracellular pH from 7.4 to 6.5 and 6.0. The 3cR10ss was initially applied at acidic pH to determine whether it can act immediately or require preincubation. Then, the compound was applied extracellularly at concentrations ranging from 0.1 pM to 25 pM, 2-3 minutes prior to the pH drop.

[0354] 3cR10ss was the first peptide to demonstrate immediate inhibitory effects upon coapplication at acidic pH (Figure 12). Neither R9 nor R18 exhibited such immediate inhibition under identical conditions. Furthermore, 3cR10ss was the first to demonstrate that slow recovery can still occur even after immediate co-application. The cR10ss also exhibited more pronounced inhibition when preapplied for two minutes (Figure 17), and the recovery period extended upto 40 minutes of washout, allowing complete recovery (Figure 18). Since all these pharmacokinetic features are novel and previously unreported, this underscores 3cR10ss as a first-in-class ASIC1 inhibitor. Moreover, the 3cR10ss peptide demonstrated similar behaviour at a lower pH (pH 6.0). Most notably, unlike R18 and Nerinetide, the 3cR10ss peptide remained stable across different conditional pH levels (pH 8.0) (Figure 28).

[0355] Conclusions:

[0356] These results substantiate the hypothesis that cyclic peptides are considerably more potent than their linear counterparts and that the length of the peptide chain is less critical than the cyclic conformation of the peptide.

[0357] These results further suggest that the peptide's conformation can elicit varying dynamics with the receptor exhibiting the attributes of small molecules to inhibit ASIC1 receptors in a millisecond timeframe, alongside the characteristics of larger toxins that lead to a gradual recovery of the receptor (Figures 16, 12, 17, 18, and 19). This phenomenon showcases a rapid effect coupled with a sustained action, resulting in a wider therapeutic window.

[0358] Without being bound by theory, the constrained arginine finger motif as described herein in SEQ ID NO:1, 2, 3 or 4 is considered to be responsible for the enhanced activity of 3cR10ss (Figure 8 - SEQ ID NO: 15 - Formula 6) which comprises 2 such motifs.

[0359] These findings contradict the conclusion presented in the study by Meloni, et al. (Neuromolecular Medicine, vol. 19, pp. 271-285, 2017), which suggests that the structural conformations associated with linear peptides do not play a critical role in contributing to the neuroprotective effect of the peptide. Furthermore, the same study also concluded that there is no significant difference between cyclic and linear peptides. Example 5: Glutamic acid

[0360] Glutamic acid was purchased with a purity of> 98.0%.

[0361] Experimental example:

[0362] HEK-293 cells were voltage-clamped at -70 mV, and acid-evoked currents were recorded in response to alterations in extracellular pH from 7.4 to 6.5 and 6.0. Glutamic acid was applied immediately and pre-infused at concentrations ranging from 500 pM to 15 mM to evaluate whether it acts instantaneously or requires prolonged exposure.

[0363] In all examined conditions, glutamic acid was demonstrated to potentiate acid-induced currents, suggesting that elevations in glutamic acid exacerbate clinical conditions through the augmentation of acidic currents mediated by ASIC1 receptors (Figure 29). Furthermore, glutamic acid has been shown to enhance acid-induced cellular death in HEK293, thereby confirming its role in ASIC1 -related cell death (Figure 29).

[0364] Conclusions:

[0365] The aforementioned findings demonstrate the significance of the polyarginine polypeptides of the disclosure in protecting against glutamate and acid-induced cellular apoptosis.

[0366] Example 6: Nicotine

[0367] Nicotine was purchased with a purity of> 99.0%.

[0368] Experimental example:

[0369] HEK-293 cells were voltage-clamped at -70 mV, and acid-evoked currents were recorded in response to alterations in extracellular pH from 7.4 to 6.5 and 6.0. Nicotine was applied immediately at concentrations ranging from 500 pM to 10 mM to evaluate whether it acts instantaneously or requires prolonged exposure.

[0370] Nicotine was demonstrated to potentiate acid-induced currents for the first time, suggesting that elevations in nicotine can augment acid-induced currents and thereby worsen clinical condition of smoking addiction (Figure 30).

[0371] Conclusions:

[0372] The findings demonstrate that ASIC1 inhibitors and in particular the polyarginine polypeptides of the disclosure can provide protection against nicotine-induced cellular injury and may facilitate treatment of smoking addiction. Example 7: 3cR10ss (SEQ ID NO: 15 - Formula 6) preserves cellular ATP levels under acidic stress in HEK 293 cells

[0373] Experimental example:

[0374] ATP content in HEK 293 cells was quantified by a luminescence assay after 24 h treatment under the following conditions: physiological pH 7.5, acidic pH 5.5, pH 5.5 + 3cR10ss 5 pM (P5), and pH 5.5 + 3cR10ss 20 pM (P20).

[0375] Acidic exposure alone (pH 5.5) caused a marked reduction in ATP levels compared to neutral pH 7.5 (p < 0.01). Co-treatment with 3cR10ss (SEQ ID NO: 15 - Formula 6) partially (5 pM) or fully (20 pM) preserved ATP content relative to the acidified control (Figure 31).

[0376] Conclusions:

[0377] The findings demonstrate that ASIC1 inhibitors and in particular the polyarginine polypeptides of the disclosure can provide protection against cellular acidic stress and may facilitate treatment of acidosis.

[0378] Example 8: 3cR10ss (SEQ ID NO: 15 - Formula 6) selectively reduces mitochondrial and total ROS in mitoPRS neuronal cultures

[0379] Experimental example:

[0380] Mitochondrial ROS levels measured by MitoSOX Red fluorescence in high- and low-mitoPRS neuronal cultures following 24 h treatment with vehicle, 3cR10ss (10 pM), exenatide (10 pM), or semaglutide (10 pM). Total intracellular ROS was quantified using DCFH-DA under the same conditions.

[0381] Conclusions:

[0382] Activity of the clinically approved GLP-1 receptor agonists exenatide and semaglutide, and 3cR10ss (SEQ ID NO: 15 - Formula 6) were compared under identical neuronal culture conditions on mitochondrial polygenic risk score (mitoPRS) iPSC-derived neuronal models from Parkinson’s Disease patients. Neither clinically approved GLP-1 receptor agonists produced statistically significant changes in mitochondrial ROS (MitoSOX fluorescence) in mitoPRS neuronal lines (p < 0.05, n = 3 independent replicates). In contrast, treatment with 3cR10ss selectively modulated mitochondrial parameters in mitoPRS neurons, reducing total (Figure 32B) and mitochondrial ROS (Figure 32A), confirming context-dependent mitochondrial protection.

[0383] These findings indicate that 3cR10ss (SEQ ID NO: 15 - Formula 6) acts through a rapid, ASIC1a-linked ion-channel mechanism, distinct from the slower transcriptional or receptor-mediated signalling of GLP-1 analogues. ASIC1a inhibition by 3cR10ss reduces basal oxidative load in metabolically vulnerable neuronal subtypes, consistent with selective mitochondrial stabilization and redox protection in a Parkinson’s-related mitochondrial risk background and may therefore facilitate treatment of Parkinson’s disease.

[0384] Example 9: 3cR12ss (SEQ ID NO: 19 - Formula 7)

[0385] Cyclic peptide 3cR12ss (Formula 7) is prepared analogously to 3cR10ss as described in Example 4. Thus, the peptide is prepared using Fmoc-based solid phase peptide synthesis (SPPS) with orthogonally protected cysteine residues (e.g., Acm, Trt) incorporated along the peptide sequence. After cleavage from the resin and global deprotection, disulfide bridges are formed by controlled air oxidation or iodine-mediated oxidation. The final product is purified by preparative RP-HPLC with >98% purity and verified by high-resolution ESI-MS. LC-MS and / or analytical HPLC may be used to confirm the molecular weight and folding state. The peptide may be stored as a lyophilised powder and then dissolved in an aqueous buffer for biological assays.

Claims

Claims1. A cyclic polyarginine polypeptide comprising at least one SEQ ID NO: 3 motifC(1) - RRR - C(6) (SEQ ID NO: 3)wherein:a. C(1) is stapled to C(6) by a stapling moiety selected from a bidentate stapling moiety, a disulfide bridge, a tridentate stapling moiety, a tetradentate stapling moiety, a pentadentate stapling moiety, a hexadentate stapling moiety or a combination thereof; andb. the arginine content of the cyclic polyarginine polypeptide is at least 40% w / w; or a pharmaceutically acceptable salt thereof.

2. The cyclic polyarginine polypeptide according to claim 1, wherein the stapling moiety is a disulfide bridge.

3. The cyclic polyarginine polypeptide according to claim 2, wherein the cyclic polyarginine polypeptide comprises a plurality of SEQ ID NO: 3 motifs, optionally wherein the cyclic polyarginine polypeptide comprises at least 2, at least 3, at least 4, at least 5 or at least 6 SEQ ID NO: 3 motifs, further optionally wherein the cyclic polyarginine polypeptide comprises at least 2 SEQ ID NO: 3 motifs.

4. The cyclic polyarginine polypeptide according to any of the preceding claims, further comprising between 3 and 50 arginine amino acid residues, optionally wherein the cyclic polyarginine polypeptide further comprises between 3 and 30 arginine amino acid residues.

5. The cyclic polyarginine polypeptide according to any of the preceding claims, wherein the polypeptide further comprises a spacer located at the N-terminus and / or C-terminus of the polypeptide, optionally the spacer is a flexible spacer comprising: S, G, GS, A, Ahx, Ado, PEG, Sar, gamma-E, repetition and / or combination thereof.

6. The cyclic polyarginine polypeptide according to any of the preceding claims, wherein the polypeptide N-terminus or the spacer located at the N-terminus of the polypeptide and the polypeptide C-terminus or the spacer located at the C-terminus of the polypeptide are covalently bound; optionally wherein they are covalently bound by an amide bond.

7. The cyclic polyarginine polypeptide according to any of the preceding claims, wherein the cyclic polyarginine polypeptide is SEQ ID NO: 15.

8. The cyclic polyarginine polypeptide according to any of the preceding claims, wherein the arginine content of the cyclic polyarginine polypeptide is at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, at least 90% w / w or at least 95% w / w, optionally wherein the arginine content of the cyclic polyarginine polypeptide is at least 80% w / w.

9. The cyclic polyarginine polypeptide according to any of the preceding claims, further comprising at least one detectable label, optionally wherein the at least one detectable label may be selected from:a. enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase, e.g., horseradish peroxidase;b. dyes;c. fluorescent labels, such as fluorescein and its derivatives, fluorochrome, rhodamine compounds and derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine;d. fluorophores such as lanthanide cryptates and chelates;e. chemoluminescent labels, such as isoluminol, luminol and the dioxetanes;f. bio-luminescent labels, such as luciferase and luciferin;g. sensitizers;h. coenzymes;i. enzyme substrates;j. radiolabels, such as bromine77, carbon14, cobalt57, fluorine8, gallium67, gallium68, hydrogen3(tritium), indium111, indium113, iodine123, iodine125, iodine126, iodine131, iodine133, mercury107, mercury203, phosphorous32, rhenium99, rhenium101, rhenium105, ruthenium95, ruthenium97, ruthenium103, ruthenium105, scandium47, selenium75, sulphur35, technetium99, technetium99, tellurium121, tellurium122, tellurium125, thulium165, thulium167, thulium168, yttrium199;k. paramagnetic labels, such as nitroxide spin labels, e.g. methanethiosulfonate spin label (MTSSL), or lanthanide-chelating tags;l. particles, such as latex or carbon particles; andm. contrast agents;optionally wherein the at least one detectable label is covalently attached via a linker, spacer and / or anchor group, e.g. a cleavable linker.

10. The cyclic polyarginine polypeptide according to any of the preceding claims, wherein the cyclic polyarginine polypeptide is an inhibitor of acid-sensing ion channels (ASICs), optionally acid-sensing ion channel 1a (ASIC1a).

11. A pharmaceutical composition comprising a cyclic polyarginine polypeptide according to any of claims 1-10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.

12. The cyclic polyarginine polypeptide according to any of claims 1-10 or the pharmaceutical composition according to claim 11 for use in therapy.

13. The cyclic polyarginine polypeptide or the pharmaceutical composition for use in the treatment of an ASIC-related disease in a subject.

14. The cyclic polyarginine polypeptide or the pharmaceutical composition for use according to claim 13, wherein the ASIC-related disease is selected from neurological disorders, neurodegenerative diseases, psychological disorders, sexual disorders, cardiovascular diseases, respiratory diseases, inflammatory diseases, renal diseases, gastrointestinal diseases, metabolic disorders, cancer, and addiction, e.g. smoking addiction.

15. The cyclic polyarginine polypeptide or the pharmaceutical composition for use according to claim 14, wherein:a. the neurological disorder is selected from the group consisting of stroke, ischemia, traumatic brain injury, spinal cord injury, neuropathic pain, and epilepsy;b. the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis;c. the psychological disorder is selected from the group consisting of anxiety, depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), and addiction;d. the cardiovascular disease is selected from the group consisting of myocardial infarction, heart failure, arrhythmia, and hypertension;e. the respiratory disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS);f. the inflammatory disease is selected from the group consisting of rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, and sepsis;g. the renal disease is selected from the group consisting of acute kidney injury, chronic kidney disease, and renal fibrosis;h. the gastrointestinal disease is selected from the group consisting of gastroesophageal reflux disease (GERD), peptic ulcer disease, and irritable bowel syndrome (IBS);i. the metabolic disorder is selected from the group consisting of diabetes, obesity, and metabolic syndrome;j. the cancer is selected from the group consisting of glioblastoma, breast cancer, lung cancer, colorectal cancer, and prostate cancer; and / ork. the addiction is smoking addiction.

16. The cyclic polyarginine polypeptide or the pharmaceutical composition for use according to claim 13, wherein the ASIC-related disease is acidosis (e.g. metabolic acidosis) or an acidosis-related (e.g. metabolic acidosis-related) condition such as a neurodegenerative disease (e.g. Parkinson’s disease), renal failure, chronic kidney disease (abbreviated as “CKD”), acute kidney injury, Addison’s disease, or diabetes.

17. The cyclic polyarginine polypeptide or the pharmaceutical composition for use according to any of claims 13-16, wherein the cyclic polyarginine polypeptide or the pharmaceutical composition is administered to the subject via oral, parenteral, subcutaneous, intravenous, intramuscular, intranasal and / or transdermal administration route.

18. A method of treatment comprising the administration of a cyclic polyarginine polypeptide according to any of claims 1-10 or a pharmaceutical composition according to claim 11 to a subject.

19. The method of treatment according to claim 18, wherein the treatment is for an ASIC- related disease in a subject.

20. Use of a cyclic polyarginine polypeptide according to any of claims 1-10 ora pharmaceutical composition according to claim 11 in the manufacture of a medicament.

21. The use according to claim 20, wherein the medicament is for the treatment of an ASIC- related disease in a subject.

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